Abstract: Rats depleted of long-chain polyunsaturated n-3 fatty acids (n-3-D) display several features of the metabolic syndrome, including obesity, liver steatosis, insulin resistance, hypertension, and cardiac hypertrophy. In this study, the heart phospholipid (PL) and triacylglycerol (TG) fatty acid content and pattern were compared between female control rats (C) and n-3-D rats. The sole n-3 fatty acids found in n-3-D rats, C22:5(n-3) and C22:6(n-3), were 10 to 20 times lower than in C. The total fatty acid content of PL was lower in n-3-D rats than C. No ectopic TG accumulation was found in n-3-D rats. In both PL and TG, the C16:0/C16:1(n-7) and C18:0/C18:1(n-9) ratios suggested increased Delta9-desaturase activity in n-3-D rats. The PL C18:2(n-6)/C20:4(n-6) and C20:4(n-6)/C22:4(n-6) ratios were also lower in n-3-D rats than C. Prior intravenous injection of a medium-chain TG:fish oil emulsion to n-3-D rats 60 to 120 minutes before killing augmented the PL content in C22:5(n-3) and C22:6(n-3), minimized the age-related decrease in the PL C18:1(n-9) relative content, and increased the TG C22:4(n-6) content. The alteration of cardiac function in n-3-D rats and its improvement after injection of medium-chain TG:fish oil emulsion coincides with parallel changes in heart lipid fatty acid content and pattern.

Abstract: Rats depleted in long-chain polyunsaturated omega3 fatty acids (omega3-depleted rats) display several features of the metabolic syndrome including hypertension and cardiac hypertrophy. This coincides with alteration of the cardiac muscle phospholipid and triacylglycerol fatty acid content and/or pattern. In the present study, the latter variables were measured in the cardiac endothelium of normal and omega3-depleted rats. Samples derived from four rats each were obtained from 16 female normal fed rats and three groups of 36-40 female fed omega3-depleted rats each aged 8-9, 15-16 and 22-23 weeks. At comparable mean age, the ratio between the square root of the total fatty acid content of phospholipids and cubic root of the total fatty acid content of triacylglycerols was lower in omega3-depleted rats than in control animals. The total fatty acid content of triacylglycerols was inversely related to their relative content in C20:4omega6. Other differences between omega3-depleted rats and control animals consisted in a lower content of long-chain polyunsaturated omega3 fatty acids in both phospholipids and triacylglycerols, higher content of long-chain polyunsaturated omega6 fatty acids in phospholipids, higher activity of delta9-desaturase (C16:0/C16:1omega7 and C18:0/C18:1omega9 ratios) and elongase [(C16:0 + C16:1omega7)/(C18:0 + C18:1omega9) and C20:4omega6/C22:4omega6 ratios], but impaired generation of C22:6omega3 from C22:5omega3 in the former rats. These findings support the view that cardiovascular perturbations previously documented in the omega3-depleted rats may involve impaired heart endothelial function.

Abstract: The catabolism of D-glucose was recently found to be impaired in pancreatic islets from rats depleted in long-chain polyunsaturated omega3 fatty acids. The specificity of this alteration was now investigated by characterizing the oxidative fate of endogenous nutrients in islets preincubated with either L-[U-14C]glutamine or [U-14C]palmitate and then incubated variously in the absence of D-glucose, presence of the hexose or presence of metabolic poisons. Relative to their radioactive content after preincubation, the production of 14CO2 by islets prelabelled with [U-14C]glutamine was higher in omega3-depleted rats than control animals. The enhancing action of D-glucose upon such production was impaired, however, in the omega3-depleted rats. The net uptake of 14C-palmitate and absolute value for 14CO2 output were both increased in omega3-depleted rats, whilst the ratio between 14CO2 output and islet radioactive content was decreased in the same animals. The inhibition of 14CO2 production by metabolic poisons was comparable in all cases. These results are consistent with recent findings on such items as the availability of endogenous amino acids and uptake of unesterified fatty acids in extrapancreatic sites of the omega3-depleted rats. They also support the view that the alteration of D-glucose metabolism in the islets of the latter animals may be attributable, in part at least, to alteration of glucokinase kinetics by high intracellular acyl-CoA levels.

Abstract: The metabolism of D-glucose was recently reported to be impaired in pancreatic islets from second generation rats depleted in long-chain polyunsaturated omega3 fatty acids. Considering the increased clearance of circulating non-esterified fatty acids prevailing in these rats, a possible inhibition of glucokinase in insulin-producing cells by endogenous long-chain fatty acyl-CoA was considered. The present study was mainly aimed at assessing the validity of the latter proposal. The activity of glucokinase in islet homogenates, as judged from the increase in D-glucose phosphorylation rate in response to a rise in the concentration of the hexose represented, in the omega3-depleted rats, was only 81.8 +/- 4.8% (n = 11; p < 0.005) of the paired value recorded in control animals. This coincided with the fact that the inclusion of D-glucose 6-phosphate (3.0 mM) and D-fructose 1-phosphate (1.0 mM) in the assay medium resulted in a lesser fractional decrease of D-glucose phosphorylation in omega3-depleted rats than in control animals. Moreover, whereas palmitoyl-CoA (50 microM) decreased the activity of glucokinase by 38.0 +/- 6.0% (n = 4; p < 0.01) in islet homogenates from normal rats, the CoA ester failed to affect significantly the activity of glucokinase in islet homogenates from omega3-depleted rats. These findings afford direct support for the view that glucokinase is indeed inhibited by endogenous long-chain fatty acyl-CoA in islets from omega3-depleted rats, such an inhibition probably participating to the alteration of D-glucose catabolism prevailing in these islets.

Abstract: The bolus intravenous injection of a novel medium-chain triglyceride:fish oil emulsion to normal subjects was recently reported to enrich within 60 min the phospholipid content of leucocytes and platelets in long-chain polyunsaturated omega3 fatty acids. The present study, conducted in second generation omega3-depleted rats, aims at investigating whether such a procedure may also increase within 60 min the phospholipid content of omega3 fatty acids in cells located outwards of the bloodstream, in this case liver cells, and whether this coincides with correction of the perturbation in the liver triglyceride fatty acid content and profile otherwise prevailing in these rats. The results indicate that such is indeed the case and further suggest a cause-to-effect relationship between the two events.

Abstract: The bolus intravenous injection of a novel medium-chain triglyceride:fish oil emulsion to normal subjects was recently reported to enrich within 60 min the phospholipid content of leucocytes and platelets in long-chain polyunsaturated omega3 fatty acids. The present study, conducted in second generation omega3-depleted rats, aimed at investigating whether such a procedure may also increase within 60 min the phospholipid content of omega3 fatty acids in cells located outwards the bloodstream, in this case liver cells, and whether this coincides with correction of the perturbation in the liver triglyceride fatty acid content and profile otherwise prevailing in these rats. This first report deals mainly with the fatty acid pattern of plasma lipids in male omega3-depleted rats that were non-injected or injected with either the omega3-rich emulsion or a control medium-chain triglyceride:olive oil emulsion. The results provide information on the fate of the exogenous lipids present in the lipid emulsions and injected intravenously 60 min before sacrifice. Moreover, in the uninjected omega3-depleted rats the comparison between individual plasma and liver measurements indicated positive correlations in the fatty acid profile of phospholipids and triglycerides.

Abstract: In order to investigate the possible link between PMCA (plasma-membrane Ca(2+)-ATPase) activity and D-glucose catabolism in insulin-producing cells, BRIN-BD11 cells were transfected with two isoforms of PMCA2. Transfection of insulin-producing BRIN-BD11 cells with PMCA2yb and PMCA2wb was documented by RT-PCR (reverse transcription-PCR), Western blot analysis, indirect immunofluorescence microscopy and (45)Ca(2+) uptake by microsomes. In the transfected cells, the overexpression of PMCA coincided with three major anomalies of D-glucose metabolism, namely a lower rate of D-[5-(3)H]glucose utilization prevailing at a low extracellular concentration of D-glucose (1.1 mM), a low ratio between D-[U-(14)C]oxidation and D-[5-(3)H]glucose utilization prevailing at a high extracellular glucose concentration (16.7 mM), and a high ratio between the net generation of (14)C-labelled acidic metabolites and amino acids and that of (3)H(2)O from D-[5-(3)H]glucose. These anomalies resulted in a decreased estimated rate of ATP generation (linked to the catabolism of the hexose) and a lowered ATP cell content, whether at low or high extracellular D-glucose concentrations. The net uptake of (45)Ca(2+) by intact cells was also decreased in the transfected cells, but to a greater extent than can apparently be attributed to the change in the ATP-generation rate. These findings document the relevance of PMCA activity to both D-glucose metabolism and Ca(2+) handling in insulin-producing cells, with emphasis on the key role of both cytosolic and mitochondrial Ca(2+) concentrations in the regulation of D-glucose catabolism. They also reveal that overexpression of PMCA leads, in insulin-producing cells, to an imbalance between ATP generation and consumption.

Abstract: Considering the changes in the fatty acid profile of liver lipids related to age, gender and nutritional status or occurring in pathological situations, this study aimed at investigating whether such changes could be judged from measurements conducted in plasma lipids. The fatty acid profile of both liver and plasma phospholipids and triglycerides was measured in 16 control animals and 26 rats depleted in long-chain polyunsaturated (n-3) fatty acids. Within each group of rats, significant correlations prevailed between the percentage of each fatty acid in liver versus plasma phospholipids or triglycerides. However, the plasma/liver ratio for the relative content of C20:5(n-3), C22:5(n-3) and C22:6(n-3) in triglycerides displayed abnormally high values in 2 control animals. The fatty acid profile of liver phospholipids and triglycerides can, as a rule, be judged from measurements made in the corresponding plasma lipids. For instance, measurements in plasma phospholipids could help to identify subjects deficient in (n-3) fatty acids and to assess the dietary correction of this defect.

Abstract: Considering the proposed preventive effect of nervonic acid on obesity- and diabetes-related coronary risk factors, the content of its precursors (oleic, 11-eicosenoic and 13-docosenoic acids) was measured in liver and plasma phospholipids and triglycerides, brain and spleen phospholipids, and adipose tissue lipids of fed or overnight fasted control and hereditarily diabetic Goto-Kakizaki female rats, as well as fed streptozotocin-induced diabetic female rats. In liver and brain phospholipids, the 11-eicosenoate/oleate ratio was significantly higher in diabetic rats than in control animals. Such was not the case in either spleen phospholipids or liver triglycerides and adipose tissue lipids. The increase in the liver phospholipid 11-eicosenoate/oleate ratio found in female diabetic rats represents a mirror image of the situation recently documented, in the same animal models of diabetes, in male rats. These contrasting findings may be relevant to the higher coronary heart disease risk prevailing in female, as compared to male, diabetic subjects.

Abstract: The bolus intravenous administration of a novel medium-chain triglyceride:fish oil emulsion to second generation rats depleted in long-chain polyunsaturated omega3 fatty acids was recently found to enrich within 60 min the content of both plasma and liver lipids in such omega3 fatty acids, this coinciding with correction of the perturbation in liver triglyceride fatty acid content and profile otherwise prevailing in these rats. The present report draws attention to cause-to-effect relationships between changes in liver phospholipid and triglyceride fatty acid content and/or pattern operative under these experimental conditions.

Abstract: Second generation rats depleted in long-chain polyunsaturated omega3 fatty acids display several features of the metabolic syndrome, including visceral obesity, liver steatosis, insulin resistance, hypertension, and cardiac hypertrophy. In the framework of an extensive study on such metabolic, hormonal and functional perturbations, the phospholipid fatty acid pattern and ex vivo metabolism of D-glucose were recently investigated in the soleus muscle of these omega3-depleted rats. The present study deals with the triglyceride fatty acid content and pattern of the soleus muscle in control animals and omega3-depleted rats. Some of the latter rats were injected intravenously 60-120 minutes before sacrifice with either an omega3 fatty acid-rich medium-chain triglyceride/fish oil emulsion (omega3-FO rats) or a control medium-chain triglyceride/olive oil emulsion (omega3-OO rats). The total fatty acid content of triglycerides was comparable in control and omega3-depleted rats and, in both cases, inversely related to their C20:4omega6 relative content. At variance with the situation found in control rats, no long-chain polyunsaturated omega3 fatty acid (C18:3omega3, C20:5omega3, C22:5omega3, C22:6omega3) was detected in the omega3-depleted rats. Unexpectedly, the triglyceride content in most long-chain polyunsaturated omega6 fatty acids (C18:2omega6, C20:3omega6, C20:4omega6 and C22:4omega6) had also decreased in the latter rats. Moreover, the activity of Delta9-desaturase was apparently increased in the omega3-depleted rats, as judged from the C16:1omega7/C16:0 and C18:1omega9/C18:0 ratios. The omega3-FO rats differed from omega3-OO rats by a lower contribution of C18:2omega6 metabolites (C18:3omega6, C20:3omega6, C20:4omega6 and C22:4omega6). These findings indicate that the prior injection of the medium-chain triglyceride/fish oil emulsion, known to increase the muscle phospholipid content in long-chain polyunsaturated omega3 fatty acids, may nevertheless accentuate the depletion in long-chain polyunsaturated omega6 fatty acids otherwise found in the triglycerides of omega3-depleted rats. Such a dual effect is reminiscent of that observed, under the same experimental conditions, for selected variables in D-glucose metabolism in the soleus muscle.

Abstract: The fatty acid pattern of spleen phospholipids and triglycerides was examined in fed or overnight fasted normal rats, streptozotocin-induced diabetic animals (type-1 diabetes) and Goto-Kakizaki rats (type-2 diabetes). In both phospholipids and triglycerides, differences were observed in the relative contribution of several fatty acids, as well as in the ratio between distinct fatty acids, when comparing fed to fasted rats, normal to diabetic animals and male to female Goto-Kakizaki rats. Diabetes increased to a greater extent the C22:6omega3 content of phospholipids in the spleen than in either the liver or the brain. However, the diabetes-induced changes in the C22:6omega3 content of triglycerides was closely comparable in the spleen, liver and brain. These findings suggest that the incorporation of fatty acids into triglycerides is controlled by comparable regulatory factor(s), e.g. insulinemia, in the spleen, liver and brain. In the case of phospholipids, however, an apparent adaptation to diabetic stress was more marked in the spleen than in the liver, and virtually absent in the brain. The proposed dichotomy in the environmental regulation of fatty acid synthesis and incorporation into phospholipids and triglycerides was further supported by distinct diabetes-related changes in the apparent activity of Delta9-desaturase in these two classes of lipids.

Abstract: Alteration of the phospholipid (PL) and triglyceride (TG) fatty acid pattern was recently documented in several organs of rats depleted in long-chain polyunsaturated omega3 fatty acid (omega3 rats). This study extends such a knowledge to the submandibular gland. The total PL and TG content of the salivary gland was not different in control and omega3 rats. The sole omega3 fatty acids found in omega3 rats (C22:5omega3 and C22:6omega3) were present at levels 3-12 times lower than in control rats. The C22:5omega3/C22:6omega3 ratio was increased threefold in omega3 rats. The PL and TG C16:0/C16:1omega7 and C18:0/C18:1omega9 ratios were decreased in omega3 rats. The conversion of C18:2omega6 to C20:4omega6 and C22:4omega6 appeared facilitated in the omega3 rats. Some of these rats were injected intravenously, 60-120 min before killing, with either a medium-chain triglyceride:fish oil emulsion or a control medium-chain triglyceride:olive oil emulsion. The former emulsion increased the PL C22:5omega3 and C22:6omega3 content and prevented the age-related decrease in C16:0/C16:1omega7 and C18:0/C18:1omega9 ratios otherwise also recorded in PL. In conclusion, these findings document an increased activity of Delta9-desaturase, a more efficient conversion of C18:2omega6 to its metabolites, and an impaired generation of C22:6omega3 from C22:5omega3 in omega3 rats.

Abstract: The presence of carbonic anhydrase (type V) was recently documented in rat and mouse pancreatic islet beta-cells by immunostaining and Western blotting. In the present study, the activity of carbonic anhydrase was measured in rat islet homogenates and shown to be about four times lower than in rat parotid cells. The pattern for the inhibitory action of acetazolamide on carbonic anhydrase activity also differed in islet and parotid cell homogenates, suggesting the presence of different isoenzymes. NaN3 inhibited carbonic anhydrase activity in islet homogenates and both D-[U-14C]glucose oxidation and glucose-stimulated insulin secretion. Acetazolamide (0.3-10.0 mM) also decreased glucose-induced insulin output but failed to affect adversely D-[U-14C]glucose oxidation, although it inhibited the conversion of D-[5-3H]glucose to [3H]OH and that of D-[U-14C]glucose to acidic metabolites. Hydrochlorothiazide (3.0-10.0 mM), which also caused a concentration-related inhibition of the secretory response, like acetazolamide (5.0-10.0 mM), decreased H(14)CO3- production from D-[U-14C]glucose (16.7 mM). Acetazolamide (5.0 mM) did not affect the activity of volume-sensitive anion channels in beta-cells but lowered intracellular pH and adversely affected both the bioelectrical response to d-glucose and its effect on the cytosolic concentration of Ca2+ in these cells. The lowering of cellular pH by acetazolamide, which could well be due to inhibition of carbonic anhydrase, might in turn account for inhibition of glycolysis. The perturbation of stimulus-secretion coupling in the beta-cells exposed to acetazolamide may thus involve impaired circulation in the pyruvate-malate shuttle, altered mitochondrial Ca2+ accumulation, and perturbation of Cl- fluxes, resulting in both decreased bioelectrical activity and insulin release.

Abstract: A depletion in long-chain polyunsaturated omega3 fatty acids may affect fuel homeostasis. In such a perspective, the present study deals mainly with the in vitro fate of D-[U-(14)C]glucose in hemidiaphragms, stretched soleus and plantaris muscle pieces obtained from normal and omega3-depleted rats (second generation) and incubated in the absence or presence of insulin. When so required, the omega3-depleted rats were injected 120 min before sacrifice with either a omega3 fatty acid-rich medium-chain triglyceride:fish oil emulsion (FO) or a control medium-chain triglyceride:olive oil emulsion (OO). The content of the soleus muscle in long-chain polyunsaturated omega3 fatty acids was severely decreased in the omega3-depleted rats, and modestly albeit significantly increased after injection of FO to these animals. In stretched soleus muscles from OO-injected omega3-depleted rats, the absolute values for glycogen synthesis measured in the absence or presence of insulin were about twice higher than in normal animals. In the OO-injected omega3-depleted rats, insulin augmented the output of (14)C-labelled amino acids, whilst such was not the case in normal animals. These and other findings suggest a lower catabolism of D-glucose relative to the anabolic process of glycogen synthesis and a lower availability of endogenous amino acids in the muscles of omega3-depleted rats, as compared to those of control animals. The prior injection of FO to the omega3-depleted rats restored a normal value for the paired ratio between the output of (14)C-labelled amino acids and acidic metabolites, but further increased glycogen net synthesis. It is proposed, therefore, that the perturbation of d-glucose metabolism in muscles from omega3-depleted rats involves a multifactorial determinism, only some of the concerned factors being susceptible to rapid correction after enrichment of cell phospholipids in long-chain polyunsaturated omega3 fatty acids.

Abstract: An intragastric D-glucose tolerance test was performed, after overnight starvation, in female rats depleted in long-chain polyunsaturated omega3 fatty acids (omega3D rats) and control rats of same age and gender. The plasma D-glucose and insulin concentrations, insulinogenic index, and HOMA for insulin resistance were all higher, after overnight starvation, in omega3D rats than in control animals. Over the 120-minute period following the intragastric administration of D-glucose, the area under the curve for the same four variables was also higher in omega3D rats than in control animals. In addition to visceral obesity, liver steatosis, hypertension, and cardiac hypertrophy, the omega3D rats thus display further features of the metabolic syndrome, namely glucose intolerance and insulin resistance, despite hyperinsulinemia.

Abstract: A number of metabolic, ionic and secretory variables were recently found to be affected in pancreatic islets obtained from second generation rats depleted in long-chain polyunsaturated omega 3 fatty acids (omega 3 rats). The present study further documents three sets of anomalies in such islets. First, after 90 min exposure to D-glucose (8.3 mM), the release of insulin from perifused islets, prelabelled with 45Ca, is lower in omega 3 rats than in control animals, despite comparable 45Ca fractional outflow rate. Second, over 15 min exposure to carbamylcholine (0.1 mM), in the presence of D: -glucose, the cytosolic concentration of Ca2+ is increased to a greater relative extent in dispersed islet cells from omega 3 rats, as compared to control animals. This coincides with a greater relative increase in insulin output from perifused islets during the second phase of the secretory response to the cholinergic agent. Last, the increase provoked by ouabain (1.0 mM) in cytosolic Ca2+ concentration, 45Ca fractional outflow rate and insulin release are all delayed in the omega 3 rats. Taking into account the decreased activity of Na+, K+-ATPase in the islets of omega 3 rats, these findings are interpreted as reflecting an impaired priming of insulin-producing cells when first exposed for 105 min to a physiological postprandial concentration of D-glucose.

Abstract: Altered D-glucose metabolism prevails in the soleus muscle of rats depleted in long-chain polyunsaturated omega3 fatty acids (omega3). In these animals, the prior intravenous injection of an omega3-rich medium-chain triglyceride:fish oil emulsion (omega3-FO rats), as compared to that of an omega3-poor medium-chain triglyceride:olive oil emulsion (omega3-OO rats), may either correct or aggravate selected metabolic variables. This study deals with the fatty acid pattern of soleus phospholipids and triglycerides in control animals versus omega3-depleted rats not injected with any lipid emulsion (omega3-NI rats) and in omega3-OO versus omega3-FO rats. In each group of omega3-depleted rats, age-related changes were also monitored. The omega3-depleted rats displayed low long-chain polyunsaturated omega3 fatty acid content, facilitated metabolism of long-chain polyunsaturated omega6 fatty acids, and increased Delta9-desaturase activity. Both the age-related changes in lipid variables and those attributable to the prior intravenous injection of the omega3-rich lipid emulsion consisted either in a move towards normalization or in the opposite direction, i.e. towards aggravation of the defect found in the omega3-depleted rats. Emphasis is placed, therefore, on the unusual situation found in the soleus muscle of omega3-depleted rats, in which both lipid and metabolic variables may be either favourably or adversely affected by the same environmental factor(s).

Abstract: Impaired glucose tolerance and overt diabetes mellitus are becoming increasingly common complications of cystic fibrosis (CF), most probably merely as a result of increased life expectancy. In order to understand the pathophysiology of cystic fibrosis-related diabetes (CFRD), knowledge on the possible expression and cell distribution of the cystic fibrosis transmembrane conductance regulator (CFTR) protein within the endocrine pancreas is required. In this report, we establish the first evidence for expression of CFTR protein in rat pancreatic islets by using independent techniques. First reverse transcriptase-polymerase chain reaction (RT-PCR) amplification showed that CFTR mRNA is present in isolated islets of Langerhans. Furthermore, the analysis of flow cytometry-separated islet cells indicated that the level of CFTR transcripts is significantly higher in the non-beta than in beta-cell populations. The expression of CFTR protein in rat islet cells was also demonstrated by Western blotting and the level of expression was also found significantly higher in the non-beta than in beta-cell populations. Last, in situ immunocytochemistry studies with two monoclonal antibodies recognizing different CFTR epitopes indicated that CFTR expression occurs mainly in glucagon-secreting alpha-cells.

Abstract: D-glucose and other nutrient insulin secretagogues have long been known to induce a transient increase in inorganic phosphate release from pancreatic islets, a phenomenon currently referred to as a "phosphate flush". The objective of this study was to explore the possible participation of volume-sensitive anion channels in such a process. Rat pancreatic islets were preincubated for 60 min in the presence of [32P]orthophosphate and then perifused for 90 min to measure 32P fractional outflow rate and insulin secretion. From minutes 46 to 70 inclusive either the concentration of D-glucose was increased from 1.1 to 8.3 mmol L-1 or the extracellular osmolarity was decreased by reducing the NaCl concentration by 50 mmol L-1. The increase in D-glucose concentration induced a typical phosphate flush and biphasic stimulation of insulin release. Extracellular hypoosmolarity caused a monophasic increase in both effluent radioactivity and insulin output. The inhibitor of volume-sensitive anion channels 5-nitro-2-(3-phenylpropylamino)benzoate (0.1 mmol L-1) inhibited both stimulation of insulin release and phosphate flush induced by either the increase in D-glucose concentration or extracellular hypoosmolarity. It is proposed that gating of volume-sensitive anion channels accounts for the occurrence of the phosphate flush and subsequent stimulation of insulin secretion in response to either an increase in D-glucose concentration or a decrease in extracellular osmolarity.

Abstract: A gating of volume-sensitive anion channels may participate in the depolarization of the plasma membrane caused by high concentrations of D-glucose in insulin-producing B-cells of the endocrine pancreas. The efflux of tritiated taurine from prelabeled cells is currently used to assess changes in the activity of such channels. The handling of [1,2-3H]taurine by isolated rat pancreatic islets was therefore investigated. The net uptake of [1,2-3H]taurine was found to represent a concentration-, time-, and temperature-dependent process. It was progressively increased in the range of D-glucose concentrations between 2.8 and 8.3 mM, but no further increase was observed at 16.7 mM D-glucose. Over 15 min incubation, the efflux of radioactivity from prelabeled islets was inhibited by MK571 (1.0 mM). It was increased in response to hypoosmolarity both in the presence and absence of extracellular Na+. Whether in salt-balanced or Na+-deprived media, the efflux of radioactivity from prelabeled islets increased in response to a rise in D-glucose concentration from 2.8 to 5.6 or 8.3 mM, but decreased when the concentration of the hexose was further increased from 8.3 to 16.7 mM. In perifused islets, however, the radioactive efflux from prelabeled islets was inhibited, in a concentration- related manner, when islets first deprived of D-glucose for 45 min were then exposed to 2.8, 5.6, or 16.7 mM D-glucose. Likewise, in prelabeled and perifused islets first exposed for 45 min to 4.0 mM D-glucose, a later rise in hexose concentration to 8.3 mM failed to affect significantly effluent radioactivity, while an increase in hexose concentration from 4.0 to 16.7 mM inhibited the radioactive outflow. In these perifusion experiments, the rise in D-glucose concentration provoked the expected changes in insulin output. The findings obtained in islets examined immediately after preincubation in the presence of [1,2-3H]taurine are consistent with the presence of volume-sensitive anion channels in islet cells and with a gating of such channels in response to a rise in D-glucose concentration from 2.8 to 5.6-8.3 mM. However, the radioactive fractional outflow rate from prelabeled islets seems to reach its highest value at about 8.3 mM D-glucose, being unexpectedly decreased at a higher concentration (16.7 mM) of the hexose. In conclusion, the pleiotropic effects of D-glucose upon tritiated taurine outflow from prelabeled rat islets, which could conceivably be ascribed to differences in the handling of this amino sulfonic acid by distinct islet cell types, indicates that the present approach is far from optimal to characterize unambiguously the regulation by the hexose of volume-sensitive anion channel activity in insulin-producing islet cells.

Abstract: The effects of D-glucose, D-mannose, D-galactose, Dglyceraldehyde, pyruvate, L-lactate, 2-ketoisocaproate, L-leucine, and/or L-glutamine on the ATP and ADP content of rat isolated pancreatic islets were reevaluated in order to compare changes evoked by these nutrient secretagogues in the islet ATP content and ATP/ADP ratio to their effects upon insulin release. Although being compatible with the fuel concept for nutrient-stimulated insulin secretion, the results of this study also argue against the monolithic view that the adenine nucleotide pattern in islet cells represents the sole coupling factor between metabolic and more distal events in the process of nutrient-stimulated insulin release.

Abstract: D-fructose (10 mM) augments, in rat pancreatic islets, insulin release evoked by 10 mM D-glucose. Even in the absence of D-glucose, D-fructose (100 mM) displays a positive insulinotropic action. It was now examined whether the insulinotropic action of D-fructose could be attributed to an increase in the ATP content of islet cells. After 30-60 min incubation in the presence of D-glucose and/or D-fructose, the ATP and ADP content was measured by bioluminescence in either rat isolated pancreatic islets (total ATP and ADP) or the supernatant of dispersed rat pancreatic islet cells exposed for 30 s to digitonine (cytosolic ATP and ADP). D-fructose (10 and 100 mM) was found to cause a concentration-related decrease in the total ATP and ADP content and ATP/ADP ratio below the basal values found in islets deprived of exogenous nutrient. Moreover, in the presence of 10 mM D-glucose, which augmented both the total ATP content and ATP/ADP ratio above basal value, D-fructose (10 mM) also lowered these two parameters. The cytosolic ATP/ADP ratio, however, was increased in the presence of D-glucose and/or D-fructose. Under the present experimental conditions, a sigmoidal relationship was found between such a cytosolic ATP/ADP ratio and either (86)Rb net uptake by dispersed islet cells or insulin release from isolated islets. These data provide, to our knowledge, the first example of a dramatic dissociation between changes in total ATP content or ATP/ADP ratio and insulin release in pancreatic islets exposed to a nutrient secretagogue. Nevertheless, the cationic and insulinotropic actions of d-glucose and/or d-fructose were tightly related to the cytosolic ATP/ADP ratio.

Abstract: The stimulus-secretion coupling for hypotonicity-induced insulin release was investigated in BRIN-BD11 cells. A 50 mM decrease in extracellular NaCl caused a twofold increase in insulin release. The release of insulin evoked by hypotonicity progressively decreased in an exponential manner. The response to extracellular hypotonicity displayed a threshold value close to 20 mOsmol/L and a maximal response at about 70 mOsmol/ L. Hypotonicity also caused a rapid increase in cell volume followed by a regulatory volume decrease (RVD), cell membrane depolarization with induction of spike activity, and a rise in cytosolic Ca2+ concentration. 5-Nitro-2-(3-phenylpropylamino)benzoate inhibited the secretory response to hypoosmolarity, failed to affect the early increase in cell volume but prevented the RVD, and suppressed the hypotonicity-induced plasma membrane depolarization. Insulin release provoked by hypotonicity was inhibited by verapamil, absence of Ca2+, thapsigargin, furosemide, tributyltin, and diazoxide. On the contrary, tolbutamide augmented modestly insulin release recorded in the hypoosmolar medium. Last, a rise in extracellular K+ concentration, while augmenting basal insulin output, failed to affect insulin release in the hypoosmolar medium. Thus, the insulin secretory response to hypotonicity apparently represents a Ca2+-dependent process triggered by the gating of volume-sensitive anion channels with subsequent depolarization and gating of voltage-sensitive Ca2+ channels.

Abstract: The presence of fructokinase (ketohexokinase) in rat pancreatic islet homogenates was previously documented. However, no information was so far available on the activity of this enzyme in islets relative to that in other tissues and on the respective contribution of insulin-producing B cells and non-B islet cells. The present study provides such an information. The activity of fructokinase, as assessed by the phosphorylation of 1.0 mM D-fructose, was compared to that of hexokinase isoenzyme(s), as measured in the presence of 1.0 mM D-glucose, and further characterized by its heat-resistance, K+ dependency and resistance to the inhibitory action of D-mannoheptulose. As judged from the results obtained in heated homogenates, the activity of fructokinase, expressed relative to protein content (nmol/min per mg protein) was highest in liver (21.5 +/- 2.5; n = 11) and lowest in parotid gland (0.16 +/- 0.09; n = 3), with in-between values in ileum (2.45 +/- 0.53; n = 3), pancreas (0.82 +/- 0.11; n = 11) and pancreatic islets (0.46 +/- 0.07; n = 6). The paired ratio between fructokinase and hexokinase isoenzyme activity was also highest in liver (548 +/- 45%; n = 8) and lowest in parotid gland (0.93 +/- 0.52%; n = 3). Such a ratio was not significantly different in pancreas, islets and purified B or non-B islet cells, with an overall mean value of 2.57 +/- 0.46% (n = 12). The present findings thus unambiguously document the presence of fructokinase activity in all cell types under consideration, except possibly parotid cells, with the following hierarchy: liver > ileum > pancreas. Relative to paired hexokinase activity, no obvious difference was found for fructokinase activity in B versus non-B islet cells.

Abstract: Considering the insufficient supply of long-chain polyunsaturated omega-3 fatty acids often prevailing in Western populations, this report deals mainly with alterations of Ca(2+) fluxes and Ca(2+)-dependent insulin secretory events in isolated pancreatic islets from omega-3-depleted rats. In terms of (45)Ca(2+) handling, the islets from omega-3-depleted rats, compared with those from normal animals, displayed an unaltered responsiveness to an increase in extracellular K(+) concentration, a lower inflow rate and lower fractional outflow rate of the divalent cation, and higher (45)Ca(2+)-labeled cellular pool(s) at isotopic equilibrium. The latter anomaly was corrected 120 min after intravenous injection of a novel medium-chain triglyceride-fish oil (MCT:FO) emulsion, distinct from a control omega-3-poor MCT-olive oil (MCT:OO) emulsion. At 8.3 mM D-glucose, insulin release was higher in islets from omega-3-depleted rats vs. control animals, coinciding with a higher cytosolic Ca(2+) concentration. The relative magnitude of the increase in insulin output attributable to a rise in D-glucose as well as extracellular Ca(2+) or K(+) concentration, to the absence vs. presence of verapamil and to the presence vs. absence of extracellular Ca(2+), theophylline, phorbol 12-myristate 13-acetate, or Ba(2+), was always more pronounced in islets from omega-3-depleted rats injected with the MCT:OO compared with the MCT:FO emulsion. A comparable situation prevailed when comparing islets from noninjected omega-3-depleted and normal rats. In light of these and previous findings, we propose that an impairment of Na(+),K(+)-ATPase activity plays a major, although not an exclusive, role in the perturbation of Ca(2+) fluxes and Ca(2+)-dependent secretory events in the islets from omega-3-depleted rats.

Abstract: In order to gain information on the determinism of the perturbation of fuel homeostasis in situations characterized by a depletion in long-chain polyunsaturated omega3 fatty acids (omega3), the metabolic and hormonal status of omega3-depleted rats (second generation) was examined. When required, these rats were injected intravenously 120 min before sacrifice with a novel medium-chain triglyceride-fish oil emulsion able to provoke a rapid and sustained increase of the omega3 content in cell phospholipids. The measurement of plasma glucose, insulin, phospholipid, triglyceride, and unesterified fatty acid concentration indicated modest insulin resistance in the omega3-depleted rats. The plasma triglyceride and phospholipid concentrations were decreased in the omega3-depleted rats with abnormally low contribution of omega3 in both circulating and pancreatic islet lipids. The protein, insulin, and lipid content of the islets, as well as their intracellular and extracellular spaces, were little affected in the omega3-depleted rats. The metabolism of D-glucose in the islets of omega3-depleted rats was characterized by a lesser increase in D-[5-3H]glucose utilization and D-[U-14C]glucose oxidation in response to a given rise in hexose concentration and an abnormally low ratio between D-glucose oxidation and utilization. These abnormalities could be linked to an increased metabolism of endogenous fatty acids with resulting alteration of glucokinase kinetics. The release of insulin evoked by D-glucose, at a close-to-physiological concentration (8.3 mM), was increased in the omega3-depleted rats, this being considered as consistent with their insulin resistance. Relative to such a release, that evoked by a further rise in D-glucose concentration or by non-glucidic nutrients was abnormally high in omega3-depleted rats, and restored to a normal level after of the intravenous injection of the omega3-rich medium-chain triglyceride-fish oil emulsion. Because the latter procedure failed to correct the perturbation of D-glucose metabolism in the islets of omega3-depleted rats, it is proposed that the anomalies in the secretory behaviour of islets in terms of their response to an increase in hexose concentration or non-nutrient secretagogues is mainly attributable to alteration in K+ and Ca2+ handling, as indeed recently documented in separate experiments.

Abstract: A low intake of long-chain polyunsaturated omega3 fatty acid often prevails in Western populations. Its consequences in terms of the control of fuel homeostasis led us to explore functional events in pancreatic islets isolated from either normal or omega3-depleted rats (second generation). In the latter rats, the inflow of K+ by both ouabain-sensitive and ouabain-resistant modalities was decreased, this coinciding with an impaired insulin secretory response to ouabain. The intravenous injection of a medium-chain triglyceride:fish oil emulsion to omega3-depleted rats 2 h before sacrifice restored a normal value for the inflow of K+ by the ouabainsensitive modality, i.e., that linked to the activity of the Na,K-ATPase, but failed to correct the entry of K+ by the ouabain-resistant modality and the defect of the insulin secretory response to ouabain. In conclusion, an impaired activity of the Na,K-ATPase in insulin-producing cells apparently represents a key determinant of altered islet function in omega3-depleted rats.

Abstract: Attention was recently drawn to differences in the fatty acid pattern of liver phospholipids and triglycerides in animal models of type 1 and type 2 diabetes. The present study extends this knowledge to epididymal or parametrial adipose tissue lipids. The fatty acid pattern of such lipids was established in four fed female normal rats, four overnight fasted female normal rats, six fed female rats rendered diabetic by an injection of streptozotocin 3 days before sacrifice (STZ rats), and four female and four male Goto-Kakizaki rats (GK rats) also examined in the fed or fasted state. In addition to the fasting-induced and diabetes-related changes in plasma D-glucose and insulin concentrations, differences in either the weight percentage of fatty acids or the paired ratio between distinct fatty acids were often encountered. For instance, in the GK rats, gender differences were observed in the weight percentage of C18:2omega6, as well as C18:2omega6/C18:3omega6, C18:3omega6/C20:4omega6, C20:5omega3/C22:5omega3 and C22:5omega3/C22:6omega3 ratios. When compared to normal rats, the activity of Delta9-desaturase was markedly increased in GK rats and, to a lesser extent, in STZ rats. Starvation also increased to some extent the activity of Delta9-desaturase. The relative content of C22:6omega3 was also higher in diabetic than in normal rats. Further differences between GK and STZ rats concerned the generation of C18:3omega6 from C18:2omega6, C20:4omega6 from C18:3omega6, and C20:5omega3 from C18:3omega3. Several differences found in the adipose tissue of GK versus STZ rats were reminiscent of those recently identified in the liver triglycerides of these two types of diabetic animals, suggesting a common regulatory mechanism, possibly linked to the higher insulinemia of GK rats versus STZ rats.

Abstract: Second generation rats depleted in long-chain polyunsaturated omega3 fatty acids are currently used as an animal model for the insufficient dietary supply of such fatty acids often prevailing in Western populations. The present study deals mainly with the effects of a novel medium-chain triglyceride: fish oil emulsion (MCT:FO), as compared to a control medium-chain triglyceride:olive oil emulsion (MCT: OO), administered as an intravenous bolus to the omega3-depleted rats 60-120 min before sacrifice upon selected biochemical and biophysical variables. The major findings consisted of a severe decrease of the omega3 fatty acid content of liver lipids in non-injected omega3-depleted rats and its partial correction after injection of the MCT:FO emulsion. The omega3-depleted rats also displayed liver steatosis, increased incorporation of long-chain polyunsaturated omega6 fatty acids in liver phospholipids and increased activity of liver Delta9-desaturase. As judged from the effects of ouabain upon 86Rb net uptake by isolated pancreatic islets, the activity of Na+,K+-ATPase was virtually abolished in the omega3-depleted rats. The latter defect was corrected by prior intravenous injection of the MCT:FO emulsion, this coinciding with suppression of the excessive secretory response to a number of insulin secretagogues otherwise observed in the islets of omega3-depleted rats injected or not with the MCT:OO emulsion.

Abstract: The liver phospholipid and triglyceride content and/or fatty acid pattern differ(s) not solely in normal versus diabetic rats, but also in distinct rat models of diabetes mellitus. The present study reveals that a comparable situation prevails in the brain. Fed and overnight fasted female normal rats (N) and Goto-Kakizaki rats (GK), as well as fed rats rendered diabetic by a prior injection 3 days before sacrifice of streptozotocin (STZ) were examined. The brain phospholipid content, expressed as milligrams of fatty acids per gram wet weight, was comparable in all groups of rats, with an overall mean value of 31.2+/-0.8 (n=22). The GK rats differed from N and STZ rats by lower C18:0/C18:1omega9 and C18:2omega6/C18:3omega6 ratios and a lower C20:5omega3 content of brain phospholipids. The total amount of fatty acids in triglycerides was 7-8 times higher in GK than N and STZ rats. The GK rats differed from N and STZ rats by lower C16:0/C16:1omega7, C18:0/C18:1omega9 and (C16:0+C16:1omega7)/(C18:0+C18:1omega9) ratios in triglycerides. These findings extend to the brain, the knowledge of alterations in phospholipid and triglyceride content and/or fatty acid pattern in GK rats, as compared to N or STZ rats. The former rats indeed displayed: (i) an apparently increased activity of Delta9- and Delta6-desaturases, as suggested by the phospholipid measurements, and a decreased C20:5omega3 content in such phospholipids; (ii) a dramatic increase in brain triglyceride content; and (iii) an increased activity of Delta9-desaturase, as well as elongase, as judged from the triglyceride data.

Abstract: It was recently proposed that in rat pancreatic islets exposed to 8.3 mM D-glucose, alpha-D-glucose-6-phosphate undergoes enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase. To explore the identity of the hexokinase isoenzyme(s) involved in such a tunnelling process, the generation of 3HOH from the alpha- and beta-anomers of either D-[2-3H]glucose or D-[5-3H]glucose was now measured over 60 min incubation at 4 degrees C in pancreatic islets exposed only to 2.8 mM D-glucose, in order to decrease the relative contribution of glucokinase to the phosphorylation of the hexose. Under these experimental conditions, the ratio for 3HOH production from D-[2-3H]glucose/D-[5-3H]glucose at anomeric equilibrium (39.7 +/- 11.6%) and the beta/alpha ratios for the generation of 3HOH from either the D-[2-3H]glucose anomers (70.9 +/- 12.6%) or the D-[5-3H]glucose anomers (59.6 +/- 12.4%) indicated that a much greater fraction of alpha-D-glucose-6-phosphate escapes from the process of enzyme-to-enzyme channelling in the islets exposed to 2.8 mM, rather than 8.3 mM D-glucose. These findings suggest, therefore, that the postulated process of enzyme-to-enzyme channelling involves mainly glucokinase.

Abstract: In diabetes-prone BioBreeding rats, an enteropathy often precedes the onset of auto-immune insulitis. The present study draws attention to quantitative and qualitative alterations of intestinal mucins in this animal model of type 1 diabetes mellitus. Male and female diabetes-resistant (BBc) and diabetes-prone (BBdp) BioBreeding rats fed, from one to two weeks after weaning onwards, either a plant-based diabetes-promoting diet (NTP) or a hydrolysed casein diabetes-protective diet (HC), were sacrificed at 11-14 weeks of age. Proteins and total mucins, as well as acid and neutral mucins, were measured in a segment of the intestinal tract, located 25-30 cm below the pylorus. No significant difference between BBc and BBdp rats was found when fed the HC diet. However, the NTP diet lowered both total and neutral mucins, whilst failing to affect significantly acidic mucins. The effects of the NTP diet were more pronounced in BBdp rats than in BBc rats. It is speculated that the quantitative and qualitative changes evoked by the NTP diet in BBdp rats may play a role in the alteration of gut permeability found under the same experimental conditions.

Abstract: The handling of 45Ca and 86Rb by aortic rings obtained from rats depleted in long-chain polyunsaturated omega3 fatty acids (second generation) was examined in vitro over 10 to 60 min incubation at either increasing concentrations of extracellular K+ (5, 3 and 60 mM) in the case of 45Ca net uptake or in the absence and presence of ouabain (50 microM) in the case of 86Rb uptake. The omega3-depleted rats were injected intravenously 120 min before sacrifice with 1.0 ml of either an omega3 fatty acid-rich medium-chain triglyceride:fish oil emulsion (MCT:FO) or a control medium-chain triglyceride:olive oil emulsion (MCT:OO). In the MCT:OO-injected rats, the rise in extracellular K+ concentration failed to stimulate 45Ca net uptake, whilst the prior injection of the MCT:FO emulsion restored the expected increase in 45Ca net uptake by aortic rings exposed to 60 mM K+. The absolute value for 86Rb net uptake after 10 or 60 min incubation and whether in the absence or presence of ouabain, which significantly decreased the uptake of 86Rb+ after 60 min incubation, only represented in the MCT:FO-injected rats 63.1+/-3.8% of the mean corresponding values found in MCT:OO-injected animals. These findings are consistent with the view that activity of cationic channels, such as the voltage-sensitive Ca2+ channel, the outflow of Ca2+ as mediated by either Na+-Ca2+ countertransport or a Ca2+-ATPase, the activity of Na+,K+-ATPase and the modality of K+ inflow by an oubain-resistant modality are all affected in aortic cells by the content of long-chain polyunsaturated omega3 fatty acids in membrane phospholipids.

Abstract: This study deals with the enteropathy recently identified in diabetes-prone BB rats (BBdp). Diabetes-resistant BB rats (BBc) and BBdp rats were fed from days 32-39 onward either a protective diabetes-retardant hydrolyzed casein diet (HC) or a plant-based diabetogenic (NTP) diet. The NTP diet decreased body weight and plasma insulin in BBc and BBdp rats. The BBdp rats displayed low intestinal invertase and increased intestinal peroxidase activity. In the BBdp rats fed the HC diet, the mucin content 30-35 cm below the pylorus was higher and the gut permeability lower than in the other three rat groups. There was a significant inverse correlation between gut permeability and the insulinogenic index in the BBdp rats fed the HC or NTP diet. Thus, in BBdp rats, the HC diet somehow prevents the increase in gut permeability and the decrease in the insulinogenic index otherwise found in some of these diabetes-prone animals.

Abstract: The metabolism of D-glucose displays anomeric specificity in rat pancreatic islets. The aim of the present report is to investigate whether such a situation implies enzyme-to-enzyme tunnelling of metabolites in the early steps of glycolysis. For such a purpose, the modelling of alpha- and beta-D-glucose catabolism, itself based on available information concerning both the utilisation of these two anomers and the intrinsic properties of phosphoglucoisomerase, was first examined. According to a theoretical model with enzyme-to-enzyme channelling, the generation of 3HOH from D-[2-3H]glucose should be higher in islets exposed to beta-D-glucose rather than alpha-D-glucose, whilst the opposite situation should prevail in the case of D-[5-3H]glucose conversion to 3HOH. Experimental data collected in rat islets incubated for 60 min at 4 degrees C in the presence of either alpha- or beta-D-glucose mixed with tracer amounts of either alpha- or beta-D-[2- 3H]glucose and alpha- or beta-D-[5-3H]glucose indicate that the beta/alpha ratio for D-[2-3H]glucose conversion to 3HOH is indeed higher than the beta/alpha ratio for D-[5-3H]glucose conversion to 3HOH. These findings are consistent with the postulated enzyme-to-enzyme tunnelling of glycolytic intermediates between hexokinase isoenzyme(s), phosphoglucoisomerase and, possibly, phosphofructokinase.

Abstract: This study aims at establishing the contribution of alpha- and beta-D-glucose to the total generation of (3)HOH by rat pancreatic islets exposed to D-[2 - (3)H]glucose or D-[5 - (3)H] glucose at anomeric equilibrium. The islets were incubated for 60 min at 4 degrees C in the presence of equilibrated D-glucose (2.8 and 8.3 mM) mixed with tracer amounts of either alpha- or beta-D-glucose labelled with tritium on either the C (2) or C (5) of the hexose. Relative to their respective concentrations, (3)HOH generation from the anomers labelled with tritium on the C (2) or C (5) of the hexose provided beta/alpha ratios comparable to those previously found at both 2.8 and 8.3 mM, when the islets were exposed to each anomer separately. The relative contributions of each anomer to the total generation of (3)HOH was also close to the theoretical values derived from mathematical models for the catabolism of D-glucose at anomeric equilibrium in rat islets at both 2.8 and 8.3 mM and in the case of both D-[2 - (3)H]glucose and D-[5 - (3)H]glucose. Thus, even in islets exposed to D-glucose at anomeric equilibrium, the metabolic fate of alpha-D-glucose differs vastly from that of beta-D-glucose, the enzyme-to-enzyme channelling between hexokinase isoenzymes, especially glucokinase, and phosphoglucoisomerase being restricted to alpha-D-glucose 6-phosphate.

Abstract: It was recently proposed that alpha-D-glucose 6-phosphate may undergo enzyme-to-enzyme channelling between glucokinase and phosphoglucoisomerase in rat pancreatic islets. The present study aims at exploring whether a different situation prevails in cells deprived of glucokinase, namely in erythrocytes. At anomeric equilibrium, the ratio between D-[2-3H]glucose and D-[5-3H]glucose conversion to 3HOH was lower in rat erythrocytes incubated for 60 min at 4 degrees C in the presence of 2.8 mM, rather than 8.3 mM, D-glucose. This coincided with both a greater relative increase in beta-D-[5-3H]glucose, as compared to alpha-D-[5-3H]glucose, conversion to 3HOH and an increase in the beta/alpha ratio for 3HOH generation from D-[5-3H]glucose in response to an increase in the anomeric concentration from 2.8 to 8.3 mM, the suppression of the difference between the beta/alpha ratios for 3HOH generation from D-[2-3H]glucose and D-[5-3H]glucose in the erythrocytes incubated at 8.3 mM, as distinct from 2.8 mM, alpha- and beta-D-glucose, and a [2-3H]/[5-3H] ratio for 3HOH generation lower than unity in erythrocytes exposed to alpha-D-glucose but not significantly different from unity in the presence of beta-D-glucose. These findings emphasize the relevance of alpha-D-glucose 6-phosphate channelling between hexokinase and phosphoglucoisomerase as a determinant of the difference between D-[2-3H]glucose and D-[5-3H]glucose conversion to 3HOH, and reveal that the regulation of such a tunnelling process by the concentration of the D-glucose represents, in rat erythrocytes, a mirror image of that observed in rat pancreatic islets. The regulation of this process thus tightly depends on the identity of the hexokinase enzyme mainly responsible for the phosphorylation of D-glucose in distinct cell types.

Abstract: Glucagon-like peptide-1 (GLP-1) is the most insulinogenic of the glucagon-like peptides secreted mainly by L cells in the small and large intestine in response to the ingestion of nutrients. It binds to a specific GLP-1 receptor (GLP-1R) on beta-cells and can increase islet neogenesis and beta-cell mass. It is not clear whether the transmission of information from the gut to islet beta-cells by messengers such as GLP-1 is different in individuals who develop autoimmune diabetes. In the present study the expression of bioactive GLP-1 protein in the gut and its receptor in the pancreas was examined in diabetes-prone BioBreeding (BBdp) rats in the period before overt diabetes and in age-matched control, non-diabetes-prone BB (BBc) rats. An N-terminal directed antibody specific for the bioactive forms of GLP-1 (GLP-1(7-37) and GLP-1(7-36amide)) was used to mea-sure GLP-1 by radioimmunoassay in proximal, median, and distal gut. Pancreas GLP-1R area fraction, GLP-1R gene expression, and insulin content were analyzed, as were plasma GLP-1, glucose, and insulin. The concentration of GLP-1 protein in the jejunum and ileum of BBdp rats was lower than in BBc rats. Although these animals maintained normal blood glucose, there was impaired pancreatic endocrine function, characterized by low baseline insulin concentration in plasma and pancreas. GLP-1R mRNA expression was threefold less in islets isolated from BBdp rats, and GLP-1R+ islet area fraction in pancreas sections was decreased. When injected iv with GLP-1, BBdp rats displayed lower second-phase insulin response (and insulin/glucose ratios) compared with BBc rats. Thus, young BBdp rats displayed decreased concentrations of bioactive GLP-1 in jejunum and ileum, reduced GLP-1R in islets, and lower second-phase insulin response to iv GLP-1 than controls. The decrease in insulinogenic and islet beta-cell mass-promoting signal from GLP-1 in BBdp rats may contribute to impaired glucoregulation and ineffective maintenance of normal islet mass that shifts islet homeostasis in favor of development of diabetes.

Abstract: Diabetes-prone BioBreeding (BBdp) rats often present an enteropathy that may precede the onset of autoimmune insulitis. The aim of the present study was to assess the influence of sex, the time course, the strain specificity, the distribution along the intestinal tract and the effect of diet for the changes in the activity of gut invertase, maltase and lactase found in BBdp rats, as compared with both Wistar-Furth (WF) and diabetes-resistant BioBreeding (BBc) rats. These hydrolases were measured, therefore, at day 10, 30, 45, 70, 95 and 120 in three intestinal segments of WF, BBc and BBdp rats fed, after weaning, either a protective hydrolysed casein diet, which decreases the incidence of diabetes in the BBdp rats, or one of two diabetogenic diets (National Toxicology Program; NTP or wheat-gluten-based; WG) [corrected]. Except for a somewhat lower lactase activity in the BBdp rats, no obvious difference in hydrolyase activity between the three strains of rats was observed at day 10. Between days 30 and 120, however, the activity of the hydrolases, especially that of invertase and lactase, was lower in the BBdp rats than in either the WF or BBc rats, at least when considering the animals fed either the NTP or WG diet. These findings support the view that BBdp rats exposed to a diabetogenic diet develop an enteropathy well before the onset of autoimmune insulitis, in a manner somehow comparable with the situation found in some type 1 diabetic patients, in whom coeliac disease may be diagnosed before diabetes onset.

Abstract: D-mannoheptulose inhibits D-glucose phosphorylation by hexokinase isoenzymes. The present study aims at investigating whether the pattern of such an inhibition differs in the case of alpha- versus beta-D-glucose. The phosphorylation of alpha- and beta-D-[U-14C]glucose was measured over 60-min incubation at 4 degrees C in the presence of bovine heart hexokinase and over 10 min at 24 degrees C in the presence of human liver glucokinase. The relative extent of the inhibitory action of D-mannoheptulose (0.02-10.0 mM) was always less marked with alpha- than beta-D-glucose. In the case of hexokinase, the experiments conducted at the high concentration of the D-glucose anomers (1.0 mM) revealed that D-mannoheptulose, at low concentrations (0.2-0.5 mM), may unexpectedly increase the phosphorylation of alpha-D-glucose. These findings thus document anomeric specificity in terms of the inhibitory action of D-mannoheptulose upon alpha- versus beta-D-glucose phosphorylation by either hexokinase or glucokinase.

Abstract: Novel information was recently provided concerning the reciprocal effects of D-glucose and D-fructose upon their respective metabolism in rat pancreatic islets. In the light of such findings, this study aims at comparing the effects of D-glucose and D-fructose on insulin, somatostatin, and glucagon release from the isolated perfused rat pancreas. A rise in D-glucose concentration from 3.3 to 5.0 or 7.3 mM or the administration of D-fructose (17 and 40 mM) in the presence of 3.3 mM D-glucose stimulated insulin release in a concentration-related manner, but failed to affect somatostatin output. The secretion of glucagon was decreased in all cases. The secretory response to L-arginine (5 mM), 25 min after restoring the basal concentration of D-glucose, was more markedly affected, in terms of potentiation of insulin and somatostatin release and reduction of glucagon output, after prior administration of D-fructose than after a prior increase in D-glucose concentration. These findings argue against any major role for a paracrine regulation of hormonal release and, instead, are consistent with a causal link between metabolic and secretory events in the islet cells. Nevertheless, the present results emphasize differences in the response of distinct pancreatic endocrine cell types to the same or distinct hexoses.

Abstract: Based on the measurement of 3HOH generation from the alpha- and beta-anomer of D-[2-3H]glucose and D-[5-3H] glucose by rat pancreatic islets, it was recently proposed that alpha-D-glucose 6-phosphate may undergo enzyme-to-enzyme channelling between hexokinase isoenzymes and phosphoglucoisomerase. Taking into account the results of such measurements, the present study aims at defining a model for the metabolism of alpha- and beta-D-glucose in islets exposed to 2.8 or 8.3 mM equilibrated D-glucose. It is proposed that, whilst keeping the activity of free phosphoglucoisomerase at its physiological value, all available experimental data can be adequately reproduced in a model in which all molecules of alpha-D-glucose 6-phosphate, whether unlabelled or tritiated on their C2 carbon atom, undergo, without isotopic discrimination, the postulated enzyme-to-enzyme channelling process. In such a model, the fractional intermolecular transfer of tritium at the phosphoglucoisomerase level amounts to 26%, suggesting that the intrinsic catalytic properties of the enzyme may be slightly different in the proposed metabolon than that prevailing for the unbound enzyme. These findings thus afford further support to the concept that alpha-D-glucose 6-phosphate, as distinct from beta-D-glucose 6-phosphate, is tunnelled, in rat pancreatic islets, in the sequence of reactions catalyzed by hexokinase isoenzymes, phosphoglucoisomerase and, possibly, phosphofructokinase.

Abstract: The enzyme-to-enzyme channeling of metabolic intermediates is not an uncommon process. The present review draws attention to recent experimental work documenting, in rat pancreatic islets, the enzyme-to-enzyme channeling of alpha-D-glucose 6-phosphate between hexokinase isoenzyme(s), mainly glucokinase, and phosphoglucoisomerase. Likewise, the possible enzyme-to-enzyme channeling of beta-D-fructose 6-phosphate between phosphoglucoisomerase and phosphofructokinase is briefly evoked. These considerations are relevant to the anomeric specificity of D-glucose metabolism, even in islets exposed to equilibrated D-glucose, to the perturbation of such an anomeric specificity in the phenomenon of so-called B-cell glucotoxicity, and to the correct interpretation of 3HOH generation from D-[2-(3H)]glucose.

Abstract: BACKGROUND: The development of immune-mediated diabetes in BB rats may involve an inflammatory lesion of the intestinal tract. METHODS: In order to further explore this issue, the activity of peroxidase was measured, in the absence and presence of either bromide or dapsone, at day 10, 30, 45, 70, 95 and 120 in three segments (top, mid and bottom) of the intestinal tract from Wistar-Furth rats and both diabetes-resistant and diabetes-prone BB rats fed after weaning either diabetes-promoting diets or a protective diet, which decreases the incidence of diabetes in the BB rats. RESULTS: In the present study, from day 30 onwards, an age-related increase in peroxidase activity was found in the intestine of diabetes-prone BB rats (BBdp rats) fed diabetogenic diets, when compared with either Wistar-Furth or diabetes-resistant BB rats (BBc rats). This increase was most pronounced in the distal segments of the intestinal tract. Even when fed a protective diet, higher peroxidase activity was found in BBdp than BBc rats. Yet, in BBc rats, and to a lesser extent in BBdp rats, the diabetogenic diets lowered peroxidase activity below the value found in rats of the same strain fed the protective diet. There was a tight correlation between the activity of peroxidase and its susceptibility to be increased by bromide. CONCLUSION: It is proposed that diabetogenic diets may decrease peroxidase activity in intestinal cells, this effect becoming masked in BBdp rats by an age-related increase in the contribution of inflammatory cells. The latter phenomenon affects preferentially the distal segments of the intestinal tract.

Abstract: The anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms was investigated. For this purpose, the hemidiaphragms were preincubated for 30 min at 37 degrees C and then incubated for 5 min at the same temperature in the presence of alpha- or beta-D-[U-14C]glucose. The concentrations of D-glucose (5.6 or 8.8 mM) and insulin (0 or 10 mU/ml) were identical during the preincubation and incubation periods. The incubation medium was prepared in D2O/H2O (3:1, v/v) in order to delay the interconversion of the D-glucose anomers. In addition to glycogen labelling, the output of radioactive acidic metabolites was also measured. Insulin caused a preferential stimulation of glycogen labelling relative to glycolysis. Such was not the case in response to a rise in D-glucose concentration. At 5.6 mM D-glucose and whether in the presence or absence of insulin, both glycogen labelling and glycolysis were lower with alpha-D-glucose than with beta-D-glucose suggesting a higher rate of beta-D-glucose than alpha-D-glucose transport across the plasma membrane. A mirror image was found at 8.8 mM D-glucose, especially in the absence of insulin. At this close-to-physiological hexose concentration, insulin lowered the alpha/beta ratio for glycogen labelling. On the contrary, the rise in D-glucose concentration increased such a ratio. Since such a rise is probably little affected by any possible anomeric difference in D-glucose transport across the plasma membrane, the present results strongly suggest that the intracellular factors regulating net glycogen synthesis, as well as glycolytic flux, display obvious preference for alpha-D-glucose.

Abstract: Thioacetamide (0.01-1.3 mM) fails to exert any significant immediate effect upon insulin release from rat isolated islets. However, when administered (4 micromol/g body wt) intraperitoneally 24 h before sacrifice, it reduced food intake and body weight and affected the secretory response of isolated islets to several secretagogues, despite unaltered insulin content of such islets. This coincided with a decrease in D-[U-14C]glucose oxidation, total islet calcium content and the ionized calcium content of secretory granules in islet B-cells, and changes in both 133Ba and 45Ca net uptake. Likewise, in islets prepared from thioacetamide-injected rats and prelabeled with 45Ca before perifusion, the cationic and insulin secretory responses to D-glucose or gliclazide, but not to the association of Ba2+ and theophylline in the absence of extracellular Ca2+, often differed from that otherwise found in islets prepared from control rats. These findings are interpreted as indicative of an impaired capacity of Ca2+ sequestration by intracellular organelles in the islet B-cells of thioacetamide-treated rats.

Abstract: A sufficient availability of the bicarbonate anion is required to allow a normal process of glucose-stimulated insulin release. Thus, both the removal of extracellular bicarbonate and the inhibition of carbonic anhydrase by acetazolamide suppress the insulinotropic action of glucose. In the present study, a comparable situation is documented in rat parotid cells. Thus, the absence of extracellular bicarbonate is shown to decrease by about 50% basal alpha-amylase secretion, as well as carbamylcholine- and isoproterenol-stimulated alpha-amylase output, without suppressing the enhancing action of the two agents. It is proposed, therefore, that the HCO3-anion participates in the secretory sequence at sites distal to the identification of secretagogues in both endocrine and exocrine cells.

Abstract: It was recently reported that alpha-D-glucose is more potent than beta-D-glucose in conferring to glucokinase positive cooperativity towards D-fructose. We have now extended pilot experiments to investigate whether a comparable situation prevails in intact rat pancreatic islets in terms of the modulation by the D-glucose anomers of the effect of D-fructose upon 45Ca efflux from prelabelled perifused islets. As expected from the effect of increasing concentrations of equilibrated D-glucose upon 45Ca efflux from the prelabelled islets, D-fructose either decreased or increased 45Ca outflow from islets perifused in the presence of either alpha- or beta-D-glucose. In all cases, the alpha-anomer of D-glucose affected more markedly than beta-D-glucose the cationic response to D-fructose. These findings indicate that the anomeric specificity of the effect of D-glucose upon D-fructose phosphorylation by glucokinase is also operative in intact islets.

Abstract: In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) or high (16.7 mM) D-glucose concentration, the labelling of both lipids and their glycerol moiety is higher in the presence of D-[1-14C]glucose than D-[6-14C]glucose. The rise in D-glucose concentration augments the labelling of lipids, the paired 14C/3H ratio found in islets exposed to both D-[1-14C]glucose or D-[6-14C]glucose and D-[3-3H]glucose being even slightly higher at 16.7 mM D-glucose than that found, under otherwise identical conditions, at 2.8 mM D-glucose. Such a paired ratio exceeds unity in islets exposed to D-[1-14C]glucose. The labelling of islet lipids by D-[6-14C]glucose is about 30 times lower than the generation of acidic metabolites from the same tracer. These findings indicate (i) that the labelling of islet lipids accounts for only a minor fraction of D-glucose catabolism in pancreatic islets, (ii) a greater escape to L-glycerol-3-phosphate of glycerone-3-phosphate generated from the C1-C2-C3 moiety of D-glucose than D-glyceraldehyde-3-phosphate produced from the C4-C5-C6 moiety of the hexose, (iii) that only a limited amount of [3-3H]glycerone 3-phosphate generated from D-[3-3H]glucose is detritiated at the triose phosphate isomerase level before being converted to L-glycerol-3-phosphate, and (iv) that a rise in D-glucose concentration results in an increased labelling of islet lipids, this phenomenon being somewhat more pronounced in the case of D-[1-14C]glucose or D-[6-14C]glucose rather than D-[3-3H]glucose.

Abstract: D-mannoheptulose was recently proposed to be transported into cells mainly at the intervention of GLUT2. In the present study, the heptose (10 mM) decreased the steady state content of dispersed rat pancreatic islet cells in D-[U-(14)C]glucose, and inhibited to a greater relative extent the utilization of D-[5-(3)H]glucose, the oxidation of D-[U-(14)C]-glucose and its conversion to radioactive amino acid when the dispersed islet cells were incubated at 16.7 mM rather than 2.8 mM D-glucose. A comparable situation was found in purified islet B-cells, whereas D-mannoheptulose only exerted minor to negligible effects upon the metabolism of D-glucose in non-B islet cells. This coincided with a much higher uptake of D-[(3)H]mannoheptulose by B, as distinct from non-B, islet cells. These findings indicate that the unexpectedly greater relative inhibitory action of D-mannoheptulose upon D-glucose metabolism by isolated islets (or dispersed islet cells) observed at high rather than low hexose concentration cannot be accounted for solely by differences in the relative contribution of non-B cells to total D-glucose metabolism by islets incubated at increasing concentrations of D-glucose. A comparable metabolic response to D-mannoheptulose is indeed observed in purified B cells. It could be attributable, in part at least, to D-glucose and D-mannoheptulose countertransport, resulting inter alia in a greater net uptake of the heptose by B cells exposed to a high concentration of the hexose.

Abstract: Cytochalasin B is known to enhance insulin release evoked by nutrient and non-nutrient secretagogues, including D-glucose, despite inhibiting D-glucose uptake and metabolism in pancreatic islets. In the present study, cytochalasin D, which failed to affect D-glucose uptake and metabolism by isolated islets, also augmented glucose-stimulated insulin release, but unexpectedly to a lesser extent than cytochalasin B. Such was not the case, however, in islets stimulated by non-glucidic nutrients such as 2-ketoisocaproate or the association of L-leucine and L-glutamine. This situation coincided with the fact that cytochalasin B inhibited more severely D-glucose metabolism in non-B, as distinct from B, islet cells and, in the former case, caused a relatively greater inhibition of hexose catabolism at 2.8 mM than at 16.7 mM D-glucose. Nevertheless, even in the presence of forskolin, cytochalasin B was more efficient than cytochalasin D in augmenting glucose-stimulated insulin secretion. Thus, although these data document that non-B islet cells are more sensitive than purified islet B cells to cytochalasin B, at least in terms of inhibition of D-glucose catabolism, such a difference and its possible consequence upon the release of glucagon and other non-insulinic hormones by non-B islet cells do not appear sufficient to account for the greater enhancing action of cytochalasin B, as distinct from cytochalasin D, upon glucose-stimulated insulin output. Likewise, the latter difference does not appear attributable to a greater efficiency of cytochalasin B, as compared to cytochalasin D, upon the mechanical events involved in nutrient-stimulated exocytosis of insulin granules. Hence, the present findings suggest a so-far-unidentified interference of cytochalasin B with the B-cell glucose-sensing device.

Abstract: This study aims at exploring specific aspects of D-glucose metabolism, so far not yet investigated, in pancreatic islets from adult control rats and animals (STZ rats) injected with streptozotocin during the neonatal period. The latter animals, which represent a current model of type-2 diabetes, displayed a lower body weight, higher plasma D-glucose concentration and lower insulinogenic index than control rats. The protein, DNA and insulin content were all also lower in islets prepared from STZ, rather than control rats. In the presence of 10.0 mM D-glucose, the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization was also decreased in the islets from STZ rats. No significant difference between control and STZ rats was observed, however, in terms of the ratios between D-[3-3H]glucose and D-[5-3H]glucose utilization, between the generation of radioactive lactate from 14C-labelled D-glucose and tritiated D-glucose utilization and between D-[1-14C]glucose and D-[6-14C]glucose oxidation. These findings reinforce the view that the previously documented preferential impairment of the oxidative modality of glycolysis in islets from STZ rats contrasts with the absence of any major anomaly in other variables of D-glucose catabolism.

Abstract: Single-strand conformational polymorphism analysis of mitochondrial FAD-linked glycerophosphate dehydrogenase (mGDH) gene has revealed mutations in both the calcium- and FAD-binding domains of this enzyme in some diabetic patients. It was now investigated whether site-directed mutations in the FAD-binding domain of the mGDH gene may affect the mitochondrial anchoring and catalytic activity of the enzyme. COS-7 cells were transfected with plasmid cDNA coding for either wild-type or mutated human mGDH (G --> A substitutions at positions 352, 355, and 364 and A --> C substitution at position 390) fused, when required, at the N-terminus of green fluorescent protein. The activity of mGDH was measured by both radioisotopic ((3)HOH production from l-[2-(3)H]glycerol 3-phosphate) and colorimetric (iodoformazan formation) procedures. In cells transfected with the mGDHwt-EGFP or mGDHmut-EGFP constructs, the fused protein was found by confocal microscopy exclusively in the mitochondria, colocalized with a mitochondrial marker. In homogenates of COS-7 cells transfected with mGDHmut, however, the catalytic activity of the enzyme was decreased, this coinciding with low ratios between both the activities measured in the absence/presence of exogenous FAD and the results obtained by the colorimetric/radioisotopic procedure. Thus, although the present site-directed mutations of the mGDH gene failed to impair the mitochondrial anchoring of the enzyme, they led to catalytic defects that were, in some respect, comparable to those previously encountered in the lymphocytes or islets of type 2 diabetic patients.

Abstract: D-mannoheptulose was recently proposed as a tool to label preferentially insulin-producing cells in the pancreatic gland in the perspective of the non-invasive imaging of the endocrine pancreas. In such a perspective, we have now synthesized 1-deoxy-1-[125I]iodo-D-mannoheptulose ([125I]MH) and examined its uptake by different rat cell types. No phosphorylation of [125I]MH by bovine heart hexokinase could be detected. The apparent distribution space of [125I]MH largely exceeded that of [U-14C]sucrose, considered as an extracellular marker, in erythrocytes, parotid cells, hepatocytes, pancreatic pieces and isolated pancreatic islets. Relative to the mean intracellular distribution space of 3HOH, that of [125I]MH was not significantly different in pancreatic pieces from either normal rats or streptozotocin-induced diabetic animals (STZ rats). In pancreatic islets, the uptake of [125I]MH was decreased at low temperature, but failed to be significantly affected by cytochalasin B. Sixty min after the intravenous injection of [125I]MH, the radioactive content of selected organs displayed the following hierarchy: muscle<pancreas<liver<parotid<kidney<plasma. In this respect, there was no obvious difference between control and STZ rats. Taken as a whole, these findings suggest that 1-deoxy-1-[125I]iodo-D-mannoheptulose does not display the same specificity towards GLUT2, as that previously documented in the case of D-[3H]- mannoheptulose.

Abstract: D-mannoheptulose is a specific inhibitor of D-glucose phosphorylation by hexokinase isoenzymes. In the present study, the phosphorylation of this heptose was investigated by either a spectrophotometric or radioisotopic procedure. Using yeast hexokinase, the phosphorylation of 25 mM D-mannoheptulose only represented 0.02% of that of 5 mM D-glucose. Such a percentage was increased to 3.93% in the case of bovine heart hexokinase. In the latter case, the Km for D-mannoheptulose was close to 0.2 mM and both D-glucose (0.1-1.0 mM) and D-glucose 6-phosphate (also 0.1-1.0 mM) inhibited the phosphorylation of the heptose (0.03-0.60 mM). Human B-cell glucokinase also catalyzed the phosphorylation of D-mannoheptulose (0.1 mM), which was now increased in a bell-shaped manner by D-glucose (1.0-20 mM). Likewise, rat parotid gland, liver and pancreatic islet homogenates catalyzed the phosphorylation of D-[3H]mannoheptulose. The results obtained in these three tissues differed from one another by their absolute values (per mg wet wt.), relative values (by reference to the phosphorylation rate of 10 mM D-glucose), and sensitivity to inhibition by D-glucose (10 mM).

Abstract: D-mannoheptulose was recently proposed as a possible tool to label preferentially insulin-producing cells in the pancreatic gland. In the present study, D-[3H]-mannoheptulose uptake by rat pancreatic islets or dispersed islet cells was found to represent a time-related and temperature-sensitive process inhibited by cytochalasin B. This mould metabolite also inhibited the efflux of D-[3H]-mannoheptulose from prelabelled islets. After 60 min incubation at 37 degrees C, the apparent intracellular distribution space of the tritiated heptose was close to or somewhat higher than that of D-[5-3H]glucose and close to 50% of the intracellular 3HOH space. It was further enhanced by D-glucose and a high concentration of 10 mM of D-mannoheptulose. The uptake of D-[3H]mannoheptulose was much lower however than that of D-[3H]mannoheptulose hexaacetate. As judged from the fate of D-mannoheptulose hexa[2-14C]acetate, the latter ester was efficiently hydrolyzed in the islet cells. The internalization of D-[3H]mannoheptulose (or its ester) coincided with the generation of tritiated acidic metabolites, reflecting phosphorylation of the heptose. The situation found in normal islet cells sharply differed from that found in tumoral islet cells of either the RINm5F or INS-1 line, in which the apparent distribution space of D-[3H]mannoheptulose represented only about 3 and 9%, respectively, of the intracellular 3HOH space. These results indicate that the entry of D-mannoheptulose into islet cells represents a carrier-mediated process, possibly mediated at the intervention of GLUT2 and, hence, provide further support to the possible use of a suitable D-mannoheptulose analog as a tool for the preferential labelling of insulin-producing cells in the pancreatic gland.

Abstract: The uptake of D-[3H]mannoheptulose by isolated pancreatic islets was recently proposed as a tool to assess the relative contribution of insulin-producing cells to the total mass of the islets. In the present study, the uptake of the tritiated heptose over 60 min incubation at 37 degrees C was about 21% lower in islets from hereditarily diabetic rats (GK rats) than in islets from control animals, this decrease being virtually identical to that documented previously by morphometric analysis of islets from the same type of rats. The intracellular 3HOH space and extracellular [U-14C]sucrose space were not significantly different in control and diabetic rats, at least when the comparison was restricted to animals of the same sex. There was a trend, however, towards a somewhat lower D-[5-3H]-glucose intracellular distribution space in islets from GK rats, as compared to control animals. These findings provide further support to the validity of D-[3H]mannoheptulose uptake as a tool to assess the density of insulin-producing cells in isolated islets.

Abstract: D-[3H]mannoheptulose (or D-[1-14C] mannoheptulose) net uptake was measured in rat erythrocytes, parotid cells and hepatocytes. In the erythrocytes and parotid cells, the intracellular distribution space of the heptose (0.1 mM) represented only about 1 and 13%, respectively, of the intracellular 3HOH space. In hepatocytes, however, it amounted to approximately 45% of the intracellular 3HOH space. In all cases, the apparent distribution space of D-[3H]mannoheptulose hexaacetate largely exceeded that of unesterified D-[3H]mannoheptulose. Relative to the intracellular water space, the generation of acidic metabolites (expressed as an apparent distribution space) from radioactive D-mannoheptulose was one order of magnitude lower in parotid cells (< or = 3%) than in hepatocytes (> or = 20%). These findings are compatible with the hypothesis that D-mannoheptulose is transported into cells mainly, if not exclusively, at the intervention of GLUT2.

Abstract: In terms of glucose sensing by pancreatic islet beta-cells, emphasis is currently placed on both the role of glucokinase, with negligible activity of low-Km hexokinase(s), and the prevalence of the oxidative over non-oxidative modality of glycolysis, a situation tentatively attributed, in part at least, to a low activity of lactate dehydrogenase. Conflicting information is available, however, on the activity of both low-Km hexokinase(s) and lactate dehydrogenase in purified beta-cell homogenates. This issue was reinvestigated, therefore, in two populations of purified rat islet beta-cells selected on the basis of their low (betaL) or high (betaH) content in reduced pyridine nucleotides. The size and protein content of betaH cells represented about twice that of betaL cells. Such was also the case for low-Km hexokinase(s), lactate dehydrogenase, mitochondrial FAD-linked glycerophosphate dehydrogenase, glutamate dehydrogenase and glutamate-alanine and glutamate-aspartate transaminases. Whether in betaH or betaL cells, the activity of low-Km hexokinase(s) was at least as high as or higher than that of glucokinase. In both betaH and betaL, the activity of lactate dehydrogenase exceeded that required to catalyze the full reduction of glucose-derived pyruvate to L-lactate, as estimated from the rate of D-glucose phosphorylation under physiological conditions. These findings thus argue against a low expression of either low-Km hexokinase(s) or lactate dehydrogenase as major determinants of the glucose-sensing device in beta-cells.

Abstract: The possible use of D-mannoheptose or D-glycero-D-gulo-heptose as substitute of D-mannoheptulose for specific inhibition of D-glucose phosphorylation, metabolism and insulinotropic action was investigated in the present study. The two aldoheptoses failed to duplicate the effect of D-mannoheptulose upon the phosphorylation of D-glucose by yeast hexokinase, bovine heart hexokinase or human B-cell glucokinase. They were poorly phosphorylated by the low-Km hexokinase isoenzymes or liver B-cell glucokinase. D-mannoheptose failed to reproduce the inhibitory action of D-mannoheptulose upon D-glucose metabolism by isolated rat pancreatic islets. Whilst D-glycero-D-gulo-heptose failed to affect glucose-induced insulin release, D-mannoheptose slightly enhanced glucose-induced insulin release when tested at low concentrations (0.75-1.5 mM) and progressively decreased insulin output at higher concentration (3. 0-20.0 mM) in islets exposed to a high (16.7 mM), but not physiological (8.3 mM), concentration of D-glucose. D-mannoheptose, however, also caused a modest inhibition of insulin release evoked by 2-ketoisocaproate. It is concluded, therefore, that neither D-mannoheptose nor D-glycero-D-guloheptose can be considered as suitable substitutes of D-mannoheptulose.

Abstract: Diadenosine polyphosphates, such as diadenosine triphosphate (A2P3) and diadenosine tetraphosphate (A2P4), were recently proposed to participate in the stimulus-secretion coupling for nutrient-stimulated insulin release. Since NaF, an inhibitor of inorganic pyrophosphatase, was reported to lower A2P3 and A2P4 content in glucose-stimulated pancreatic islets, its effects upon metabolic, cationic, biosynthetic and secretory variables in rat pancreatic islets were investigated in the present study. Up to a concentration close to 0.1 mM, NaF failed to affect most of these variables, except for a decrease in 45Ca net uptake. Much higher concentrations of NaF (e.g. 5.0 mM) were required to cause inhibition of the metabolic, ionic, biosynthetic and secretory responses of the islets to nutrient secretagogues. Yet, even at this high concentration, NaF failed to lower the islet content in tritiated A2P3 and A2P4 in islets prelabelled with [2,8-3H]adenosine and failed to prevent the glucose-induced increase in such a content. It is concluded, therefore, that NaF may not represent a suitable tool to assess the participation of diadenosine polyphosphates in the process of nutrient-induced insulin secretion.

Abstract: The anomeric specificity of the wild-type recombinant forms of human liver and B-cell glucokinase was investigated using radioactive anomers of d-glucose as tracers. With d-glucose at anomeric equilibrium and at 30 degrees C, the maximal velocity, Hill number, and K(s) amounted, respectively, to 16 micromol min(-1) mg(-1), 1.8 and 6.9 mM in the case of liver glucokinase, and 7.3 micromol min(-1) mg(-1), 2.0 and 7.1 mM in the case of B-cell glucokinase. Whether at 20-22 or 30 degrees C, the maximal velocity, Hill number, and K(m) were significantly lower with alpha-d-glucose than with beta-d-glucose in both liver and B-cell glucokinase. As a result of these differences, the reaction velocity was higher with alpha-d-glucose at low hexose concentrations, while the opposite situation prevailed at high hexose concentrations. In the presence of 0.2 mM d-fructose 6-phosphate, the glucokinase regulatory protein caused a concentration-related inhibition of d-glucose phosphorylation, such an effect fading out at high concentrations of either d-glucose or glucokinase relative to that of its regulatory protein. The phosphorylation of alpha-d-glucose by liver glucokinase appeared more resistant than that of beta-d-glucose to the inhibitory action of d-fructose 6-phosphate, as mediated by the glucokinase regulatory protein. Such a phenomenon failed to achieve statistical significance in the case of the B-cell glucokinase. It is proposed that this information, especially the novel findings concerning the anomeric difference in both Hill number and sensitivity to the glucokinase regulatory protein, should be taken into account when considering the respective contributions of alpha- and beta-d-glucose to the overall phosphorylation of equilibrated d-glucose by glucokinase.

Abstract: The present studies were performed to determine if a protective diet has different effects on the metabolic activity or function of islet cells, as well as the metabolic activity of mesenteric lymph node (MLN) cells and spleen cells, from BioBreeding (BB) rats. Diabetes-prone BB (BBdp) rats and control non-diabetes-prone BB (BBc) rats were fed for about 20 days either a mainly plant-based diabetogenic diet, NIH-07 (NIH), or a protective semipurified diet with hydrolyzed casein (HC) as the amino acid source. At 6 to 8 weeks of age, BBdp rats had high plasma D-glucose and low insulin concentrations, low insulin content, and low metabolic and secretory responses to D-glucose in isolated pancreatic islets. Islet metabolism, as measured by accumulation of 14C-acidic metabolites, amino acids, and the ratio of D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization was increased in control rats fed HC (P < .05); a similar trend in BBdp rats was not significant. Feeding the HC diet increased islet insulin content (P < .01) by 13% in BBdp and 23% in BBc rats; other metabolic and hormonal variables were unaffected. Compared with BBc rats, BBdp rats displayed higher rates of L-[U-14C]glutamine oxidation, D-[5-3H]glucose utilization, and D-[U-14C]glucose oxidation in MLN cells, but not in splenocytes. There was a dramatic decrease of L-[U-14C]glutamine oxidation in MLN cells from BBc and BBdp rats fed HC. Glycolysis was decreased in control rats. We conclude that the protection afforded by feeding BBdp rats a HC diet is associated with increased insulin in target beta cells and downregulation of metabolic activity in gut-associated MLN cells. Metabolic activity in splenocytes, cells representative of the systemic immune system, was less affected. These data suggest that diet-induced metabolic changes occur in the islets and nearby cells of the gut immune system in the period before classic insulitis. Changes in the islets were smaller in comparison to the dramatic remodeling of nutrient catabolism in MLN cells. MLN downregulation may reflect baseline metabolic activity in the absence of diabetogenic (or other) food antigens and further highlights an important interaction between diabetogenic food antigens and the gut immune tissues.

Abstract: D-mannoheptulose was recently found to inhibit D-glucose metabolism in hepatocytes and pancreatic islets, whilst failing to do so in parotid cells, erythrocytes and the exocrine pancreas. In the latter three systems, however, the hexaacetate ester of D-mannoheptulose efficiently inhibits D-glucose metabolism. It was proposed, therefore, that the transport of unesterified D-mannoheptulose into cells may be mediated by GLUT2. Since cytochalasin B is known to inhibit D-glucose transport into pancreatic islet cells, it was now investigated whether the mould metabolite (0.02 mM) also impairs the inhibitory action of D-mannoheptulose (1.0 mM) upon D-glucose metabolism in rat pancreatic islets. The relative extent of D-mannoheptulose inhibitory action on D-[5-3H]glucose utilization and D-[U-14C]glucose conversion to 14CO2, as well as radioactive amino acids and acidic metabolites, was indeed much less marked in the presence of cytochalasin B (13+/-4% inhibition) than in its absence (40+/-3% inhibition). A comparable situation was not observed, however, in the case of glucose-stimulated insulin secretion, cytochalasin B augmenting insulin output to the same relative extent in the absence or presence of D-mannoheptulose. These findings support the view that the entry of D-mannoheptulose into cells may be mediated by a cytochalasin B-sensitive transport system, such as the GLUT2 carrier.

Abstract: The apparent distribution space of 6-deoxy-6-[125I]iodo-D-glucose, recently proposed as a tracer of D-glucose transport, was measured in rat isolated islets, acinar tissue, and pieces of pancreas. While such a space reached a steady-state value corresponding to the 3HOH volume in pancreatic islets within 5 min, it slowly increased in pieces of pancreas and, even after 60-min incubation, remained lower than the 3HOH volume. Moreover, the net uptake of 6-deoxy-6-[125I]iodo-D-glucose by pancreatic pieces was inhibited by unlabeled 6-deoxy-6-iodo-D-glucose, D-glucose, and cytochalasin B, while being less or not affected by these agents in isolated islets. A preferential labeling of the endocrine, relative to exocrine, moiety of the pancreas was documented both by comparing, after 2 min incubation, the uptake of 6-deoxy-6-[125I]iodo-D-glucose by pieces of pancreas from normal vs streptozotocin-injected rats and by comparing the radioactive content of pancreatic islets and acinar tissue obtained from normal rats injected intravenously 3 min before sacrifice with 6-deoxy-6-[125I]iodo-D-glucose. It is proposed, therefore, that advantage could conceivably be taken from the vastly different time course for the uptake of selected monosaccharides by pancreatic islets vs acinar cells in the perspective of imaging of the endocrine pancreas by a non invasive method.

Abstract: The present studies were undertaken to examine concomitant diet-induced changes in pancreatic islets and cells of the gut immune system of diabetes-prone BB rats in the period before classic insulitis. Diabetes-prone (BBdp) and control nondiabetes prone (BBc) BB rats were fed for approximately 17 days either a mainly plant-based standard laboratory rodent diet associated with high diabetes frequency, NIH-07 (NIH) or a protective semipurified diet with hydrolyzed casein (HC) as the amino acid source. By about 7 weeks of age, NIH-fed BBdp rats had lower plasma insulin and insulin/glucose ratio, lower insulin content of isolated islets, lower basal levels of NO but higher responsiveness of NO production to IL-1beta in cultured islets, and higher Con A response and biosynthetic activities in mesenteric lymphocytes than control rats fed the same diet. In control rats, the HC diet caused only minor changes in most variables, except for a decrease in oxidation of L-[U-14C]glutamine in Peyer's patch (PP) cells and an increase in protein biosynthesis in mesenteric lymphocytes. In BBdp rats, however, the HC diet increased plasma insulin concentration, islet insulin/protein ratio, and tended to normalize the basal and IL-1beta-stimulated NO production by cultured islets. The HC diet decreased oxidation of L[U-14C]glutamine in BBdp pancreatic islets, whereas oxidation of L-[U-14C]glutamine in PP cells was increased, and the basal [Methyl-3H]thymidine incorporation in mesenteric lymphocytes was decreased. These findings are compatible with the view that alteration of nutrient catabolism in islet cells as well as key cells of the gut immune system, particularly changes in mitotic and biosynthetic activities in mesenteric lymphocytes, as well as basal and IL-1beta stimulated NO production, participate in the sequence of events leading to autoimmune diabetes in BB rats. Thus, the protection afforded by feeding a hydrolysed casein-based diet derives from alterations in both the target islet tissue and key cells of the gut immune system in this animal model of type 1 diabetes.

Abstract: The apparent distribution space of 6-deoxy-6-[125I]iodo-D-glucose, recently proposed as a tracer of D-glucose transport, was measured in rat isolated islets, acinar tissue, and pieces of pancreas. While such a space reached a steady-state value corresponding to the 3HOH volume in pancreatic islets within 5 min, it slowly increased in pieces of pancreas and, even after 60-min incubation, remained lower than the 3HOH volume. Moreover, the net uptake of 6-deoxy-6-[125I]iodo-D-glucose by pancreatic pieces was inhibited by unlabeled 6-deoxy-6-iodo-D-glucose, D-glucose, and cytochalasin B, while being less or not affected by these agents in isolated islets. A preferential labeling of the endocrine, relative to exocrine, moiety of the pancreas was documented both by comparing, after 2 min incubation, the uptake of 6-deoxy-6-[125I]iodo-D-glucose by pieces of pancreas from normal vs streptozotocin-injected rats and by comparing the radioactive content of pancreatic islets and acinar tissue obtained from normal rats injected intravenously 3 min before sacrifice with 6-deoxy-6-[125I]iodo-D-glucose. It is proposed, therefore, that advantage could conceivably be taken from the vastly different time course for the uptake of selected monosaccharides by pancreatic islets vs acinar cells in the perspective of imaging of the endocrine pancreas by a non invasive method.

Abstract: The role currently ascribed to the accumulation of L-arginine in the pancreatic islet B-cell as a determinant of its insulinotropic action was reevaluated by comparing the uptake and the metabolic, ionic, electric, and secretory effects of the cationic amino acid with those of its more positively charged methyl ester in rat pancreatic islets. The response to L-arginine methyl ester differed from that evoked by the unesterified amino acid by a lower uptake and oxidation, lack of inhibitory action on D-glucose metabolism, more severe inhibition of the catabolism of endogenous L-glutamine, inhibition of 45Ca net uptake, decrease in both 86Rb outflow from prelabeled islets perifused at normal extracellular Ca2+ concentration and 45Ca efflux from prelabeled islets perifused in the absence of extracellular Ca2+, and delayed and lesser insulinotropic action. These findings reinforce the view that the carrier-mediated entry of L-arginine into the islet B-cells, with resulting depolarization of the plasma membrane, represents the essential mechanism for stimulation of insulin release by this cationic amino acid.

Abstract: D-mannoheptulose was recently proposed to be transported into cells at the intervention of GLUT2. Since GLUT1, rather than GLUT2, represents the major carrier system for the transport of monosaccharides across the islet B-cell plasma membrane in human subjects, the uptake of D-mannoheptulose and its metabolic and secretory effects were investigated in human islets. The uptake of D-glucose reached much more rapidly a close-to-equilibrium value in isolated islets than in pieces of pancreas obtained from the same donor. The distribution space of D-[3H]mannoheptulose in the human islets largely exceeded that of [U-14C]sucrose, considered as an extracellular marker, and did not differ significantly from that of 3HOH. In the human islets, the heptose (10.0 mM) inhibited both D-[5-3H]glucose utilization and D-[U-14C] glucose oxidation, and decreased glucose-stimulated insulin release to the same extent as D-mannoheptulose hexaacetate. These findings indicate that a suitable radioactive analog of D-mannoheptulose could be used, in human like in rat islets, for preferential labelling of the endocrine moiety of the pancreatic gland.

Abstract: The analog of D-glucose, 3-O-methyl-D-glucose, is thought to delay the equilibration of D-glucose concentration across the plasma membrane of pancreatic islet B-cells, but not to exert any marked inhibitory action upon the late phase of glucose-stimulated insulin release. In this study, however, 3-O-methyl-D-glucose, when tested in high concentrations (30-80 mM) was found to cause a rapid, sustained and not rapidly reversible inhibition of glucose-induced insulin release in rat pancreatic islets. In relative terms, the inhibitory action of 3-O-methyl-D-glucose was more marked at low than high concentrations of D-glucose. It could not be attributed to hyperosmolarity and appeared specific for the insulinotropic action of D-glucose, as distinct from non-glucidic nutrient secretagogues. Although 3-O-methyl-D-glucose and D-glucose failed to exert any reciprocal effect upon the steady-state value for the net uptake of these monosaccharides by the islets, the glucose analog inhibited D-[5-3H]glucose utilization and D-[U-14C]glucose oxidation. This coincided with increased 86Rb outflow and decreased 45Ca outflow from prelabelled islets, as well as decreased 45Ca net uptake. A preferential effect of 3-O-methyl-D-glucose upon the first phase of glucose-stimulated insulin release was judged compatible with an altered initial rate of D-glucose entry into islet B-cells. The long-term inhibitory action of the glucose analog upon the metabolic and secretory response to D-glucose, however, may be due, in part at least, to an impaired rate of D-glucose phosphorylation. The phosphorylation of the hexose by beef heart hexokinase and human B-cell glucokinase, as well as by parotid and islet homogenates, was indeed inhibited by 3-O-methyl-D-glucose. The relationship between insulin release and D-glucose utilization or oxidation in the presence of 3-O-methyl-D-glucose was not different from that otherwise observed at increasing concentrations of either D-glucose or D-mannoheptulose. It is concluded, therefore, that 3-O-methyl-D-glucose adversely affects the metabolism and insulinotropic action of D-glucose by a mechanism largely unrelated to changes in the intracellular concentration of the latter hexose.

Abstract: The pentaacetate ester of alpha-D-glucose was recently introduced as a new insulin secretagogue. Its insulinotropic action appears mainly attributable to the catabolism of its hexose moiety in islet cells, but a direct effect of the ester itself upon a receptor apparently displaying analogy with that involved in the recognition of bitter agents by taste buds may also be operative. In the present study, the secretory response of rat isolated pancreatic islets to alpha-D-glucose pentaacetate (1.7 mM) was found to be preserved, except in the absence of any other exogenous nutrient, when the extracellular pH was raised from about 7.4 to 8.0. Inversely, however, when the extracellular pH was lowered to about 7.0, alpha-D-glucose pentaacetate inhibited both basal and D-glucose- or L-leucine-stimulated insulin output. These findings are interpreted to support a dual mode of action of alpha-D-glucose pentaacetate upon insulin secretion, a lowering of extracellular pH revealing a negative component of the islet B-cells functional response to such a monosaccharide ester.

Abstract:

Abstract: Six middle-aged (42-59 years old) and six aged (71-75 years old) subjects received each, on separate days, an oral administration of gliquidone (30 mg), glibenclamide (5 mg), gliclazide (80 mg) and glipizide (5 mg). The plasma concentration of the drugs was measured before and at eight times (60 min to 24 h) thereafter. The half-life of gliclazide was higher than that of the other three hypoglycemic agents in middle-aged subjects and was the sole to be significantly increased in aged subjects. There is no obvious difference between sulfonylureas eliminated mainly by either the kidney (glibenclamide, gliclazide, glipizide) or the liver (gliquidone) in terms of the influence of aging upon their clearance.

Abstract: Lactate is released in large quantity from sites of sepsis and inflammation. We asked whether the increased lactate production found in sepsis can be explained by the augmented glycolysis of inflammatory cells. The glycolytic metabolism of rat peritoneal leukocytes was measured following cecal ligation and perforation (CLP) or sham laparotomy. CLP augmented glucose uptake, the pentose phosphate pathway, and glucose oxidation. Lactate output increased from 1.03 +/- 0.05 to 1.20 +/- 0.05 fmol x cell(-1) x min(-1) (P < .001). Total lactate output of peritoneal lavage fluid increased from 7.94 +/- 2.59 to 28.12 +/- 5.60 nmol L x min(-1) (P < .005). The effect of lipopolysaccharide (LPS) on the lactate output of whole blood from 31 critically ill patients was measured. Leukocyte lactate production was calculated by multiple linear regression analysis. Following exposure to LPS, human leukocyte lactate output increased from 0.20 +/- 0.09 to 1.22 +/- 0.14 fmol x cell(-1) x min(-1) (P < .001). This rate of production is so high that it suggests that the lactate output of different tissue beds in sepsis may be affected by their different cell populations and state of activation. This study supports the hypothesis that lactate may be more a product of inflammation than a marker of tissue hypoxia in sepsis.

Abstract: In the light of recent findings on the effect of D-glucose upon D-fructose phosphorylation by human B-cell glucokinase, the influence of the aldohexose upon the metabolism of the ketohexose was investigated in rat pancreatic islets. D-glucose, although slightly decreasing D-[5-(3)H]fructose utilization, augmented the oxidation of the ketohexose, indicating that the aldohexose stimulates preferentially the oxidative, as distinct from anaerobic, modality of glycolysis. Such was not the case in parotid cells, taken as representative of functionally nonglucose-responsive cells. In the islets exposed to D-fructose, D-glucose also decreased the fractional contribution of the pentose shunt to the generation of CO2 and D-glyceraldehyde 3-phosphate from the ketohexose, and increased the inflow into the Krebs cycle of dicarboxylic metabolites relative to that of fructose-derived acetyl-CoA. This glucose-induced remodeling of D-fructose metabolism may optimize the insulin secretory response of islet cells to these hexoses, e.g. after food intake.

Abstract: The metabolic and secretory responses to D-glucose and/or D-fructose were measured in pancreatic islets prepared from either control rats or animals that had been injected with streptozotocin during the neonatal period (STZ rats). The STZ rats displayed higher plasma D-glucose concentrations, but lower plasma insulin concentrations, islet insulin content, as well as basal and nutrient-stimulated insulin release. This coincided with lower rates of D-[U-(14)C]hexose oxidation and D-[5-(3)H]hexose utilization. In both control and STZ rats, D-fructose failed to affect significantly the metabolism of d-glucose, while the aldohexose increased the ratio between D-[U-(14)C]fructose oxidation and D-[5-(3)H]fructose conversion to (3)HOH. Such a ratio was higher than that found with radioactive D-glucose in islets exposed to both hexoses, whether in control or STZ rats, indicating a far-from-negligible contribution of fructokinase to the phosphorylation of D-fructose. Despite these analogies between both the respective fate of D-glucose and D-fructose and the reciprocal metabolic effects of the two hexoses in islets from control and STZ rats, the secretory response to the ketohexose in islets from STZ rats was preferentially suppressed, relative to that evoked by the aldohexose. This gives support to the idea that the insulinotropic action of D-fructose may not be entirely accounted for by its nutritional value in islet cells.

Abstract: The effects of substituting a plant-based control diabetogenic diet (NIH diet) by a protective hydrolyzed casein diet (HC diet) upon selected metabolic and functional variables were recently investigated in Peyer's patch cells, splenocytes, mesenteric lymph node cells, and pancreatic islets from either control (BBc) or diabetes-prone (BBdp) BB rats. In the present work, the plasma d-glucose and insulin concentrations, the protein and insulin content of pancreatic islets, the metabolism of d-glucose, and its insulinotropic action in islets first cultured for 24 h in the absence or presence of IL-1beta, the production of IFN-gamma and IL-10 by mesenteric lymph node cells cultured for 48 h in the absence or presence of concanavalin A, the mitogenic activity of Peyer's patch cells and pancreatic lymph node cells in the absence or presence of the same lectin, and the biosynthetic activity of Peyer's patch cells were measured in the BBc and BBdp rats fed either the NIH or the HC diet. Two major novel findings emerged from this study. First, in immune cells, diet HC increased to a greater extent the responsiveness to concanavalin A of certain metabolic and functional variables in BBdp rats than in BBc rats. Second, pancreatic islet cells of BBdp rats were less sensitive to IL-1beta than those of BBc rats and this difference was further accentuated when the animals were fed the HC rather than the NIH diet. These findings afford further support to the view that, in BB rats, changes in the biological behavior of Peyer's patch cells, mesenteric and pancreatic lymph node cells, and pancreatic islet cells participate in the pathogenesis of insulin-dependent diabetes mellitus and its prevention by a suitable dietary manipulation.

Abstract: The metabolism of D-glucose and/or D-fructose was investigated in both pancreatic islets and parotid cells of control and hereditarily diabetic Goto-Kakizaki (GK) rats. In the islets from GK rats, a preferential alteration of the oxidative response to D-glucose coincided with an impaired secretory response to the aldohexose. Such a metabolic alteration was not found in the parotid cells of GK rats. Whether in islet or parotid cells, D-fructose little affected the catabolism of glucose in either control or GK rats. The metabolism of D-fructose and the effect of D-glucose thereupon were essentially comparable in control and GK rats in both pancreatic islets and parotid cells. In both cell types, the comparison between the metabolism of D-glucose and D-fructose in cells simultaneously exposed to the two hexoses suggested a far from negligible contribution of fructokinase to the phosphorylation of D-fructose. Although the catabolism of the ketohexose and its modulation by D-glucose were closely comparable in islets from control and GK rats, the insulinotropic action of the ketohexose, relative to that of the aldohexose, was severely impaired in the GK rats. The present work thus emphasizes the specificity of the alteration in D-glucose metabolism in islets, as opposed to extrapancreatic cells, of GK rats. It also reveals in the islets of GK rats a further secretory anomaly apparently not attributable to the impairment of nutrient catabolism in the islet cells of these diabetic animals.

Abstract: A rise in D-glucose concentration may augment insulin release independently of changes in K(+) conductance or Ca(2+) influx in pancreatic islet cells, the insulinotropic action of the hexose remaining dependent on an increased generation of high-energy phosphates. In the present study, therefore, it was investigated to which extent the procedures currently used to assess the modalities of the secretory response to D-glucose independent of its effect on ATP-sensitive K(+) channels and Ca(2+) inflow may themselves affect the catabolism of the hexose in isolated rat pancreatic islets. A rise in the extracellular K(+) concentration from 5 to 30 or 60 mM failed to significantly affect the metabolism of D-glucose. At 90 mM K(+), however, the maximal velocity of the glycolytic flux was decreased and the apparent K(m) for D-glucose lowered, without an obvious alteration of the preferential stimulation of oxidative mitochondrial events in response to a rise in D-glucose concentration. Such a preferential stimulation was abolished, however, either by diazoxide at a low, but not high, K(+) concentration or by Ca(2+) deprivation, in the absence or presence of diazoxide, at a high K(+) concentration. It is speculated that these metabolic changes may be attributable, in part at least, to an altered activity of key cytosolic (e.g. pyruvate kinase) and mitochondrial (e.g. FAD-linked glycerophosphate dehydrogenase) enzymes.

Abstract: 1. The effect of N-[(trans-4-isopropylcyclohexyl)-carbonyl]-D-phenylalanine (A-4166) on nutrient metabolism was investigated in isolated rat pancreatic islets. 2. At a 10-microM concentration, the meglitinide analogue caused a modest increase in 14CO2 output from islets prelabeled with L-[U-14C]glutamine but failed to affect D-[5-3H]glucose utilization, D-[U-14C]glucose oxidation and conversion into 14C-labeled acidic metabolites and amino acids, L[1-14C]leucine and L-[U-14C]leucine oxidation, the generation of 2-ketoisocaproate and further acidic metabolites from the branched-chain amino acid and the production of 14CO2 by islets prelabeled with [U-14C]palmitate. 3. These findings indicate that the insulinotropic action of A-4166 is not attributable to any sizeable increase in the metabolism of exogenous or endogenous nutrients.

Abstract: The metabolism and metabolic effects of alpha-D-glucose pentaacetate were investigated in isolated rat pancreatic islets. Several findings were compatible with the view that the insulinotropic action of alpha-D-glucose pentaacetate is causally related to its capacity to act as a fuel in the islet B-cell. First, the ester was efficiently taken up and hydrolyzed with resulting accumulation of D-glucose in the islet cells. Second, the conversion of alpha-D-[5-3H]glucose pentaacetate to 3HOH and that of alpha-D-[U-14C]glucose pentaacetate to 14CO2 exceeded those found at an equimolar concentration (1.7 mM) of D-glucose and were both inhibited by 2-deoxy-D-glucose (16.7 mM). Last, the ester inhibited the catabolism of both exogenous D-glucose or endogenous fatty acids. Yet, an apparent dissociation between the metabolic and secretory responses to the ester was suggested by the failure of alpha-D-glucose pentaacetate to increase O2 uptake by the islets. Moreover, there were striking differences between the catabolism of the ester and that of unesterified D-glucose, such as a much higher intracellular D-glucose content and an insensitiveness to the inhibitory action of D-mannoheptulose in islets exposed to alpha-D-glucose pentaacetate. Likewise, the ratio between hexose oxidation and utilization was lower for alpha-D-glucose pentaacetate than for unesterified D-glucose in islets concomitantly exposed to the hexose and its ester. It is proposed, therefore, that the insulinotropic action of alpha-D-glucose pentaacetate, although probably linked to the intracellular generation of D-glucose from the ester, may not involve the same coupling process between metabolic and functional events as that currently implied in the process of glucose-stimulated insulin release.

Abstract: The effects of two aldose reductase inhibitors, ARI 509 (4.0 microM) and tolrestat (40.0 microM), upon sorbitol output, D-[5-3H]glucose and D-[U-14C]glucose metabolism and insulin release were investigated in pancreatic islets prepared from normal rats or hereditarily diabetic animals (Goto-Kakizaki rats) and incubated in the presence of 16.7 mM D-glucose. At this hexose concentration, the output of sorbitol, the utilization of D-[5-3H]glucose, the oxidation of D-[U-14C]glucose and its conversion to 14C-labelled acidic metabolites and amino acids and the secretion of insulin were all much higher than those found in islets exposed to only 2.8 mM D-glucose. In both normal and diabetic rats, the aldose reductase inhibitors suppressed glucose-stimulated sorbitol output, but failed to affect the metabolism of D-[5(-3H]glucose or D-[U-14C]glucose and the secretory response to the hexose. These findings document the efficiency and specificity of ARI 509 and tolrestat as inhibitors of aldose reductase in islet cells, whilst arguing against any major role of sorbitol formation in the stimulus-secretion coupling process for glucose-induced insulin release and any major perturbation of those factors regulating the generation and output of sorbitol in islets of Goto-Kakizaki rats.

Abstract: D-mannoheptulose, but not its hexaacetate ester, inhibits, in a competitive manner, D-glucose phosphorylation by either purified beef heart hexokinase or crude parotid gland homogenates. Yet, D-mannoheptulose hexaacetate, but neither the unesterified heptose nor acetate or its methyl ester, inhibits D-[5-3H]glucose utilization and D-[U-14C]glucose conversion to 14CO2 and 14C-labelled acidic metabolites and amino acids in intact isolated parotid cells. It is proposed, therefore, that D-mannoheptulose hexaacetate crosses efficiently the plasma membrane of parotid cells and, after intracellular hydrolysis, allows inhibition of D-glucose phosphorylation by the unesterified heptose. The ester of D-mannoheptulose could thus represent a useful tool to inhibit hexose phosphorylation and interfere with cell growth in cells otherwise resistant to the heptose.

Abstract: The polyacetate esters of selected monosaccharides were recently found to either stimulate insulin release or inhibit glucose-stimulated insulin secretion in islets from normal rats. The present study extends such findings both to new combinations of either D-glucose or L-leucine and some polyacetate esters and to hereditarily diabetic, as distinct from normal, rats. In the normal animals, 2-deoxy-D-glucose tetraacetate (1.7 mM) increased both glucose- and leucine-stimulated insulin output. The secretory response to L-leucine was also increased by beta-D-glucose pentaacetate, but inhibited by alpha-D-galactose pentaacetate and D-mannoheptulose hexaacetate (1.7 mM) in the islets of normal rats. In the diabetic rats, the secretory response to D-glucose (8.3 mM) was increased by alpha- or beta-D-glucose pentaacetate and 2-deoxy-D-glucose tetraacetate (1.7 mM), inhibited by alpha-D-galactose pentaacetate and D-mannoheptulose hexaacetate, and unaffected by beta-L-glucose pentaacetate, all esters being tested at 1.7 mM concentration. L-Leucine-stimulated insulin release was also increased by alpha-D-glucose pentaacetate, but not significantly affected by beta-D-galactose pentaacetate, beta-L-glucose pentaacetate, 2-deoxy-D-glucose tetraacetate and D-mannoheptulose hexaacetate in the islets of diabetic animals. These findings suggest a dual mode of action of the esters in the pancreatic islet B-cell, involving both the metabolic response to their sugar moieties and a direct effect of the esters themselves upon a specific receptor system. It is proposed that selected esters could be used as insulinotropic tools in non-insulin-dependent diabetes mellitus.

Abstract: Rat pancreatic islets were found to display a much lower content of immunoreactive CD38 and a much lower ADP-ribosyl cyclase activity than rat spleen or brain. Cyclic ADP-ribose was also measured by a radioimmunological procedure in rat pancreatic islets. In fed rats, the cyclic ADP-ribose content appeared higher after isolation of the islets in the presence of 2.8 mM D-glucose rather than in the absence of the hexose, progressively increased during incubation of the islets for 5-60 min at 37 degrees C, but failed to be affected by the concentration of D-glucose (zero to 20.0 mM) in the incubation medium. In rats fasted for 24 hours, the cyclic ADP-ribose islet content also increased during incubation, but again failed to be affected by the concentration of D-glucose in the incubation medium. Although these findings indicate that the islet cyclic ADP-ribose content is influenced by nutritional and environmental factors, they do not support the view that the insulinotropic action of D-glucose involves major change in the islet cell content of the cyclic nucleotide.

Abstract: This study aims at gaining further insight into the mode of action of repaglinide in pancreatic islet B-cells. At a 1.0 mumol/L concentration, the meglitinide analog failed to affect the metabolism of exogenous D-glucose and that of endogenous nutrients in islets prelabeled with either L-[U-14C]glutamine or [U-14C]palmitate. Likewise, repaglinide (1.0 mumol/L) failed to modify significantly the incorporation of L-[4-3H]phenylalanine into TCA-precipitable material in islets exposed to a close-to-physiological concentration of D-glucose (7.0 mmol/L). The threshold concentration for the insulinotropic action of repaglinide was close to 0.1-1.0 mumol/L and a maximal response was reached at 10.0 mumol/L in islets incubated in the presence of 5.6-8.3 mmol/L D-glucose. At a higher hexose concentration (16.7 mmol/L), however, an enhancing action of repaglinide (10 mumol/L) upon glucose-stimulated insulin release was only observed over 25 min stimulation in perifused islets, no significant increase in insulin output being detected when islets were exposed to repaglinide (0.1 mumol/L to 0.1 mmol/L) over 90 min incubation at the high D-glucose level. The increase in insulin output evoked by repaglinide in the islets perifused at 16.7 mmol/L D-glucose coincided with a modest increase in 86Rb outflow and a marked stimulation of 45Ca efflux from prelabeled islets, suggesting stimulation of Ca2+ influx into the islet cells and subsequent activation of Ca(2+)-responsive K+ channels. When the administration of repaglinide was halted, the reversibility of its cationic and secretory effects was more pronounced in islets perifused at a high (16.7 mmol/L), rather than a low (6.0 mmol/L), D-glucose concentration. These findings support the view that the primary site of action of repaglinide consists in a remodeling of cationic fluxes, and document that this drug displays favorable attributes as an insulinotropic agent for the treatment of non-insulin-dependent diabetes, such as its lack of interference with nutrient metabolism and biosynthetic activity in isolated islets, the low threshold concentration for its insulin-releasing action and its capacity to augment, at least transiently, insulin release at a high concentration of D-glucose.

Abstract: The modality of the insulinotropic action of 1,1-dimethyl-2-[2-morpholinophenyl]guanidine fumarate (BTS 67 582), a new antidiabetic agent, was investigated in rat pancreatic islets. At a 0.1 mM concentration, which was sufficient to cause a close-to-maximal secretory response, BTS 67 582 failed to affect the utilization and oxidation of exogenous D-glucose, but slightly augmented 14CO2 production from islets prelabelled with either L-[U-14C]glutamine or [U-14C]palmitate. BTS 67 582 (0.1 mM) also failed to affect biosynthetic activity in islets incubated with L-[4-3H]phenylalanine. It augmented insulin release from islets incubated for 90 min in the absence or presence of D-glucose (2.8 to 16.7 mM), this coinciding with stimulation of 45Ca net uptake. In perifused islets deprived of extracellular D-glucose for 45 min, BTS 67 582 (0.1 mM) decreased 86Rb outflow from prelabelled islets, but failed to increase 45Ca efflux and insulin release. In the presence of D-glucose (7.0 mM), BTS 67 582, whilst failing to decrease 86Rb+ outflow, provoked rapid, sustained and rapidly reversible increases of both 45Ca2+ efflux and insulin output. The latter increases were attenuated, but not totally suppressed, in the absence of extracellular Ca2+. BTS 67 582 (0.1 mM) suppressed the inhibitory action of diazoxide (0.25 mM) upon glucose-stimulated insulin release, but nevertheless augmented insulin output from islets incubated in the presence of 90 mM K+. These findings support the view that the insulinotropic action of BTS 67 582 is mainly attributable to the inactivation of ATP-sensitive K+ channels. An intracellular redistribution of Ca2+ ions may also participate, however, to the islet functional response to BTS 67 582.

Abstract: At concentrations in excess of about 80 mmol/l, D-fructose stimulates insulin release from rat islets incubated in the absence of any other exogenous nutrient, an optimal secretory response being recorded in the 240 to 320 mmol/l range. D-galactose and 3-O-methyl-D-glucose fail to reproduce the insulinotropic action of D-fructose. At a concentration of D-fructose close to the threshold value for such an insulinotropic action (80 mmol/l), as little as 1.0-4.0 mmol/l D-glucose is sufficient to increase insulin release, with a sigmoidal concentration-response relationship similar to that otherwise evoked by much higher concentrations of the aldohexose. The release of insulin evoked by D-fructose (240 mmol/l) is abolished in the absence of Ca2+ or presence of KCN (2.0 mmol/l), partially inhibited by 3-O-methyl-D-glucose (80 mmol/l) or D-mannoheptulose (1.0 mmol/l), and potentiated by forskolin (10 micromol/l), theophylline (1.4 mmol/l), cytochalasin B (21 micromol/l) and glibenclamide (5 micromol/l). These findings indicate that the stimulation of insulin release by high concentrations of D-fructose corresponds to an active secretory process modulated by the metabolic fate of the hexose, the availability of ATP, the activity of ATP-sensitive K+ channels, the extracellular concentration of Ca2+, the cell content in cyclic AMP and the motile events under the control of the microfilamentous cell web.

Abstract: The metabolism of beta-L-glucose pentaacetate and its interference with the catabolism of L-[U-14C]glutamine, [U-14C]palmitate, D-[U-14C]glucose, and D-[5-3H]glucose were examined in rat pancreatic islets. Likewise, attention was paid to the effects of this ester on the biosynthesis of islet peptides, the release of insulin from incubated or perifused islets, the functional behavior of individual B cells examined in a reverse hemolytic plaque assay of insulin secretion, adenylate cyclase activity in a membrane-enriched islet subcellular fraction, cAMP production by intact islets, tritiated inositol phosphate production by islets preincubated with myo-[2-3H]inositol, islet cell intracellular pH, 86Rb and 45Ca efflux from prelabeled perifused islets, and electrical activity in single isolated B cells. The results of these experiments were interpreted to indicate that the insulinotropic action of beta-L-glucose pentaacetate is not attributable to any nutritional value of the ester but, instead, appears to result from a direct effect of the ester itself on a yet unidentified receptor system, resulting in a decrease in K+ conductance, plasma membrane depolarization, and induction of electrical activity.

Abstract: D-mannoheptulose is currently used as a tool to inhibit, in a competitive manner, D-glucose phosphorylation, metabolism and functional effects in the pancreatic islet B-cell. In order to better understand the mode of action of the heptose, we have explored its effect upon D-glucose phosphorylation in liver, parotid cells and islet homogenates, this allowing to characterize the interference of the heptose with glucokinase and/or hexokinase. The effect of D-mannoheptulose upon the metabolism of D-glucose was also examined in both intact parotid cells and pancreatic islets. Last, the effect of D-mannoheptulose upon glucose-stimulated insulin release was reinvestigated over large concentration ranges of both the heptose and hexose. The experimental data revealed a mixed type of D-mannoheptulose inhibitory action upon D-glucose phosphorylation, predominantly of the non-competitive and competitive type, in liver and parotid homogenates, respectively. Despite efficient inhibition of hexose phosphorylation in both parotid cell and islet homogenates, the heptose suppressed the metabolic and functional responses to D-glucose only in pancreatic islets, whilst failing to affect adversely D-glucose catabolism in parotid cells. These findings suggest that factors such as the intracellular transport and availability of the heptose may interfere with the expression of its antagonistic action upon D-glucose metabolism.

Abstract: 99mTc-sesta-(2-methoxy-isobutyl-isonitrile)(Tc-MIBI) is currently used for imaging of several organs. In the present study, its uptake by rat pancreatic islets, rat parotid cells, and human breast adenocarcinoma cells (MCF-7 cells) was found to be grossly proportional to its concentration (up to 0.1 microM), time-related (with a fractional turnover rate close to 2-3 10(-2).min(-1)), and stimulated by D-glucose. Comparable values for the fractional turnover rate were found in prelabeled islets and MCF-7 cells, D-glucose failing to affect Tc-MIBI efflux from prelabeled islets. In the islets, the uptake of Tc-MIBI was decreased at low temperature, in the presence of mitochondrial poisons and at high extracellular K+ concentration, unaffected by the absence of extracellular Ca2+, and increased by nutrient secretagogs, such as 2-ketoisocaproate and the association of L-leucine and L-glutamine. These findings are consistent with the view that Tc-MIBI uptake is ruled by its extracellular concentration, and the polarization of both plasma and mitochondrial membranes. It is proposed that this lipophilic cation may be useful to detect alteration of nutrient metabolism in pancreatic islets deprived of any exogenous fuel.

Abstract: The pentaacetate esters of selected hexoses were recently found to stimulate insulin release. The kinetics of their hydrolysis was now investigated in both rat pancreatic islet homogenates and intact islets. In islet homogenates, the hydrolysis of alpha-d-glucose pentaacetate, as judged from the measurement of acetate production, displayed a pH optimum of 7.4 and a Km for the ester of 0.95 mM. At pH 7.4, the reaction velocity was about 5 times higher than the rate of alpha-d-glucose pentaacetate hydrolysis by intact islets, as judged from the ester-induced increase in the acetate content of both the islet and surrounding incubation medium. Comparable results were obtained in intact islets exposed to either beta-l-glucose pentaacetate or beta-d-galactose pentaacetate. The ester content of the islets after 120 min incubation was close to 0.1 nmol/islet, yielding an apparent intracellular concentration at least one order of magnitude higher than the extracellular concentration (1.7 mM). These findings indicate that hexose esters that either stimulate insulin release or fail to do so are equally well taken up and hydrolyzed by islet cells. They are compatible, therefore, with the view that the insulinotropic action of some of these esters may be favored by the catabolism of their hexose moiety, although some other mechanisms for stimulation of insulin release must be operative in the case of beta-l-glucose pentaacetate.

Abstract: It was recently, and surprisingly, found that D-mannoheptulose did not affect D-glucose metabolism and insulinotropic action in pancreatic islets incubated at a low concentration of D-glucose. To explain this finding, the metabolism and secretory response to the hexose were investigated in rat islets exposed to D-mannoheptulose hexaacetate, which was recently found to inhibit D-glucose catabolism in cells that are otherwise fully resistant to the heptose. At a high concentration of D-glucose (16.7 mmol/l), the utilisation of D-[5-(3)H]glucose and oxidation of D-[U-14C]glucose, as well as the insulinotropic action of the hexose, were affected less by D-mannoheptulose tetraacetate than by unesterified D-mannoheptulose. This coincided with a reduced uptake of the ester by intact islets and a lower rate of hydrolysis of the ester in islet homogenates compared with findings in other monosaccharide esters such as D-glucose pentaacetate. At a low concentration of D-glucose (2.8 mmol/l), D-mannoheptulose hexaacetate was slightly more efficient than the unesterified heptose in reducing D-glucose catabolism, but still failed to suppress the secretory response to the hexose. These findings do not necessarily mean that unesterified D-mannoheptulose enters beta-cells more efficiently at high than at low extracellular D-glucose concentrations, especially if possible differences in the respective contributions of distinct islet cell types to the overall catabolism of D-glucose by whole islets is allowed for. These data do not rule out the possibility that D-glucose phosphorylation is more resistant to D-mannoheptulose in beta cells incubated at a low than a high concentration, independently of any difference in the intracellular concentration of the heptose. However, the mechanism of this resistance is still not explained.

Abstract: Control rats and diabetic animals injected with streptozotocin during the neonatal period were either maintained on a standard diet or given access to food supplemented with dehydroepiandrosterone (DHEA, 0.2%) for 11 days before sacrifice. In both control and diabetic rats, DHEA feeding augmented the activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase and cytosolic NADP-linked malate dehydrogenase in liver, but not so in either the parotid gland or pancreatic islets. DHEA lowered, in both control and diabetic rats, the ratio between D-glucose oxidation and utilization and the rate of insulin release in pancreatic islets exposed to a high concentration of D-glucose, as well as the insulin concentration and insulin/glucose ratio in plasma. These findings support the view that, in diabetes, DHEA, by increasing sensitivity to insulin, may allow islet B-cells to avoid the otherwise unfavorable consequences of chronic hyperactivity.

Abstract: The Ca(2+)-sensitive and mitochondrial enzyme FAD-linked glycerophosphate dehydrogenase (m-GDH) represents an essential component of the pancreatic B-cell glucose-sensing device. This report deals with the first identified case of mutation in the calcium-binding domain of the m-GDH gene in a patient with type-2 diabetes and his glucose-intolerant half sister. Single strand conformation polymorphism analysis indeed revealed an abnormal mobility of the 32P-labelled polymerase chain reaction product in these two subjects. The corresponding base pair mutations and amino acid changes were documented. In the diabetic proband, the relative extent of the Ca(2+)-induced activation of m-GDH in CD3+ T-lymphocytes was lower than in his brother with a normal m-GDH gene sequence.

Abstract: Cyclic ADP-ribose was measured by a radioimmunological procedure in rat pancreatic islets. The cyclic ADP-ribose content of the islets was much lower immediately after isolation (< or = 2.3 +/- 0.2 fmol/islet) than after a further incubation of 90 min in a salt-balanced medium (26.4 +/- 1.3 fmol/islet). Under the latter condition, the islet cyclic ADP-ribose content was not affected by the concentration of D-glucose and/or D-fructose in the incubation medium and, when expressed per micrograms protein, was 3- to 30-fold higher than that found in other biological samples such as liver, whole pancrease or tumoral islet cells. Although these findings might suggest a physiological role for cyclic ADP-ribose in islet cells, they argue against the view that the cyclic nucleotide acts as a coupling factor in the process of hexosestimulated insulin release.

Abstract: Insulin release from rat pancreatic islets was stimulated by alpha-D-glucose pentaacetate (1.7 mM), but not by an equimolar concentration of beta-D-galactose pentaacetate. The secretory response to alpha-D-glucose pentaacetate was not reproduced by D-glucose and/or acetate, tested at concentrations equimolar to that of the hexose ester, and failed to be adversely affected by 3-O-methyl-D-glucose, even when used at a concentration sufficient to inhibit glucose-stimulated insulin release. These findings suggest that the insulinotropic action of alpha-D-glucose pentaacetate is attributable, in part at least, to the intracellular generation of D-glucose from the ester. The present work thus introduces selected esters of D-glucose as tools for the cellular supply of the hexose by a process that does apparently not involve its carrier-mediated transport across the plasma membrane.

Abstract: It was recently reported that selected esters of D-glucose are more efficiently metabolized and display a greater insulinotropic action in pancreatic islets than the unesterified hexose. Likewise, in the present study, the inhibitory action of 2-deoxy-D-glucose tetraacetate upon D-glucose metabolism in rat erythrocytes and pancreatic islets was found to be more pronounced than that of unesterified 2-deoxy-D-glucose. At a concentration of 10 mM, the ester also inhibited more severely than unesterified 2-deoxy-D-glucose the release of insulin evoked by D-glucose in isolated islets. It is proposed that 2-deoxy-D-glucose tetraacetate represents a useful tool to facilitate the intracellular delivery of the glucose analog and to increase its efficiency as a potential therapeutic agent.

Abstract: Succinic acid esters are currently under investigation as possible insulinotropic tools in the treatment of noninsulin-dependent diabetes mellitus. The present article introduces three novel nutrient esters and aims mainly to explore, in both normal and GK rats, the secretory response to such esters when tested alone or in combination. It documents that in pancreatic islets from normal rats, methyl acetate (10 mM), which fails to augment basal insulin output, potentiates the secretory response to succinate dimethyl ester (also 10 mM). It also reveals that alpha-D-glucose pentaacetate (alpha GPA) (1.7 mM) stimulates insulin release in the absence of any other exogenous nutrient and even more so in the presence of succinate methyl ester. Moreover, the methyl esters of succinic acid (10 mM), when used together with either methyl acetate or alpha GPA, provoked insulin secretion in islets from diabetic GK rats incubated in the absence of D-glucose, although no significant secretory response of such islets could be detected when each of these agents was tested separately. These findings thus draw attention to the insulinotropic potential in type 2 diabetes of selected combinations of nutrient esters, including a D-glucose ester presumably able to enter into islet cells without requiring the intervention of a hexose carrier.

Abstract: The functional determinants of the insulinotropic action of alpha-D-glucose pentaacetate were investigated in rat pancreatic islets. The ester mimicked the effect of nutrient secretagogues by recruiting individual B cells into an active secretory state, stimulating proinsulin biosynthesis, inhibiting 86Rb outflow, and augmenting 45Ca efflux from prelabeled islets. The secretory response to the ester was suppressed in the absence of Ca2+ and potentiated by theophylline or cytochalasin B. The generation of acetate from the ester apparently played a small role in its insulinotropic action. Thus acetate, methyl acetate, ethyl acetate, alpha-D-galactose pentaacetate, and beta-D-galactose pentaacetate all failed to stimulate insulin release. The secretory response to alpha-D-glucose pentaacetate was reproduced by beta-D-glucose pentaacetate and, to a lesser extent, by beta-L-glucose pentaacetate. It differed from that evoked by unesterified D-glucose by its resistance to 3-O-methyl-D-glucose, D-mannoheptulose, and 2-deoxy-D-glucose. It is concluded that the insulinotropic action of alpha-D-glucose pentaacetate, although linked to the generation of the hexose from its ester, entails a coupling mechanism that is not identical to that currently implied in the process of glucose-induced insulin release.

Abstract: It was recently proposed that stimulation of pancreatic islet by D-glucose results in the translocation of glucokinase from the perinuclear area to the cell periphery, where the enzyme might conceivably interact with either the glucose transporter GLUT-2 or some other proteins and, by doing so, become better able to express its full catalytic activity. To explore the possible interaction between glucokinase and the cell boundary, dispersed rat pancreatic islet cells were preincubated for 60 min at a low (2.8 mM) or high (16.7 mM) concentration of D-glucose, then exposed for 1 min to digitonin (0.5 mg/ml) and eventually centrifuged through a layer of oil for separation of the cell pellet from the supernatant fraction containing the material released by digitonin. Under these conditions, the bulk of lactate dehydrogenase and glutamate dehydrogenase activities were recovered in the supernatant fraction and cell pellet, respectively. The measurement of hexokinase isoenzyme activities in the two subcellular fractions, as conducted at low or high hexose concentrations and in either the absence or presence of exogenous hexose phosphates (3.0 mM glucose 6-phosphate and 1.0 mM fructose 1-phosphate) indicated a preferential location of the low-Km hexokinase in the cell pellet and of the high-Km glucokinase in the cytosolic fraction. Such a distribution pattern failed to be significantly affected by the concentration of D-glucose used during the initial incubation of the dispersed islet cells. These findings argue against the view that the glucose-induced translocation of glucokinase would result in any sizeable binding of the enzyme to a plasma membrane-associated protein.

Abstract: Human B-cell glucokinase displays sigmoidal kinetics towards D-glucose or D-mannose, but fails to do so towards D-fructose. Yet, D-glucose, D-mannose and 2-deoxy-D-glucose confer to the enzyme positive cooperativity towards D-fructose. For instance, in the presence of 5 mM D-[U-14C]fructose, its rate of phosphorylation is increased to 214.3 +/- 11.0%, 134.0 +/- 4.3% and 116.5 +/- 3.0% of paired control value by D-glucose, D-mannose and 2-deoxy-D-glucose (each 6 mM), respectively. D-glucose and, to a lesser extent, D-mannose also display reciprocal kinetic cooperativity. D-fructose, however, fails to affect D-glucose or D-mannose phosphorylation under conditions in which positive cooperativity is otherwise observed. These findings are relevant to the reciprocal effects of distinct hexoses upon their phosphorylation by B-cell glucokinase and, as such, to the metabolic and functional response evoked in pancreatic islet B-cells by these sugars, when tested either separately or in combination.

Abstract: Mitochondrial NAD+, NADH, NADP+ and NADPH were measured in dispersed pancreatic islet cells incubated in the absence or presence of D-glucose and then exposed for 20 s to 0.5 mg/ml digitonin. The latter treatment resulted in the full release of lactate dehydrogenase without any detectable loss of glutamate dehydrogenase. The permeabilized cells were separated from the incubation medium by centrifugation through an oil layer and their content in pyridine nucleotides measured by a radioisotopic procedure coupled to the classical cycling technique. Relative to basal value, D-glucose, in concentrations of 2.8 and 16.7 mM, caused a concentration-related increase in both the NADH/NAD+ and NADPH/NADP+ ratio. These findings provide the first direct evidence for the induction of a more reduced mitochondrial redox state in glucose-stimulated pancreatic islets.

Abstract: Western blotting of pancreatic islet extracts from either hereditarily diabetic Goto-Kakizaki rats (GK rats) or animals injected with streptozotocin during the neonatal period (STZ rats) demonstrated a pronounced decrease of immunoreactive mitochondrial glycerophosphate dehydrogenase (m-GDH), when compared to results obtained in islets from control rats. By contrast, the islet glucokinase protein content was either unaffected (GK rats) or much less severely decreased than that of m-GDH (STZ rats). These findings indicate that the impaired activity of m-GDH previously documented in islet homogenates from diabetic rats coincides with a decreased content of this enzyme in the endocrine pancreas.

Abstract: The activities of FAD-linked glycerophosphate dehydrogenase (m-GDH), glutamate dehydrogenase (GlDH), glutamate-pyruvate transaminase (GPT) and glutamate-oxalacetate transaminase (GOT) were measured in purified populations of CD3+ lymphocytes from 55 control subjects, 62 type-2 diabetics and 50 non-diabetic relatives of the latter patients. The activity of m-GDH was measured by both a radioisotopic procedure and colourimetric technique. As judged from these measurements and relative to the paired value for GlDH, the incidence of abnormally low m-GDH activity was significantly higher in type-2 diabetics than in control subjects. Moreover, the paired ratio in reaction velocity between the colourimetric and radioisotopic assay of m-GDH was abnormally high in patients with low m-GDH activity. Low m-GDH activity often coincided with increased GPT activity in plasma or high GPT/GOT ratio in lymphocytes. No obvious clustering of these anomalies was found in relatives of diabetic patients. These findings suggest that an inherited or acquired genomic defect of m-GDH in lymphocytes, and possibly in pancreatic B-cells, may participate to the pathogenesis of non-insulin-dependent diabetes mellitus.

Abstract: The anomeric specificity of wild-type human beta-cell glucokinase and six of its mutant forms toward alpha- and beta-D-glucose was examined over 6-min incubation at 30 degrees C. When D-[U-14C]glucose at anomeric equilibrium was used as substrate, the wild-type form yielded a maximal velocity of 76 U/mg, a K(m) of 4-5 mM, and a Hill coefficient close to 1.2. The maximal velocity (2 to 89 U/mg) and K(m) (2.4 to 209.8 mM) of the mutant forms both covered a range of about two orders to magnitude. Wild-type glucokinase displayed a higher affinity for alpha-D-glucose but greater maximal velocity with beta-D-glucose. A variance, however, in four mutant forms, the maximal velocity was higher with alpha- than beta-D-glucose. These findings indicate that the higher insulinotropic efficiency of alpha- than beta-glucose cannot be ascribed to the intrinsic catalytic properties of human beta-cell glucokinase. They also suggest that the perturbation of the anomeric specificity of glucose-stimulated insulin release in type-2 diabetes could conceivably be attributable, on occasion and at least in part, to a mutation of the glucokinase gene.

Abstract: The activities of hexokinase isoenzymes, lactate dehydrogenase, cytosolic NAD-linked glycerophosphate dehydrogenase, mitochondrial FAD-linked glycerophosphate dehydrogenase, and glutamate dehydrogenase were measured in homogenates of rat purified pancreatic B and non-B islet cells. In B cell homogenates, the maximal activity of hexokinase and glucokinase was one to two orders of magnitude lower than that of lactate dehydrogenase. The activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase was also much lower than that of the cytosolic NAD-linked glycerophosphate dehydrogenase . A comparable hierarchy in the activity of these enzymes was observed in non-B islet cells. These findings reinforce the view that the preferential stimulation of oxidative glycolysis observed in insulin-producing cells, when exposed to high concentrations of D-glucose, is attributable to a Ca2+-induced activation of the mitochondrial FAD-linked glycerophosphate dehydrogenase, rather than to saturation of the catalytic activity of lactate dehydrogenase.

Abstract: The present study reevaluates the relevance of human B-cell glucokinase activity to the process of hexose-induced insulin release. Taking into account a phenomenon of positive cooperativity (Hill number: 1.34), the Km of the enzyme for glucose ( < or = 5.1 mM) was lower than the concentration of the hexose required to cause half-maximal stimulation of insulin release in intact islets. Likewise, there were obvious discrepancies between the kinetics of glucose, mannose and fructose phosphorylation by B-cell glucokinase, e.g. in terms of maximal velocity, and the secretory and metabolic responses to these hexoses in intact islets. Glucose 6-phosphate decreased, modestly but significantly, B-cell glucokinase activity, such an inhibitory action being of the non-competitive type. Mannoheptulose caused competitive inhibition of B-cell glucokinase. It is concluded that the intrinsic catalytic properties of B-cell glucokinase cannot fully account for the concentration dependency and sugar specificity of the secretory response to D-glucose or other hexoses in pancreatic islets.

Abstract: This study examines the effects of chronically elevated glucose levels on the survival and function of purified rat beta-cells. Prolonged exposure (9 days) of beta-cell aggregates to 20 mmol/l glucose did not lead to cell losses, but reduced the amount of insulin secreted in response to glucose. This decrease was not caused by cellular desensitization but resulted from the lower cellular insulin content after a prolonged imbalance between stimulated rates of insulin synthesis and release. Virtually all beta-cells exhibited a state of metabolic and biosynthetic activation, which was maintained for at least 2 h in glucose-depleted media. Their rates of protein and insulin synthesis were amplified by glucose, reaching (half-) maximal stimulation at lower glucose concentrations (2 and 5 mmol/l, respectively) than control cells cultured at 10 mmol/l glucose (5 and 10 mmol/l, respectively). As for insulin release, the net glucose effect on insulin synthesis was markedly reduced as compared with that in control cells. This was also the case after culture at 6 mmol/l glucose. In the latter condition, the lower glucose-inducible activities were caused by cellular desensitization, with 50% of the beta-cells unresponsive to glucose and the other 50% responding with a lower sensitivity (half-maximal stimulation at 7 mmol/l glucose). Comparison of beta-cells cultured at the three glucose concentrations indicated that prolonged exposure to elevated glucose levels increases the number of degranulated cells, of cells with a high proportion of immature insulin granules, and of cells with glycogen deposition-morphologic features previously described in conditions of hyperglycemia. It is concluded that chronic exposure (9 days) of rat beta-cells to elevated glucose levels induces a prolonged state of beta-cell activation and glucose hypersensitivity rather than a glucotoxicity or glucose desensitization. This shift in the functional state of the beta-cell population is responsible for a reduced insulin release in response to glucose, as observed in other conditions of prolonged exposure to high glucose levels.

Abstract: It was recently speculated that leptin may exert a direct inhibitory effect upon insulin release from the pancreatic B-cell. This proposal meets, however, with two objections. First, although the message for leptin receptors is indeed detected in rat pancreatic islets, the short form of this receptor, for which no signalling function is known, represents the major species present in islet cells. Second, in the isolated perfused rat pancreas, leptin (1.0 nM) fails to affect the release of either insulin or glucagon.

Abstract: The metabolism of methyl pyruvate was compared to that of pyruvate in isolated rat pancreatic islets. Methyl pyruvate was found to be more efficient than pyruvate in supporting the intramitochondrial conversion of pyruvate metabolites to amino acids, inhibiting D-[5-3H]glucose utilization, maintaining a high ratio between D-[3,4-14C]glucose or D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization, inhibiting the intramitochondrial conversion of glucose-derived 2-keto acids to their corresponding amino acids, and augmenting 14CO2 output from islets prelabeled with L-[U-14C]glutamine. Methyl pyruvate also apparently caused a more marked mitochondrial alkalinization than pyruvate, as judged from comparisons of pH measurements based on the use of either a fluorescein probe or 14C-labeled 5,5-dimethyl-oxazolidine-2,4-dione. Inversely, pyruvate was more efficient than methyl pyruvate in increasing lactate output and generating L-alanine. These converging findings indicate that, by comparison with exogenous pyruvate, its methyl ester is preferentially metabolized in the mitochondrial, rather than cytosolic, domain of islet cells. It is proposed that both the positive and the negative components of methyl pyruvate insulinotropic action are linked to changes in the net generation of reducing equivalents, ATP and H+.

Abstract: In rat pancreatic islets, D-fructose causes a concentration-related shift to the left of the sigmoidal relationship between insulin release and D-glucose concentration. For instance, when D-fructose is tested at a 80 mM concentration, which is close to the threshold value for stimulation of insulin release by the ketohexose in the absence of D-glucose, a close-to-maximal secretory response is recorded in islets concomitantly exposed to as little as 6.0 to 8.3 mM D-glucose. Under these conditions, however, D-fructose fails to affect the utilization of D-[5-3H]glucose, the oxidation of D-[U-14C]glucose, or its conversion to either 14C-labeled acidic metabolites or amino acids. Under the same experimental conditions, the oxidation of D-[U-14C]fructose and its conversion to 14C-labeled amino acids represent no more than 80-85% of the corresponding values found with 6 mM D-[U-14C]glucose. Actually, the total output of 14CO2 attributable to the oxidation of both D-[U-14C]glucose (6 mM) and D-[U-14C]fructose (80 mM) remains lower than that found in the sole presence of 8.3 mM D-[U-14C]glucose, despite the much higher rate of insulin secretion found in the former compared to the latter situation. These findings suggest that the insulinotropic action of D-fructose cannot be fully accounted for by its capacity to act as a fuel in islet cells, as if it were to involve the generation of a second messenger distinct from those coupling factors currently implied in the process of nutrient-stimulated insulin release.

Abstract: Methyl pyruvate was found to exert a dual effect on insulin release from isolated rat pancreatic islets. A positive insulinotropic action prevailed at low concentrations of D-glucose, in the 2.8 to 8.3 mM range, and at concentrations of the ester not exceeding 10.0 mM. It displayed features typical of a process of nutrient-stimulated insulin release, such as decreased K+ conductance, enhanced Ca2+ influx, and stimulation of proinsulin biosynthesis. A negative insulinotropic action of methyl pyruvate was also observed, however, at a high concentration of D-glucose (16.7 mM) and/or at a high concentration of the methyl ester (20.0 mM). It was apparently not attributable to any adverse effect of methyl pyruvate on ATP generation, but might be due to hyperpolarization of the plasma membrane. The ionic determinant(s) of the latter change was not identified. The dual effect of methyl pyruvate probably accounts for an unusual time course of the secretory response, including a dramatic and paradoxical stimulation of insulin release upon removal of the ester.

Abstract: The metabolism of D-[5-3H]glucose, D-[3,4-14C]glucose, [2-3H]glycerol, L-[U-14C]glutamine, L-[1-14C]-leucine, and L-[U-14C]leucine was investigated in pancreatic islets isolated from either control rats or animals fed a low-protein isocaloric diet, containing 8% instead of 20% protein, during both fetal and postnatal life. In the latter animals, decreases in body weight, plasma insulin concentration, and insulinogenic index were associated with two major anomalies of islet nutrient metabolism. First, an imbalance between oxidative and anaerobic glycolysis was found in the islets of rats fed the low-protein isocaloric diet. It coincided with a decreased circulation in the glycerol phosphate shuttle, as judged by the generation of 3HOH from [2-3H]glycerol, and was probably attributable to the deficiency of mitochondrial FAD-linked glycerophosphate dehydrogenase previously documented in islet homogenates of the rats fed low protein. Second, the transamination of L-leucine to 2-ketoisocaproate was decreased in the low-protein-fed rats, while the oxidative decarboxylation of the 2-keto acid and the further catabolism of isovaleryl CoA occurred at normal rates when expressed relative to the initial transamination rate. These metabolic anomalies may account, in part at least, for the impairment of insulin release in protein malnutrition.

Abstract: Two cases of factitious hypoglycaemia due to intentional or inadvertent intake of glipizide by non-diabetic subjects were identified through the measurement of this sulphonylurea in plasma by a modified assay procedure.

Abstract: A standardized procedure for the assay and identification of hypoglycemic sulfonylureas in plasma is presented. External standards and plasma extracts containing glibenclamide, gliquidone, glipizide, or gliclazide were examined by high-performance liquid chromatography. The four sulfonylureas could be identified and measured with a precision of 5%-7% and a limit of detection close to 1-2 ng in injected external standards and 10-40 ng/ml in plasma samples. This method is suitable to study the pharmacokinetics of hypoglycemic sulfonylureas, for blood drug monitoring in diabetic patients, for diagnostic purposes in factitious hypoglycemia, and in cases relevant to forensic medicine.

Abstract: An iodide channel has been previously identified in the plasma membrane of bovine throcytes [Golstein et al., Am. J. Physiol. 263 (Cell Physiol. 32): C590-C597, 1992]. The plasma membrane proteins were solubilized and ultrafiltered, and the protein fraction collected above 100 kDa was inserted in liposomes. Voltage-sensitive uptake of radiolabeled I- by these proteoliposomes was studied. To this end, an outward I- gradient was set up by loading the proteoliposomes with KI and removing extraliposomal I-. I- exit from the proteoliposome induces an inside positive membrane potential, which leads to the uptake of 125I- added to the incubation medium. This uptake was abolished by valinomycin, which in the presence of K+ short circuits the liposomal membrane potential, demonstrating the conductive nature of this uptake. A double reciprocal plot of I- influx over I- concentration suggests the existence of a single population of channels in these proteoliposomes with a Michaelis-Menten constant for I- of approximately 9 microM. When the proteoliposomes were loaded with KCl or KSCN instead of I-, no conductive uptake occurred anymore, suggesting that these anions are unable to diffuse through the I- conductance, hence do not generate a diffusion potential. I- uptake by KI-loaded proteoliposomes was not inhibited in the presence of a 1,000-fold excess of extraliposomal Cl- but was completely inhibited by a 1,000-fold excess of extraliposomal SCN-, indicating that Cl- does not permeate the I- channel, whereas SCN- inhibits it. SCN- and flufenamate were both shown to be competitive inhibitors of the I- channel with an inhibitor constant of approximately 10 and 750 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: In rats that received a low protein isocaloric diet (protein content of the diet: 8 instead of 20%) during fetal life and thereafter up to the time of sacrifice at 12-13 weeks of age, a low plasma insulin concentration, a decreased insulin content of isolated pancreatic islets, and an impaired secretory response of the islets to either D-glucose or the association of L-leucine and L-glutamine coincided, in islet homogenates, with a low activity of the mitochondrial glycerophosphate dehydrogenase and an abnormally high ratio between glutamate-alanine and glutamate-aspartate transaminase activities. Opposite enzymatic changes were found in liver extracts of the same rats. No obvious change in these hormonal, secretory, and enzymatic variables were observed when the period of protein deficiency was restricted to fetal life. These findings support the view that, in protein malnutrition, an impaired activity of pancreatic B-cell mitochondrial glycerophosphate dehydrogenase contributes, possibly in association with other enzymatic anomalies, to the perturbation of islet function.

Abstract: The mitochondrial NADH/NAD+ ratio for free nucleotides in rat pancreatic islets was judged from the cell content in L-glutamate and L-alanine, 2-ketoglutarate and pyruvate, and NH4+. At a physiological concentration of D-glucose, such a ratio averaged 9.6 +/- 1.1%. A rise in hexose concentrations, above a threshold value in excess of 5.6 mM, caused a rapid, sustained and rapidly reversible decrease in the mitochondrial NADH/NAD+ ratio. It is speculated that in the process of glucose-stimulated insulin release, the latter change participates in the coupling between metabolic and secretory events by favouring both the activity of key mitochondrial dehydrogenases and the translocation of Ca2+ from the mitochondria into the cytosol.

Abstract: In rat pancreatic islets, D-glucose in concentrations exceeding 5.6 mM caused a concentration-related decrease of the mitochondrial NADH/NAD+ ratio, as judged from the changes in the islet content of glutamate, NH4+, and 2-ketoglutarate, and assuming that the glutamate dehydrogenase reaction is near equilibrium with the mitochondrial NAD system. The concentration dependency of the response to D-glucose was vastly different in islet and parotid cells, respectively. L-Leucine, 2-ketoisocaproate, BCH (a nonmetabolized but insulinotropic analog of L-leucine) and 3-phenylpyruvate also lowered the mitochondrial NADH/NAD+ ratio. In the presence of D-glucose, the latter ratio was also decreased by NH4+ or the absence of extracellular Ca2+, but dramatically increased by aminooxyacetate. Taking into account prior metabolic findings, the nutrient-induced fall in the mitochondrial redox state is thought to reflect an increased clearance of mitochondrial NADH through both the respiratory chain and malate-aspartate shuttle. The nutrient-induced decrease in the mitochondrial NADH/NAD+ ratio might favor both the circulation of metabolites in the Krebs cycle and the exit of Ca2+ from the mitochondria.

Abstract: A sensitive assay is developed to assess the existence of an iodide channel in a fraction of solubilized membrane proteins. This step is critical when considering various procedures for purification of this channel. Sodium cholate is used as a detergent as it does not denature the iodide channel. A simple and rapid method involving gel-filtration chromatography is used simultaneously to remove the detergent and to adjust the buffer composition, before protein insertion into liposomes. The presence of an iodide channel is investigated by measuring the iodide conductance of these proteoliposomes at 4 degrees C. An outward iodide gradient is set up across the proteoliposomal membrane by anion-exchange chromatography, allowing uptake of radiolabelled iodide. This uptake is conductive as it is abolished by valinomycin in the presence of potassium. It is specifically mediated by a thyroid plasma-membrane protein inserted into liposomes, as its denaturation before insertion totally abolished uptake. It was observed only within a well-defined fraction of thyroid membrane proteins collected by size-exclusion chromatography (molecular mass between 100 and 200 kDa). Furthermore, it was not observed with other membrane proteins such as ileal brush-border-membrane proteins or bacteriorhodopsin. Like many anion channels, this conductance was also inhibited by N-phenylanthranilic acid. Optimization of the assay is described, validating the measurement of conductive iodide uptake at 30 s by proteoliposomes reconstituted in a ratio of 10 micrograms of protein to 90 micrograms of lipid, with an outward iodide gradient (KI 15 mM inside and 1 microM outside). This assay provides a test of the biological activity of the iodide channel at each step of the purification; it can be applied to any anionic channel.

Abstract: Formycin A (1.0 mM) caused a rapid, sustained and rapidly reversible inhibition of effluent radioactivity in rat pancreatic islets prelabelled with myo-[2-3H]inositol and perifused in the presence of 8.3 mM D-glucose. This coincided with a progressive decrease in islet ATP content and transient inhibition of insulin release. Thereafter, however, formycin A increased glucose-induced insulin release. Moreover, in islets that were preincubated with myo-[2-3H]inositol and then exposed during perifusion to a rise in D-glucose concentration from 2.8 to 16.7 mM, the release of insulin and 3H fractional outflow rate at both the low and high hexose concentrations were much higher when both the preincubation and perifusion were conducted in the presence, rather than absence, of formycin A. It is concluded that formycin A first inhibits and later enhances both the hydrolysis of phosphoinositides and release of insulin, these effects being possibly related to changes in the islet cell content of adenosine and/or formycin A triphosphates.

Abstract: Conflicting data were previously reported concerning the effect of starvation upon the glucokinase protein and glucokinase mRNA content of pancreatic islets. In the present study, conducted in fed rats and animals starved for 48 h, the catalytic activity of glucokinase in islet homogenates and the content of this enzyme in islets were both decreased by starvation to the same relative extent. These findings support the view that the altered metabolic and secretory response to D-glucose found in islets from starved rats may be attributable, in part at least, to an apparent repression of glucokinase.

Abstract: Pancreatic islets isolated from control rats, Goto-Kakizaki rats and adult rats that were injected with streptozotocin during the neonatal period were incubated for two successive period of 90 min each in the presence of D-glucose (11.1 mM) with or without formycin A (1.0 mM), and in the presence of the dimethyl ester of succinic acid (SAD, 10.0 mM) with or without palmitate (1.0 mM). Although formycin A augmented glucose-stimulated insulin release in both control and diabetic rats, it failed to compensate for the impaired secretory response to D-glucose in the latter animals. Likewise, non-glucidic nutrients such as SAD and/or palmitate failed to display a more efficient insulinotropic action, relative to basal insulin output, in diabetic than control rats. These results indicate that both formycin A and non-glucidic nutrients are unable, through their immediate insulinotropic action, to restore a normal output of insulin in islets of animals with inherited or acquired non-insulin-dependent diabetes.

Abstract: The hydrolysis of the dimethyl ester of [1,4-14C]succinic acid and/or [2,3-14C]succinic acid was measured in homogenates of rat pancreatic islets, liver, jejunum, brain, BC3H1 mouse myocytes, NG108-19 mouse neuroblastoma x rat glioma hybrid cells, and Caco-2 human colon adenocarcinoma cells. The specific activity of the enzyme was much higher in liver, jejunum, and Caco-2 cells than in the other cell types. The affinity of the enzyme for succinic acid dimethyl ester (SAD) was also much higher in liver than in islet homogenates. In the latter case, both particulate and cytosolic activity were observed upon subcellular fractionation. The activity found in islet homogenates was commensurate with the rate of SAD hydrolysis in intact cells. While the intracellular pool of acidic metabolites generated from SAD remained fairly stable over a 15- to 120-min incubation and was mainly located in the cytosolic compartment, the amount of acidic metabolites released in the extracellular milieu progressively increased with the length of incubation. Such metabolites included both monocarboxylic and dicarboxylic acids, the latter consisting mainly of succinic acid and, to a much lesser extent, of fumaric acid and malic acid. However, at variance with SAD, succinic acid failed to be taken up by intact islets. There was no close parallelism between the specific activity of the SAD esterase and the extent of SAD utilization in distinct cell types.

Abstract: Palmitate and oleate (0.5 to 1.0 mM) caused a time- and concentration-related augmentation of insulin release evoked by D-glucose (6.0 to 16.7 mM) in rat isolated pancreatic islets. This contrasted with an inhibitory action of the fatty acids upon L-[4-3H]phenylalanine incorporation into TCA-precipitable material, but coincided with an increased biosynthesis of proinsulin relative to that of other islet peptides. The failure of palmitate to cause an immediate increase in insulin output at a low glucose concentration (6.0 mM) coincided with an unchanged rate of O2 uptake over a 10- to 15-min exposure to this fatty acid. Over prolonged incubation (90 min), however, both palmitate and oleate (1.0 mM) stimulated 45Ca net uptake by islets exposed to 6.0 mM D-glucose. Like their insulinotropic effect, the time course for the oxidation of [U-14C]palmitate and [U-14C]oleate was characterized by a progressive buildup in 14CO2 production rate. Moreover, palmitate and oleate decreased D-[5-3H]glucose conversion to 3HOH and D-[U-14C]glucose conversion to radioactive acidic metabolites over short (30 min) but not prolonged (120 min) incubation periods. The two fatty acids also interfered with the generation of 14CO2 from islets prelabeled with [U-14C]palmitate, but not L-[U-14C]glutamine. It is concluded that, at least during prolonged exposure to either palmitate or oleate, the secretory, cationic, and metabolic response to these fatty acids displays features comparable to those usually found in islets stimulated by nutrient secretagogues.

Abstract: The mitochondrial FAD-linked enzyme glycerophosphate dehydrogenase plays a key role in the pancreatic B-cell glucose sensing device. In the present study, the activity of this enzyme was examined in islets of fa/fa rats in which inherited diabetes mellitus is associated with obesity, hyperinsulinism and severe insulin resistance. The specific activity of both FAD-linked glycerophosphate dehydrogenase and glutamate dehydrogenase were decreased in islet and liver homogenates prepared from fa/fa, as compared to Fa/Fa, rats, this coinciding with a low ratio between glutamateoxalacetate and glutamate-pyruvate transaminase activity in both islet and liver extracts, islet hyperplasia, hyperinsulinemia and hepatic steatosis in the hyperglycemic fa/fa rats. It is speculated that a low activity of FAD-linked glycerophosphate dehydrogenase in the pancreatic B-cell may participate to the perturbation of glucose homeostasis in fa/fa rats, like in other animal models of non-insulin-dependent diabetes mellitus.

Abstract: Islets were isolated by automatic digestion from non-diabetic cadaveric organ donors and from Type 2 (non-insulin-dependent) diabetic subjects. The activity of FAD-glycerophosphate dehydrogenase, but not that of either glutamate dehydrogenase, glutamate-oxalacetate transaminase or glutamate-pyruvate transaminase, was lower in Type 2 diabetic patients than control subjects. Hexokinase, glucokinase and glutamate decarboxylase activities were also measured in islets from control subjects. The utilization of D-[5-3H]glucose, oxidation of D-[6-14C]glucose and release of insulin evoked by D-glucose were all lower in Type 2 diabetic patients than control subjects. The secretory response to the combination of L-leucine and L-glutamine appeared less severely affected. Islets from Type 2 diabetic patients may thus display enzymatic, metabolic and secretory anomalies similar to those often observed in animal models of Type 2 diabetes, including a deficiency of beta-cell FAD-linked glycerophosphate dehydrogenase, the key enzyme of the glycerol phosphate shuttle.

Abstract: Preincubation of rat pancreatic islets with AICA riboside (0.1 to 1.0mM) caused a concentration-related stimulation of both 45Ca net uptake and insulin release evoked by 8.3 mM D-glucose, but failed to affect the conversion of D-[5-3H]glucose to 3HOH, the generation of 14CO2 and 14C-labelled amino acids or acidic metabolites from D-[U-14C]glucose, and the islet content in ATP, ADP or AMP. The secretory response to AICA riboside was not suppressed in islets preincubated with methotrexate. AICA riboside caused a progressive decrease in 86Rb outflow from prelabelled islets perifused at 2.8 or 6.0mM D-glucose. This effect faded out at a higher concentration of D-glucose (16.7 mM), in which case AICA riboside nevertheless provoked a delayed, progressive and not rapidly reversible enhancement of insulin output. At concentrations up to 0.4 mM, ZTP only exerted a modest effect on the activity of KATP-channels in inside-out patches of dispersed mouse islet cells. These findings raise the question whether the insulinotropic action of AICA riboside may be attributable to the sequential generation of ZMP, ZDP and ZTP from the nucleoside.

Abstract: UDP-glucose pyrophosphorylase was measured in rat pancreatic islets, the generation of D-glucose 1-phosphate from UDP-glucose and PPI being eventually coupled to the generation of L-[U-14C]glutamate from 14C-labelled alpha-ketoglutarate. The activity of the enzyme was about one order of magnitude lower in islet than liver homogenates. The affinity of the enzyme for either UDP-glucose or PPi was comparable, however, in liver and islets. The activity of UDP-glucose pyrophosphorylase was somewhat lower in islets from animals with inherited or acquired diabetes mellitus than in those from control rats. These findings are considered in connection with the accumulation of glycogen in islets of hyperglycemic animals.

Abstract: Succinic acid methyl esters are currently under investigation as potential insulinotropic tools in animal models of non-insulin-dependent diabetes mellitus. The in vivo administration of these esters may result in the undesirable generation of methanol through their intracellular hydrolysis. As a first attempt to circumvert this drawback, we have now investigated whether the esterification of the carboxylic group of succinic acid monomethyl ester by D-glucose or 3-O-methyl-D-glucose affects its insulin-otropic action. Both the 6-O-D-glucosyl and 6-O-(3-O-methyl)-D-glucosyl esters were found to stimulate insulin release in pancreatic islets and the isolated perfused pancreas. The 6-O-D-glucosyl ester also stimulated insulin release after intravenous administration to anaesthetized rats. These findings suggest that the undersirable generation of methanol from the methyl esters of succinic acid could eventually be avoided by using other esters of this dicarboxylic acid, whilst keeping the benefit of their insulinotropic action.

Abstract: This study aimed to compare the metabolic and secretory responses of pancreatic islets from animals with non-insulin-dependent diabetes to D-glucose with the effects of the methyl esters of succinic acid (SME) and glutamic acid (GME). The insulin secretory response to D-glucose was impaired in islets from rats with diabetes which was either inherited (Goto-Kakizaki (GK) rats) or acquired (streptozotocin-treated (STZ) rats). This coincided with a preferential alteration of oxidative relative to total glycolysis in intact islets and a selective defect of FAD-linked mitochondrial glycerophosphate dehydrogenase (m-GDH) in islet homogenates. This enzymatic defect was also found in purified B cells from STZ rats. It contrasted both with unaltered activities of glutamate dehydrogenase and succinate dehydrogenase in the islets of diabetic animals and with a normal or even increased activity of m-GDH in the livers of GK and STZ rats. The oxidation of [1,4-14C]SME and [U-14C]GME appeared decreased in islets of GK or STZ animals when compared with control rats, but no significant difference between control and diabetic rats was observed when the oxidative data were expressed relative to the rate of [U-14C]GME hydrolysis. Nevertheless, the absolute values for insulin release evoked by a non-metabolized analogue of L-leucine (BCH), by SME and by the association of BCH with either SME or GME were invariably lower in islets of GK and STZ rats than in those of control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Glutamic acid dimethyl ester (GME; 3.0-10.0 mM) enhanced insulin release evoked by 6.0-8.3 mM D-glucose, 1.0-10.0 mM L-leucine, or 5.0-10.0 mM 2-amino-bicyclo(2,2,1)heptane-2-carboxylic acid, causing a shift to the left of the sigmoidal relationship between insulin output and D-glucose concentration. In the absence of D-glucose, GME also unmasked the insulinotropic potential of glibenclamide. In islets exposed to L-leucine, the insulinotropic action of GME coincided with an early fall and later increase in 86Rb outflow and augmentation of 45Ca outflow from prelabeled islets. The measurement of O2 uptake, NH4+ output, production of 14CO2 from islets prelabeled with [U-14C]palmitate, generation of 14C-labeled amino acids and 14CO2 from the dimethyl ester of either L-[1-14C]glutamic acid or L-[U-14C]glutamic acid, and D-[2-14C]glucose as well as D-[6-14C]glucose oxidation in the presence or absence of GME indicated that the latter ester was efficiently converted to L-glutamate and its further metabolites. The overall gain in O2 uptake represented the balance between GME oxidation and its sparing action on the catabolism of endogenous fatty acids and exogenous D-glucose. It is proposed that GME might represent a new tool to bypass beta-cell defects in D-glucose transport, phosphorylation, and further metabolism and, hence, to stimulate insulin release in experiments conducted in animal models of non-insulin-dependent diabetes mellitus.

Abstract: The monomethyl ester of succinic acid (SME) was recently proposed as a novel tool for stimulation of proinsulin biosynthesis and insulin release in animal models of non-insulin-dependent diabetes mellitus. In the present study, either saline or SME (14 mmol/day) was infused for 3 days to control rats, animals injected with streptozotocin during the neonatal period, and Goto-Kakizaki rats with inherited diabetes. The infusion of SME failed to correct the anomalies found in the islets of diabetic rats, namely, a decreased activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase, a low insulin content, and an impaired secretory response to various nutrient secretagogues including D-glucose, 2-ketoisocaproate, and the combination of L-leucine and L-glutamine. These findings raise the question of whether a more prolonged administration of SME is required to raise the insulin store and improve the secretory potential of the endocrine pancreas in animals with type 2 diabetes.

Abstract: The generation of C2- and C3-deuterated lactic acid produced by rat parotid cells exposed to [1-13C] glucose, [2-13C]glucose, and [6-13C]glucose in the presence of D2O was assessed by 13C NMR. The results indicated that the escape from deuteration amounted to about 46% at the phosphoglucoisomerase level, 100% at the phosphomannoisomerase level, 65% in the reactions catalyzed by phosphofructoaldolase and triose phosphate isomerase, and 58% at the level of glutamate pyruvate transaminase. Such high values are considered to support a possible enzyme-to-enzyme tunneling of metabolic intermediates at selected sites in the glycolytic pathway.

Abstract: The metabolic fate and metabolic effects of succinic acid methyl esters were examined in rat isolated hepatocytes. Both the monomethyl ester (SAM) and dimethyl ester (SAD) of succinic acid inhibited D-glucose metabolism. Such an inhibition affected, in order of increasing severity, the direct incorporation of D-glucose into glycogen and futile cycling between the hexose and its 6-phosphate ester, the phosphorylation of D-glucose, the generation of triose phosphates from the hexose, and the production of 14C-labeled lactate, pyruvate, and amino acids from D-[U-14C]glucose and its oxidation. The dimethyl ester of [1,4-14C]succinic acid ([1,4-14C]SAD) or [2,3-14C]-succinic acid ([2,3-14C]SAD) was efficiently converted to acidic metabolites. The oxidation of [1,4-14C]SAD largely exceeded that of [2,3-14C]SAD. Inversely the generation of newly formed radioactive D-glucose and glycogen appeared higher in cells exposed to [2,3-14C]SAD, rather than [1,4-14C]SAD. It is proposed that SAM and SAD are suitable nutrients both to cover the energy need of hepatocytes and to act as gluconeogenic precursors.

Abstract: The monomethyl ester of succinic acid (SME) was recently found to protect pancreatic islet B-cells against the impairment of glucose-stimulated insulin release caused by either glucopenia or starvation. The possible metabolic determinants of such a protective action are now scrutinized. After 180 min preincubation at 2.8 mM D-glucose in the presence of SME (10 mM), the oxidation of D-[U-14C]glucose, relative to either the utilization of D-[5-3H]glucose or the generation of 14C-labeled acidic metabolites, was higher than that after preincubation in the absence of SME and became close to that otherwise found after preincubation at 16.7 mM D-glucose. Likewise, after 3 days of culture at a low concentration of D-glucose (2.8 mM), the presence of SME in the culture medium tended to increase the subsequent oxidation of D-[6-14C]glucose and utilization of D-[5-3H]glucose. These two variables increased as a function of the concentration of D-glucose in the culture medium, this coinciding with a modest increase in hexokinase activity and a more pronounced increase in glucokinase activity. The presence of SME in the culture medium failed, however, to exert any obvious effect upon the respiration of the islets, suggesting that the protective action of the ester against glucopenia may also involve variables distinct from the metabolism of either endogenous or exogenous nutrients. Likewise, the fact that SME infusion to starved rats prevents the impairment of glucose-induced insulin release otherwise attributable to starvation may involve enzymatic determinants, such as a less severe decrease in glucokinase activity, metabolic variables, such as a greater relative increase in D-[U-14C]glucose oxidation relative to D-[5-3H]glucose utilization in response to a rise in extracellular D-glucose concentration, and other factors yet to be identified that participate in the secretory sequence at a site distal to those metabolic events triggered by D-glucose in the islet cells.

Abstract: The adenosine analogue formycin A is phosphorylated to its triphosphate ester in a sequence of reactions catalyzed by adenosine kinase and adenylate kinase. Formycin A triphosphate is an ATP analogue that is currently used to probe for ATP binding sites. Considering the key role ascribed to ATP in the coupling of metabolic to cationic events in the process of glucose-stimulated insulin release, we investigated whether formycin A displays insulinotropic action in rat pancreatic islets. Formycin A (10 microM to 1.0 mM) caused a concentration-related increase of insulin release evoked by 8.3 mM D-glucose and prevented the fall in insulin output otherwise observed over two successive incubations of 90 min each. Formycin A (1.0 mM) also augmented insulin secretion at low (5.6 mM) and high (16.7 mM) concentrations of D-glucose. At the low hexose concentration, the secretory response to formycin A was comparable to that evoked by either glibenclamide or glipizide. At higher concentrations of D-glucose, however, formycin A was more potent than the hypoglycemic sulfonylureas in enhancing insulin output. These findings support the role of ATP in glucose-stimulated insulin release and, therefore, suggest that ATP mimetics represent a new class of insulinotropic agents that have potential utility in the treatment of non-insulin-dependent diabetes mellitus.

Abstract: In islets from both adult rats injected with streptozotocin during the neonatal period and spontaneously diabetic rats obtained by repeated selective breedings (GK rats), the ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose conversion to 3HOH was 25% lower than in islets from control rats, indicating an impaired contribution of oxidative to total glycolysis. No primary defect in the Krebs cycle was found in the islets of diabetic rats, as judged from the ratio between either D-[2-14C]glucose or D-[6-14C]glucose and D-[3,4-14C]glucose oxidation. Therefore, we propose that a preferential alteration of oxidative glycolysis in the pancreatic beta cell may contribute to the impairment of glucose-induced insulin release not only in a cytotoxic but also in a spontaneous model of non-insulin-dependent diabetes mellitus.

Abstract: In islets from adult rats injected with streptozotocin during the neonatal period, both a nonmetabolized analog of L-leucine and 3-phenylpyruvate augmented 14CO2 output from islets either prelabeled with L-[U-14C]glutamine or exposed to D-[2-14C]glucose and D-[6-14C]glucose, in a manner qualitatively comparable to that found in islets from control rats. The islets of diabetic rats differed, however, from those of control rats by their unresponsiveness to both the L-leucine analog and a high concentration of D-glucose in terms of increasing 3HOH generation from [2-3H]glycerol, an impaired sparing action of the hexose upon 14CO2 output from islets prelabeled with [U-14C]palmitate, and, most importantly, by a decreased rate of D-[2-14C]glucose and D-[6-14C]glucose oxidation when either incubated at a high concentration of the hexose (16.7 mM) or stimulated by nonglucidic nutrient secretagogues at a low concentration of D-glucose (2.8 mM). In islet homogenates, the activity of glyceraldehyde phosphate dehydrogenase, glutamate decarboxylase, and NADP-malate dehydrogenase was lower in diabetic than control islets. Such was not the case for glutamate-alanine transaminase, glutamate-aspartate transaminase, or glutamate dehydrogenase. The neonatal injection of streptozotocin thus affected, in the adult rats, the activity of several islet enzymes. Nevertheless, the metabolic data suggest that an impaired circulation in the glycerol phosphate shuttle, as observed in response to stimulation of the islets by either a high concentration of D-glucose or nonglucidic nutrient secretagogues, represents an essential determinant of the preferential impairment of glucose-induced insulin release in this model of non-insulin-dependent diabetes.

Abstract: When pancreatic islets isolated from rats infused for 48-72 h with a hypertonic solution of D-glucose were incubated for two successive periods of 10 min each, in the presence first of 16.7 mM and then 2.8 mM D-[U-14C]glucose, the total output of L-lactic acid during the second incubation was as high as that recorded during the first incubation, while the specific radioactivity of L-lactic acid dramatically decreased during the second incubation. In islets from normoglycemic rats, however, the total output of L-lactic acid decreased and its specific radioactivity modestly increased as the concentration of D-glucose was lowered from 16.7 to 2.8 mM. Such contrasting results indicate that in the glycogen-rich islets isolated from glucose-infused rats, the fall in extracellular D-glucose concentration was not accompanied by a parallel fall in glycolytic flux, the decreased utilization of exogenous D-[U-14C]glucose coinciding with stimulation of glycogenolysis. This unusual metabolic situation also coincided with a transient and paradoxical stimulation of insulin release in response to the decrease in extracellular D-glucose concentration. It is proposed, therefore, that the interference of glycogenolysis with glycolysis in pancreatic islets from glucose-infused rats participates in the paradoxical changes in insulin output which represent a typical feature of B-cell glucotoxicity.

Abstract: The time-course for the generation of 3HOH from D-[5-3H]glucose and for the production of 14CO2, 14C-labelled acidic metabolites and radioactive amino acids from D-[3,4-14C]glucose, D-[2-14C]glucose and D-[6-14C]glucose was monitored, over 60 to 120 min incubation, in both rat pancreatic islets and parotid cells exposed to a low or high concentration of the hexose. In islets stimulated by D-glucose, a progressive increase in the oxidation rate of glucose-derived acetyl residues was observed. Such a phenomenon was not observed in islets exposed to a low concentration of D-glucose, concerned specifically the oxidation of acetyl residues in the Krebs cycle as distinct from their generation in the reaction catalyzed by pyruvate dehydrogenase, and failed to occur in parotid cells. It is concluded that the increase in the oxidation rate of glucose-derived acetyl residues found in pancreatic islets represents an unusual phenomenon not encountered in other cell types and specifically regulated in terms of its time-course, concentration dependency and relationship to other oxidative events.

Abstract: The mitochondrial enzyme FAD-linked glycerophosphate dehydrogenase plays a key role in the glucose-sensing device of the insulin-producing pancreatic B-cell. Its activity was found to be decreased in islet, but not liver, homogenates of BL/Ks-db/db mice, in which diabetes mellitus represents an inherited disease. The decreased activity of FAD-linked glycerophosphate dehydrogenase contrasted with a normal activity of glutamate dehydrogenase and 2-ketoglutarate dehydrogenase in the islets of db/db mice. It is proposed that a site-specific defect of FAD-linked glycerophosphate dehydrogenase in the pancreatic B-cell might represent a far-from-uncommon causal or contributing factor in the pathogenesis of non-insulin-dependent diabetes mellitus.

Abstract: The metabolic effects and the catabolism of succinate methyl esters were examined in rat pancreatic islets. The esters augmented 14CO2 production from islets prelabeled with L-[U-14C]-glutamine but inhibited NH4+ output, suggesting that they do not activate glutamate dehydrogenase. They decreased 14CO2 output from islets prelabeled with [U-14C]palmitate. They had little effect on the oxidation of exogenous D-[3,4-14C]glucose, D-[2-14C]glucose, D-[6-14C]glucose, or D-[1-14C]glucose, suggesting unaltered ratio between the input of acetyl residues and four- or five-carbon metabolites, such as succinate, into the Krebs cycle. By following the fate of both [1,4-14C]succinate dimethyl ester and [2,3-14C]succinate dimethyl ester, data were obtained to indicate that succinate is efficiently formed from the ester and further metabolized, leading to the generation of 14C-labeled acidic metabolites including pyruvate and L-lactic acid, CO2, and amino acids. It is proposed that a concerted increase of both succinate and acetyl residue influx into the Krebs cycle accounts for the increase in O2 uptake caused by the succinate methyl esters and, hence, for stimulation of both pro-insulin biosynthesis and insulin release.

Abstract: In perifused pancreatic islets from euglycemic rats, the secretory response to either glibenclamide or glimepiride (1.0 microM each) increases as a function of the concentration of D-glucose (2.8-16.7 mM) present in the perifusion medium. On the contrary, the sulfonylurea-induced increment in 45Ca efflux from prelabeled islets decreases at increasing concentrations of the hexose. Neither glibenclamide nor glimepiride affect D-glucose metabolism in isolated islets, as judged from the production of 3HOH from D-[5-3H]glucose or the generation of 14CO2, as well as 14C-labeled amino acids and acidic metabolites, from D-[3,4-14C]glucose, D-[2-14C]glucose and D-[6-14C]glucose. The insulinotropic action of the hypoglycemic sulfonylureas is not impaired in islets prepared from rats infused for 48 hr with a hypertonic solution of D-glucose. The dimethyl ester of succinic acid is more efficient than D-glucose in supporting the insulin-releasing effect of glibenclamide or glimepiride. Thus, although the insulinotropic action of hypoglycemic sulfonylureas appears unaffected in a model of B-cell glucotoxicity, a potentiation of their secretory effects might be expected, in non-insulin-dependent diabetes, from the combined administration of succinic acid methyl ester.

Abstract: The metabolism of endogenous nutrients was examined in pancreatic islets of control and Goto-Kakizaki (GK) rats. At the ultrastructural level, no glycogen was found in the islet cells of GK rats, a situation similar to that prevailing in normal islets. Likewise, by measuring the output of L-lactate from islets first incubated at 16.7 mM D-glucose and then at 2.8 mM D-glucose, no evidence of glycogenolysis was found in the islets of GK rats. The production of NH4+ and that of 14CO2 from islets prelabelled with either L-[U-14C]glutamine or [U-14C]palmitate were higher, however, in GK than in control rats. The changes in NH4+ and 14CO2 production evoked by D-glucose, by a non-metabolized analogue of L-leucine (2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid; BCH) and by 3-phenylpyruvate were qualitatively comparable in control and GK rats. The secretory response to these three secretagogues was severely decreased in the islets of GK rats. This coincided with an impaired enhancing action of D-glucose on the conversion of [2-3H]glycerol into 3HOH. It is concluded that the catabolism of endogenous amino and fatty acids in islets is greater in GK than in control rats, especially at low D-glucose concentration. This may account, in part at least, for the altered secretory response to BCH and 3-phenylpyruvate. For glucose-induced insulin release, however, an impaired acceleration of the glycerol phosphate shuttle apparently also participates in the secretory defect.

Abstract: In thyroidectomized rats, the activity of FAD-linked glycerophosphate dehydrogenase was severely diminished in liver homogenates but not affected significantly in pancreatic islet homogenates, whilst the activity of 2-ketoglutarate dehydrogenase was decreased modestly in both liver and islet homogenates. Likewise, in intact islets of thyroidectomized rats, the generation of 3HOH from [2-3H]glycerol was not decreased, and the ratio between oxidative and total glycolysis not significantly lower than in islets from sham-operated rats, at least in the presence of a high concentration of D-glucose. Nevertheless impaired oxidation of both D-[3,4-14C]glucose and D-[6-14C]glucose was observed in islets of thyroidectomized rats, the relative magnitude of such a decrease being more pronounced at a low than at a high D-glucose concentration. Such metabolic anomalies coincided with a lower level of plasma insulin and decreased output of insulin by islets incubated at low (2.8 mM), but not higher, concentrations of D-glucose. It is concluded that hypothyroidism does not mimic the deficiency in islet FAD-linked glycerophosphate dehydrogenase activity found in rats with inherited or acquired non-insulin-dependent diabetes.

Abstract: In 12 out of 32 non-insulin-dependent diabetic subjects, the activity of FAD-linked glycerophosphate dehydrogenase in T lymphocyte homogenates was abnormally low when measured by both a colorimetric and radioisotopic procedure. A comparable situation characterized by a deficient activity of FAD-linked glycerophosphate dehydrogenase in both the colorimetric and radioisotopic assays was only observed once among 26 other subjects including 11 healthy volunteers, 9 non-diabetic patients, 5 type-1 (insulin-dependent) diabetics, and 1 pancreatectomized diabetic. By analogy, it is speculated that an impaired activity of FAD-linked glycerophosphate dehydrogenase in the insulin-producing pancreatic B-cell could represent a far-from-uncommon contributive factor in the pathogenesis of non-insulin-dependent diabetes mellitus.

Abstract: The metabolic, ionic, and secretory response to D-glucose was investigated in islets of adult rats either injected with streptozotocin during the neonatal period (STZ rats) or presenting with inherited diabetes (GK rats). At a high concentration of D-glucose (16.7 mM), the ATP/ADP ratio was lower in islets from STZ and GK than control rats. This coincided with an impaired response of perifused islets to a rise in D-glucose concentration in terms of stimulation of insulin release, suppression of effluent radioactivity from islets prelabeled with [2-3H]adenosine, reduction in 86Rb efflux, and induction of a phosphate flush in islets prelabeled with 32P(i). The ratio in either D-[5-3H]glucose utilization or D-[2-14C]glucose oxidation at high/low hexose concentration, as well as the paired ratio between D-[2-14C]glucose oxidation and D-[5-3H]glucose utilization in islets incubated at a high concentration of the hexose, was also lower in STZ and GK rats than in control rats. Such was not the case, however, from the oxidation of [2-14C]pyruvate. Instead, the latter 2-keto acid, when tested at a 5.0 mM concentration, improved more efficiently the overall oxidative response of the islets to a rise in D-glucose concentration in STZ and GK rats than in control animals. It is proposed, therefore, that in both STZ and GK rats, the B-cell secretory defect is primarily attributable to an anomaly in oxidative glycolysis. In islets exposed to a high concentration of D-glucose, this metabolic deficiency results in impaired ATP generation, altered closing of ATP-responsive K+ channels, and, hence, diminished insulin output.

Abstract: In pancreatic islet extracts of rats with hereditary non-insulin-dependent diabetes mellitus (GK rats), the activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase, as measured by either a radioisotopic or colorimetric procedure, only represented 30 to 40% of that found in control rats. This decrease in enzymic activity was not attributable to any sizeable change in either islet DNA content or the relative contribution of insulin-producing beta cells to total islet mass. It contrasted with a normal activity of other mitochondrial dehydrogenases and hexokinase isoenzymes. It coincided, however, with an increased activity of glutamate-pyruvate transaminase, as already observed in adult rats injected with streptozotocin during the neonatal period. The decreased activity of islet FAD-linked glycerophosphate dehydrogenase also contrasted with an increased activity of the same enzyme in the liver of GK, as compared to control rats. In the light of these findings and recent metabolic data collected in intact islets of GK rats, it is proposed that a deficiency of beta-cell FAD-linked glycerophosphate dehydrogenase, the key enzyme of the glycerol phosphate shuttle, may represent a cause of inherited non-insulin-dependent diabetes.

Abstract: The activity of FAD-linked glycerophosphate dehydrogenase (m-GDH), as well as that of glutamate dehydrogenase and both glutamate-oxalacetate and glutamate-pyruvate transaminases, were measured in islet, liver, and splenocyte homogenates from 6- to 7-week-old female nonobese diabetic mice (NOD) and age- and sex-matched control mice. Despite incipient insulitis and euglycemia, the NOD mice displayed both high islet insulin content and elevated insulinemia. The activity of m-GDH, expressed relative to protein content, was not decreased in islets of NOD mice, despite the fact that such a specific activity is lower in splenic lymphocytes than islet cells. In liver homogenates, the activity of m-GDH was even higher in NOD than control mice. It is proposed, therefore, that in this model of insulin-dependent diabetes no primary decrease in islet m-GDH activity occurs, at variance with the situation recently documented in several animal models of non-insulin-dependent diabetes.

Abstract: The activity of FAD-glycerophosphate dehydrogenase, as measured through the generation of either 3HOH from L-[2-3H]glycerol-3-phosphate in the presence of FAD or iodoformazan from iodonitrotetrazolium, displayed comparable values in islet homogenates of lean and obese (ob/ob) mice. In the liver of the obese animals, the results obtained by the colorimetric and radioisotopic assays yielded a paired ratio twice higher than in control mice. Although isoforms of the mitochondrial enzyme could be present in variable proportions depending on the cell type and genetic background, the present results suggest that, in ob/ob mice, the increased secretory responsiveness of the islet B-cell to D-glucose coincides with an unaltered activity of FAD-glycerophosphate dehydrogenase. This contrasts with the situation recently documented in db/db mice, in which an impaired secretory response of the B-cell to D-glucose is associated with a decreased activity of FAD-glycerophosphate dehydrogenase.

Abstract: A radioisotopic procedure for the assay of myo-inositol is presented. It is based on the generation of NADH from NAD+ in the reaction catalyzed by myo-inositol dehydrogenase and the subsequent NADH-dependent conversion of 2-[U-14C]ketoglutarate to 14C-labeled L-glutamate in the reaction catalyzed by glutamate dehydrogenase. This method was applied to the measurement of myo-inositol in rat pancreatic islets. The myo-inositol islet content was decreased when the animals were fed a diet deprived of myo-inositol. When incubated in the absence of exogenous D-glucose, pancreatic islets, like parotid cells, released myo-inositol in the incubation medium. Over 90 min of incubation, a rise in extracellular D-glucose concentration increased the myo-inositol islet content, which was decreased, however, after incubation in the presence of carbamylcholine. These findings indicate that the myo-inositol content of islets is affected by nutritional and other environmental factors.

Abstract: The activities of aspartate and alanine aminotransferases in biological samples were assessed through a novel and sensitive procedure, based on the conversion of [U-14C]2-ketoglutarate to L-[U-14C]glutamate. In human plasma, the generation of L-[U-14C]glutamate was proportional to the volume of plasma (20-60 microL) and to the length of incubation (30-90 min). The reaction velocity was related to the temperature with a Q10 close to 1.7 for aspartate aminotransferase and 2.0 for alanine aminotransferase. At 37 degrees C, the 95% confidence interval in healthy subjects ranged from 5.1-18.8 U/mL (mean value 11.9 U/L) for aspartate aminotransferase and from zero to 20.1 U/L (mean value 9.9 U/L) for alanine aminotransferase. The intra-assay coefficient of variation did not exceed 2.5%. The present method was also applied to homogenates prepared from rat pancreatic islets, liver, heart, parotid glands, and erythrocytes, using no more than 40 micrograms wet weight of tissue per sample, and could thus be used in small biological samples, such as those obtained by needle biopsy.

Abstract: At 3-4 degrees C, the transport of 3-O-methyl-D-glucose (30 mM) was severely impaired in islets prepared from adult rats injected with streptozotocin during the neonatal period. However, at 37 degrees C, the first and second phase of glucose-stimulated insulin release were decreased to the same relative extent in perifused islets of diabetic, as compared to control, animals. Moreover, the time-related increase in the oxidative response of the islets to 16.7 mM D-glucose was less pronounced in diabetic than control rats. The activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase in islet homogenates of diabetic rats only represented one-fifth of that found in control rats, whereas the activity of the cytosolic NAD-glycerophosphate dehydrogenase was comparable in both types of rats. This coincided with the fact that a rise in D-glucose concentration from 2.8 to 16.7 mM failed to increase significantly L-[2-3H]glycerol conversion to 3HOH in islets from diabetic rats, in contrast to the situation found in control animals. The activity of 2-ketoglutarate dehydrogenase in islet homogenates when expressed per microgram protein was not different in control and diabetic rats. Likewise, the ratio between D-[6-14C]glucose oxidation and D-[3,4-14C]glucose oxidation and the capacity of either a non-metabolized analog of L-leucine or 3-phenylpyruvate to preferentially stimulated D-[6-14C]glucose oxidation relative to D-[5-3H]glucose utilization were both unaffected in islets from diabetic rats. These findings argue against the existence of a primary defect in the Krebs cycle of diabetic rats. It is proposed that, despite an obvious alteration of the hexose transport system in the islet cells of diabetic rats, the preferential impairment of the B-cell secretory response to D-glucose, as distinct from other secretagogues, in this model of non-insulin-dependent diabetes is mainly attributable to the low activity of FAD-linked glycerophosphate dehydrogenase, resulting in a decreased metabolic flow through the glycerol phosphate shuttle and a reduced rate of aerobic glycolysis.

Abstract: The fate of the C1 and C2 of glucose-derived acetyl residues was examined in rat pancreatic islets. The production of 14CO2 from D-[2-14C]glucose exceeded that from D-[6-14C]glucose, in the same manner as the oxidation of [1-14C]acetate exceeded that of [2-14C]acetate. The difference in 14CO2 output from D-[2-14C]glucose and D-[6-14C]glucose was matched by complementary differences in the generation of 14C-labeled acidic metabolites and amino acids. Even the production of 14C-labeled L-lactate was somewhat higher in the case of D-[6-14C]glucose than D-[2-14C]glucose. The ratio between D-[2-14C]glucose and D-[6-14C]glucose oxidation progressively decreased at increasing concentrations of the hexose (2.8, 7.0, and 16.7 mM), was higher after 30 than 120 min incubation, and was decreased in the presence of a nonmetabolized analogue of L-leucine. These findings are consistent with the view that the difference between D-[6-14C]glucose and D-[2-14C]glucose oxidation is mainly attributable to the inflow into the Krebs cycle of unlabeled metabolites generated from endogenous nutrients, this being compensated by the exit of partially labeled metabolites from the same cycle. The present results also indicate that the oxidation of glucose-derived acetyl residues relative to their generation in the reaction catalyzed by pyruvate dehydrogenase is higher than that estimated from the ratio between D-[6-14C]glucose and D-[3,4-14C]glucose conversion to 14CO2.

Abstract: A preferential impairment of the pancreatic B cell secretory response to D-glucose occurs in adult rats injected with streptozotocin during the neonatal period. Three possible explanations for such a preferential defect were investigated in the present study. First, the time course for 3-O-methyl-D-glucose uptake by islets suggested that the anomaly in hexose transport was mainly attributable to a decrease in the space accessible to the D-glucose analog commensurate with the decrease in B cell mass, rather than to a delayed equilibration of hexose concentration across the B cell plasma membrane. Second, the activity of glucose-6-phosphatase was found to be equally low in islets from diabetic and control rats, ruling out the futile cycling between D-glucose and D-glucose 6-phosphate as a cause for the preferential alteration of the secretory response to the hexose. Third, the activity of flavine adenine dinucleotide-linked glycerophosphate dehydrogenase was found to be decreased to a greater relative extent than the B cell mass. This coincided with an impaired generation of 3HOH from L-[2-3H] glycerol in intact islets. It is proposed, therefore, that an altered circulation in the glycerol phosphate shuttle may play a major role in the impaired process of glucose-stimulated insulin release in this model of noninsulin-dependent diabetes.

Abstract: Aldolase and triose phosphate isomerase both display strict specificity towards the enantiomers of [1-3H]glycerone 3-phosphate. The enantiomer generated from D-[1-3H]glyceraldehyde 3-phosphate produces 3HOH in the aldolase reaction, whilst the other enantiomer generated from D-[3-3H]fructose 1,6-bisphosphate is solely detritiated in the reaction catalyzed by triose phosphate isomerase. Advantage was taken of such a specificity to assess, in human erythrocytes exposed to either D-[3-3H]glucose or D-[3,4-3H]glucose, the extent of D-glyceraldehyde 3-phosphate sequential conversion to glycerone 3-phosphate and D-fructose 1,6-bisphosphate, relative to net glycolytic flux. At 37 degrees C and in the presence of 5.6 mM D-glucose, only 55% of the metabolites of D-[4-3H]glucose underwent detritiation in the reactions catalyzed by triose phosphate isomerase and aldolase. Such a percentage was further decreased at low temperature (8 degrees C) or lower concentrations of D-glucose (0.2 and 1.0 mM). However, when the erythrocytes were exposed to menadione, the increase in 3HOH production from either D-[3-3H]glucose or D-[3,4-3H]glucose indicated that the majority of the 3H atoms initially located on the C4 of D-glucose were recovered as 3HOH upon circulation through the pentose phosphate pathway. These findings suggest that, under physiological conditions, a large fraction of D-glyceraldehyde 3-phosphate generated from exogenous D-glucose may undergo enzyme-to-enzyme channelling in the glycolytic pathway.

Abstract: The presence of the purine nucleotide cycle is investigated in rat pancreatic islets. Adenylosuccinase, adenylate deaminase, and adenylosuccinate synthetase activities are characterized in islet homogenates. In the assay of the latter enzyme, evidence is obtained for operation of the full cycle in islet extracts. The activities of the three enzymes are not vastly different in islet and brain. These findings are discussed in the light of the role currently ascribed to the purine nucleotide cycle in producing ammonia from amino acids, in adjusting the concentration of Krebs cycle intermediates, in regulating the relative concentrations of ATP, ADP, and AMP, and in controlling the activity of phosphofructokinase.

Abstract: Rat pancreatic islets cultured for 1 to 5 days in the presence of 20 to 80 mmol/L D-glucose accumulate glycogen in a time- and concentration-dependent manner. When the glycogen-rich islets are incubated for 6 to 10 minutes in the absence of D-glucose, the rate of glycogenolysis is grossly proportional to the glycogen content. Exogenous D-glucose (7 to 20 mmol/L) inhibits glycogenolysis. This inhibitory effect opposes the increase in glycolytic flux attributable to the utilization of exogenous glucose. Both the inhibitory effect of D-glucose on glycogenolysis and the utilization of exogenous hexose tend to be higher with alpha- than with beta-D-glucose. In light of these findings, it is proposed that the interference of D-glucose with glycogenolysis might play a role in the paradoxical changes in insulin output and its altered anomeric specificity in response to D-glucose administration, as is often encountered in non-insulin-dependent diabetic subjects and experimental models of B-cell glucotoxicity.

Abstract: A multifactorial quantitative analysis of oscillations in glycolysis was conducted in the postmicrosomal supernatant of rat muscle homogenates incubated in the presence of yeast hexokinase. Oscillations in adenine nucleotides, D-fructose 1,6-bisphosphate, triose phosphates, L-glycerol 3-phosphate, 3HOH generation from D-[5-3H]glucose, NADH and L-lactate production were documented. The occurrence of such oscillations were found to depend mainly on the balance between the consumption of ATP associated with the phosphorylation of D-glucose, as catalyzed by both yeast and muscle hexokinase, and the net production of ATP resulting from the further catabolism of D-fructose 6-phosphate, as initiated by activation of phosphofructokinase. The oscillatory pattern was suppressed in the presence of D-fructose 2,6-bisphosphate. It is proposed that the quantitative information gathered in this study may set the scene for further studies in extracts of cells other than myocytes, e.g. hepatocytes and pancreatic islet cells, in which no oscillation of glycolysis was so far observed.

Abstract: A method is proposed for the measurement of the flux through the glycerol phosphate shuttle in pancreatic islets. Such a flux is taken as the ratio between the production of 3HOH and the specific radioactivity of L-[2-3H]glycerophosphate in islets exposed to [2-3H]glycerol. D-Glucose and non-glucidic nutrient secretagogues, such as 2-ketoisocaproate and 2-aminobicyclo[2,2,1]heptane-2-carboxylate, stimulate, in a Ca(2+)-dependent manner, circulation in the glycerol phosphate shuttle. The shuttle flux is commensurate with the fraction of pyruvate generation which is not coupled with L-lactate production. These findings support the view that a rise in D-glucose concentration leads to activation of the FAD-linked mitochondrial glycerophosphate dehydrogenase through an increase in cytosolic Ca2+ concentration.

Abstract: Glucose-6-phosphatase activity was measured in rat liver or pancreatic islet crude homogenates and microsomes. The data recorded in the liver were comparable to those reported in prior studies. However, in the islets, the hydrolysis of D-glucose 6-phosphate by disrupted microsomes represented, when expressed relative to the protein content, less than 2% of the value recorded in liver microsomes. Moreover, no phosphotransferase activity was detected in the islets. These findings impose reservation on both the presence of glucose-6-phosphatase in rat islets and its participation to stimulus-secretion coupling.

Abstract: In rat pancreatic islets, a rise in D-glucose concentrations increases the oxidation of hexose-derived acetyl residues relative to glycolytic flux, an effect possibly attributable, in part at least, to the activation of key mitochondrial dehydrogenases by Ca2+ accumulated in the mitochondria of glucose-stimulated islet cells. The effects of non-nutrient insulinotropic agents upon D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization were investigated. At an intermediate concentration of D-glucose (6 mM), the oxidation of D-[6-14C]glucose was unaffected by hypoglycemic sulfonylureas, an organic Ca2+ agonist, a cholinergic agent, forskolin, theophylline and cytochalasin B. At a higher concentration of the hexose (17 mM), however, the 14CO2/3H2O production rate was decreased by organic and inorganic Ca(2+)-antagonists and by ouabain, whilst being increased by NH+4 (10 mM) and aminooxyacetate. These findings suggest that the preferential stimulation of oxidative events in the Krebs cycle is largely independent of the rate of insulin release, and not merely consequential to the stimulation of Ca2+ inflow into the B-cell. It might be regulated, in a feedback process, by the rate of ATP utilization and, both directly and indirectly, by the mitochondrial redox state. The glucose-induced mitochondrial accumulation of Ca2+ and subsequent activation of the Krebs cycle appear to require an increase in both cytosolic Ca2+ activity and ATP availability.

Abstract: In islets from adult rats injected with streptozocin during the neonatal period, the oxidative and secretory responses to D-glucose are more severely affected than those evoked by L-leucine. A possible explanation for such a preferential defect was sought by comparing the rate of aerobic glycolysis, taken as the sum of D-[3,4-14C]glucose conversion to labeled CO2, pyruvate, and amino acid, with the total glycolytic flux, as judged from the conversion of D-[5-3H]glucose to 3H2O. A preferential impairment of aerobic relative to total glycolysis was found in islets from diabetic rats incubated at either low or high D-glucose concentration. This coincided in islet mitochondria of diabetic rats with a severe decrease in both the basal (no-Ca2+) generation of 3H2O from L-[2-3H]glycerol-3-phosphate and the Ca2(+)-induced increment in [3H]glycerophosphate detritiation. The mitochondria of diabetic rats were also less efficient than those of control animals in generating 14CO2 from [1-14C]-2-ketoglutarate. The diabetes-induced alteration of 2-ketoglutarate dehydrogenase in islet mitochondria was less marked, however, than that of the FAD-linked glycerophosphate dehydrogenase and was not associated with any change in responsiveness to Ca2+. Sonicated islet mitochondria of diabetic rats displayed normal to slightly elevated glutamate dehydrogenase activity. We propose, therefore, that the preferential impairment of the oxidative and secretory responses of islet cells to D-glucose in this experimental model of diabetes may be at least partly attributable to an altered transfer of reducing equivalents into the mitochondria as mediated by the glycerol phosphate shuttle.

Abstract: 1. D-Glucose (0.5-16.7 mM) preferentially stimulates aerobic glycolysis and D-[3,4-14C]glucose oxidation, relative to D-[5-3H]glucose utilization in rat pancreatic islets, the concentration dependency of such a preferential effect displaying a sigmoidal pattern. 2. Inorganic and organic calcium antagonists, as well as Ca2+ deprivation, only cause a minor decrease in the ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose utilization in islets exposed to a high concentration of the hexose (16.7 mM). 3. Non-glucidic nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate (BCH), 2-ketoisocaproate and 3-phenylpyruvate fail to stimulate aerobic glycolysis and D-[3,4-14C]glucose oxidation in islets exposed to 6.0 mM D-glucose. Nevertheless, BCH augments [1-14C]pyruvate and [2-14C]pyruvate oxidation. 4. The glucose-induced increment in the paired ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose utilization is impaired in the presence of either cycloheximide or ouabain. 5. These findings suggest that the preferential effect of D-glucose upon aerobic glycolysis and pyruvate decarboxylation is not attributable solely to a Ca(2+)-induced activation of FAD-linked glycerophosphate dehydrogenase and/or pyruvate dehydrogenase, but may also involve an ATP-modulated regulatory process.

Abstract: The possible relevance of changes in extracellular and/or intracellular pH to the insulinotropic action of L-arginine and L-homoarginine was investigated in rat pancreatic islets. A rise in extracellular pH from 7.0 to 7.4 and 7.8 augmented the secretory response to these cationic amino acids whilst failing to affect the uptake of L-arginine by islet cells and whilst decreasing the release of insulin evoked by D-glucose. Under these conditions, a qualified dissociation was also observed between secretory data and 45Ca net uptake. Moreover, at high extracellular pH, the homoarginine-induced increase in 86Rb outflow from prelabelled islets rapidly faded out, despite sustained stimulation of insulin release. The cationic amino acids failed to affect the intracellular pH of islet cells, whether in the absence or presence of D-glucose and whether at normal or abnormal extracellular pH. These findings argue against the view that the secretory response to L-arginine would be related to either a change in cytosolic pH or the accumulation of this positively charged amino acid in the beta-cell. Nevertheless, they suggest that the yet unidentified target for L-arginine and its non-metabolized analogue in islet cells displays pH-dependency with optimal responsiveness at alkaline pH.

Abstract: When rat pancreatic islets were incubated in the presence of unlabelled D-glucose (16.7 mM) and 3HOH, the production of 3H-labelled material susceptible to be phosphorylated by yeast hexokinase and then detritiated by yeast phosphoglucoisomerase did not exceed 2.66 +/- 0.21 pmol/islet per 180 min, i.e. about 1% of the rate of exogenous D-[5-3H]glucose utilization. Such a material accounted for 43 +/- 4% of the total radioactivity, associated with tritiated hexose(s). It is proposed, therefore, that the futile cycling of D-glucose in the reactions catalyzed in the islet cells by the hexokinase isoenzymes and glucose-6-phosphatase represents a negligible fraction of the total rate of D-glucose phosphorylation.

Abstract: L-Arginine and L-ornithine, which stimulate amylase release, are taken up by rat parotid cells. L-Arginine is converted, in an NADPH-dependent manner and to a limited extent to L-citrulline in parotid cell homogenates, despite the absence of ornithine transcarbamylase activity. L-Arginine is largely converted to urea and L-ornithine. The generation of putrescine and polyamines from L-ornithine occurs at a very low rate, relative to the cell content in performed amines. The major fate of exogenous or arginine-derived ornithine consists in its conversion to L-glutamate, which is then further metabolized. These findings raise several hypotheses for the secretory response of the parotid cells to cationic amino acids, including their accumulation as positively charged molecules inside the cell and the generation of either NO, amines, substrates for a transglutaminase-catalyzed reaction, or ATP through oxidative catabolism. However, each of these hypotheses meets with objections, the modality for the stimulation of amylase release by cationic amino acids being eventually considered as an unsettled matter.

Abstract: A radioisotopic procedure for the assay of 3-hydroxybutyrate is presented. It is based on the measurement of NADH, generated in the 3-hydroxybutyrate dehydrogenase reaction, through the conversion of 2-[U-14C]ketoglutarate to 14C-labeled L-glutamate in the presence of beef liver glutamate dehydrogenase. The assay is linear in the range of 2.5 to 20.0 pmole/sample and about 100-times more sensitive than previous methods. The procedure proved useful for the measurement of 3-hydroxybutyrate in liver samples not exceeding 25 micrograms wet weight.