Christophe BINNERT

Abstract: The effects of a 7 d high-fructose diet (HFrD) or control diet on lipid metabolism were studied in a group of six healthy lean males. Plasma NEFA and beta-hydroxybutyrate concentrations, net lipid oxidation (indirect calorimetry) and exogenous lipid oxidation (13CO2 production) were monitored in basal conditions, after lipid loading (olive oil labelled with [13C]triolein) and during a standardised mental stress. Lactate clearance and the metabolic effects of an exogenous lactate infusion were also monitored. The HFrD lowered plasma concentrations of NEFA and beta-hydroxybutyrate as well as lipid oxidation in both basal and after lipid-loading conditions. In addition, the HFrD blunted the increase in plasma NEFA and exogenous lipid oxidation during mental stress. The HFrD also increased basal lactate concentrations by 31.8 %, and lactate production by 53.8 %, while lactate clearance remained unchanged. Lactate infusion lowered plasma NEFA with the control diet, and net lipid oxidation with both the HFrD and control diet. These results indicate that a 7 d HFrD markedly inhibits lipolysis and lipid oxidation. The HFrD also increases lactate production, and the ensuing increased lactate utilisation may contribute to suppress lipid oxidation.

Abstract: OBJECTIVE: Recent observations indicate that the delivery of nitric oxide by endothelial nitric oxide synthase (eNOS) is not only critical for metabolic homeostasis, but could also be important for mitochondrial biogenesis, a key organelle for free fatty acid (FFA) oxidation and energy production. Because mice deficient for the gene of eNOS (eNOS(-/-)) have increased triglycerides and FFA levels, in addition to hypertension and insulin resistance, we hypothesized that these knockout mice may have decreased energy expenditure and defective beta-oxidation. RESEARCH DESIGN AND METHODS: Several markers of mitochondrial activity were assessed in C57BL/6J wild-type or eNOS(-/-) mice including the energy expenditure and oxygen consumption by indirect calorimetry, in vitro beta-oxidation in isolated mitochondria from skeletal muscle, and expression of genes involved in fatty acid oxidation. RESULTS: eNOS(-/-) mice had markedly lower energy expenditure (-10%, P < 0.05) and oxygen consumption (-15%, P < 0.05) than control mice. This was associated with a roughly 30% decrease of the mitochondria content (P < 0.05) and, most importantly, with mitochondrial dysfunction, as evidenced by a markedly lower beta-oxidation of subsarcolemmal mitochondria in skeletal muscle (-30%, P < 0.05). Finally, impaired mitochondrial beta-oxidation was associated with a significant increase of the intramyocellular lipid content (30%, P < 0.05) in gastrocnemius muscle. CONCLUSIONS: These data indicate that elevated FFA and triglyceride in eNOS(-/-) mice result in defective mitochondrial beta-oxidation in muscle cells.

Abstract: BACKGROUND: After major burns, patients exhibit an intense catabolism, and the wounds require surgery and grafting for closure. Complications, such as weight loss and delayed wound healing, are worsened by trace element (TE) deficiencies. OBJECTIVE: We aimed to assess the effects of TE supplements on systemic substrate turnover and local protein metabolism during wound healing after major burns. DESIGN: This was a prospective, randomized, placebo-controlled trial in 21 patients aged 35 +/- 11 y with burns on 45 +/- 16% of their body surface area; 12 had skin biopsies performed on days 3, 10, and 20, and 10 patients underwent a stable-isotope investigation on day 10. Intravenous copper, selenium, and zinc (TE group) or vehicle (V group) was given with a saline solution for 14-21 d. On day 10, [(13)C]phenylalanine (600-microg/kg bolus followed by 12 microg x kg(-1) x min(-1)) plus 6-[(2)H(2)]glucose and [(2)H(5)]glycerol were infused for 6 h to determine skin protein turnover. Biopsies were performed 1 and 6 h after the start of infusion to determine [(13)C]phenylalanine enrichment. RESULTS: The patients' mean age and burn severity did not differ significantly between the groups nor between the skin investigations subgroups. Plasma TE concentrations were significantly higher in the TE group. In the burned areas, the skin contents of selenium (P=0.02) and zinc (P=0.03) increased by day 20. The supernatant-to-plasma (13)C enrichment ratio in burned skin was 0.363 +/- 0.094 (TE group) and 0.286 +/- 0.130 (V group) after 1 h (NS) and 0.592 +/- 0.153 (TE group) and 0.262 +/- 0.171 (V group) after 6 h, which reflected lower catabolism in the TE group (P=0.03). No significant differences in whole-body substrate turnover were found between the groups. CONCLUSION: TE supplementation was associated with an increased skin tissue content of selenium and zinc and with a reduction in skin protein catabolism.

Abstract:

Abstract: Obesity, lipid disorders, type 2 diabetes, high blood pressure and coronary heart disease are frequently encountered in wealthy populations. All these disorders frequently occur as clusters, constituting the metabolic syndrome. It is currently admitted that insulin resistance plays a central role in the pathogenesis of this syndrome. Stress responses include activation of the sympathetic nervous system and stimulation of epinephrine and cortisol release. These hormones may over the long term reduce insulin sensitivity. Cortisol may also favour the development of central obesity. In healthy individuals, mental stress increases heart rate, but simultaneously decreases vascular resistance in skeletal muscle. This results in a moderate increase in blood pressure, and an acute increase in insulin-mediated glucose disposal. In obese patients, mental stress elicits responses which differ widely from those of healthy individuals. While mental stress enhances catecholamine-mediated energy expenditure in obese patients to the same extent as in lean subjects, it fails to decrease systemic vascular resistance due to endothelial dysfunction. This leads to enhanced blood pressure responses and the absence of stimulation of glucose disposal in obese subjects during mental stress. It can be hypothesized that repeated professional or social stress may activate the sympathoadrenal system, resulting in high cortisol levels, stimulation of the sympathetic nervous system, and epinephrine secretion. All these factors may eventually lead to the development of central obesity and insulin resistance. Furthermore, the blood pressure responses to mental stress may be enhanced in insulin-resistant individuals, favouring the development of vascular complications.

Abstract: OBJECTIVE: To assess the effect of a possible interaction between dietary fat and physical inactivity on whole-body insulin sensitivity and intramyocellular lipids (IMCLs). RESEARCH DESIGN AND METHODS: Eight healthy male volunteers were studied on two occasions. After 2 days of an equilibrated diet and moderate physical activity, participants remained inactive (bed rest) for 60 h and consumed either a high-saturated fat (45% fat, of which approximately 60% was saturated fat [BR-HF]) or a high-carbohydrate (70% carbohydrate [BR-HCHO]) diet. To evaluate the effect of a high-fat diet alone, six of the eight volunteers were restudied after a 2-day equilibrated diet followed by 60 h on a high-saturated fat diet and controlled physical activity (PA-HF). Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp and IMCL concentrations by (1)H-magnetic resonance spectroscopy. RESULTS: Insulin-mediated glucose disposal was decreased by BR-HF condition (-24 +/- 6%, P < 0.05) but did not change with BR-HCHO (+19 +/- 10%, NS). BR-HF and BR-HCHO increased IMCL levels (+32 +/- 7%, P < 0.05 and +17 +/- 8%, P < 0.0011, respectively). Although the increase in IMCL levels with PA-HF (+31 +/- 19%, P = 0.12) was similar to that during BR-HF, insulin-mediated glucose disposal (-7 +/- 9%, NS) was not decreased. CONCLUSIONS: These data indicate that physical inactivity and a high-saturated fat diet may interact to reduce whole-body insulin sensitivity. IMCL content was influenced by dietary lipid and physical inactivity but was not directly associated with insulin resistance.

Abstract: Ripglut1;glut2-/- mice have no endogenous glucose transporter type 2 (glut2) gene expression but rescue glucose-regulated insulin secretion. Control of glucagon plasma levels is, however, abnormal, with fed hyperglucagonemia and insensitivity to physiological hypo- or hyperglycemia, indicating that GLUT2-dependent sensors control glucagon secretion. Here, we evaluated whether these sensors were located centrally and whether GLUT2 was expressed in glial cells or in neurons. We showed that ripglut1;glut2-/- mice failed to increase plasma glucagon levels following glucoprivation induced either by i.p. or intracerebroventricular 2-deoxy-D-glucose injections. This was accompanied by failure of 2-deoxy-D-glucose injections to activate c-Fos-like immunoreactivity in the nucleus of the tractus solitarius and the dorsal motor nucleus of the vagus. When glut2 was expressed by transgenesis in glial cells but not in neurons of ripglut1;glut2-/- mice, stimulated glucagon secretion was restored as was c-Fos-like immunoreactive labeling in the brainstem. When ripglut1;glut2-/- mice were backcrossed into the C57BL/6 genetic background, fed plasma glucagon levels were also elevated due to abnormal autonomic input to the alpha cells; glucagon secretion was, however, stimulated by hypoglycemic stimuli to levels similar to those in control mice. These studies identify the existence of central glucose sensors requiring glut2 expression in glial cells and therefore functional coupling between glial cells and neurons. These sensors may be activated at different glycemic levels depending on the genetic background.

Abstract: The mechanism underlying hypertriglyceridemia-associated insulin resistance in humans remains poorly understood. It has been proposed that hypertriglyceridemia only produces insulin resistance when associated with an increased lipid delivery to muscle. Accordingly, hypertriglyceridemia secondary to a decreased clearance of triglyceride-rich particles should not cause insulin resistance. To verify this hypothesis, we assessed whole body and adipose tissue insulin sensitivity in 15 healthy male volunteers before and after a 5-day administration of isotretinoin (1 mg/kg/d), a vitamin A derivative that decreases the clearance of triglyceride-rich particles. Whole body insulin-mediated glucose disposal (6,6 (2)H(2)glucose), glucose oxidation (indirect calorimetry), lipolysis ((2)H(5) glycerol), and subcutaneous adipose lipolysis (microdialysis) were evaluated during a 3-step hyperinsulinemic euglycemic clamp. Isotretinoin increased plasma triglyceride from 0.97 +/- 0.15 to 1.30 +/- 0.22 mmol/L (P <.02), but did not change whole body insulin-mediated glucose disposal and lipolysis. These observations are consistent with an isotretinoin-induced inhibition of very-low-density lipoprotein (VLDL)-triglyceride clearance. The suppression of endogenous glucose production and the reduction in subcutaneous adipose glycerol concentrations by insulin remained equally unaffected after isotretinoin administration. We conclude that the impaired clearance of triglyceride-rich particles secondary to a 5-day isotretinoin administration does not impair insulin-mediated antilipolysis or glucose disposal. The data support the concept that hypertriglyceridemia-associated insulin resistance develops primarily when triglyceride production is increased.

Abstract: The role of the gluco-incretin hormones GIP and GLP-1 in the control of beta cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double- as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R(-/-) but not in Gipr(-/-) mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on beta cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a beta cell autonomous secretion defect when both receptors are inactivated.

Abstract: PURPOSE OF REVIEW: Various threatening stimuli, such as pain, low blood pressure, or infection, elicit a set of neuroendocrine responses that include an increased secretion of catecholamines and glucocorticoid from the adrenal gland and activation of the sympathetic nervous system. These hormonal secretions allow a "fight or flight" response by mobilizing endogenous substrate. They also exert anti-insulin actions, and may in the long term induce a state of insulin resistance. In addition, stress stimulates inflammatory mediators in mononuclear cells. Given the possible role of low-grade inflammation in chronic metabolic disorders, this suggests that stress may be a factor in the development of insulin resistance and the metabolic syndrome. RECENT FINDINGS: Studies reviewed in this article cover: (1) the metabolic and haemodynamic effects of stress in healthy and insulin-resistant individuals; (2) the relationship between stress and inflammation and the role of the autonomic nervous system; and (3) some factors known to modulate the neuroendocrine responses to stress. Future perspectives, together with some hints regarding the role of neurotrophins such as brain-derived neurotrophic factor, are delineated. SUMMARY: Recent work performed in the field has indicated that stress may be a significant factor in the pathogenesis of metabolic disorders. Nutritional intervention or pharmacological agents targeted at modulating stress should be investigated.

Abstract: OBJECTIVE: Recent reports suggest that lipid-induced insulin resistance is more pronounced in men than in women. Whether such gender difference exists for other factors known to induce insulin resistance in healthy individuals remains unknown. We therefore assessed whether glucocorticoid-induced insulin resistance differs in men and women. METHODS: The insulin sensitivity and insulin secretion of 8 women and 7 men, all non obese and healthy, were evaluated with or without administration of dexamethasone (2 mg/day during 2 days) by means of a two-step hyperglycemic clamp. RESULTS: Dexamethasone decreased insulin sensitivity to the same extent in men and women. The relative increases in insulin concentration observed after dexamethasone in the basal state, during the first phase of insulin release and at the two steps of hyperglycemia were similar in men and women. The hyperinsulinemia thus attained allowed to fully compensate for insulin resistance in both genders. CONCLUSIONS: The effects of glucocorticoids on insulin sensitivity and insulin secretion show no gender difference in healthy humans.

Abstract: OBJECTIVES: Metformin is recognized as the treatment of chronic obese, insulin-resistant type 2 diabetic patients. Whether it improves insulin sensitivity in obese patients with normal glucose tolerance remains unknown. METHODS: Eight obese female patients with normal glucose tolerance were studied during a double blinded, randomized cross-over study including a 2-week administration of metformin and a 2-week administration of placebo. Insulin secretion and insulin sensitivity were assessed after metformin and placebo by means of a 3-hour hyperglycemic clamp. RESULTS: The plasma insulin and C-peptide concentrations during the hyperglycemic clamp were identical after placebo or metformin (both first and second phases). Insulin-mediated glucose disposal, stimulation of glucose oxidation and suppression of endogenous glucose production were identical after metformin and placebo. CONCLUSIONS: Metformin does not improve insulin sensitivity nor insulin secretion in obese female patients with normal glucose tolerance.

Abstract: OBJECTIVES: A diet rich in n-3 fatty acids (fish oils) is associated with reduced risks of cardiovascular and metabolic diseases, but the mechanisms remain incompletely understood. Sympathoadrenal activation is postulated to be involved in the pathogenesis of these diseases, and may be inhibited by n-3 fatty acids. We therefore evaluated the effects of a diet supplemented with n-3 fatty acids on the stimulation of the sympathetic nervous system and of stress hormones elicited by a mental stress. METHODS: Seven human volunteers were studied on two occasions, before and after 3 weeks of supplementation with 7.2 g/day fish oil. On each occasion, the concentrations of plasma cortisol, and catecholamines, energy expenditure (indirect calorimetry), and adipose tissue lipolysis (plasma non esterified fatty acid concentrations) were monitored in basal conditions followed by a 30 min mental stress (mental arithmetics and Stroop's test) and a 30 min recovery period. RESULTS: In control conditions, mental stress significantly increased heart rate, mean blood pressure, and energy expenditure. It increased plasma epinephrine from 60.9 +/- 6.2 to 89.3 +/- 16.1 pg/ml (p<0.05), plasma cortisol from 291 +/- 32 to 372 +/- 37 micromol/l (p<0.05) and plasma non esterified fatty acids from 409 +/- 113 to 544 +/- 89 micromol/l (p<0.05). After 3 weeks of a diet supplemented with n-3 fatty acids, the stimulation by mental stress of plasma epinephrine, cortisol, energy expenditure, and plasma non esterified fatty acids concentrations, were all significantly blunted. CONCLUSION: Supplementation with n-3 fatty acids inhibits the adrenal activation elicited by a mental stress, presumably through effects exerted at the level of the central nervous system.

Abstract: Regular physical exercise and endurance training are associated with low body weight and low body fat mass. The relationship between exercise and body-weight control is complex and incompletely understood. Regular exercise may decrease energy balance through an increase in energy expenditure or an increase in fat oxidation. It may also contribute to weight loss by modulating nutrient intake. An intriguing question that remains unresolved is whether changes in nutrient intake or body composition secondarily affect spontaneous physical activity. If this were the case, physical activity would represent a major adaptative mechanism for body-weight control.

Abstract: Microdialysis is a minimally invasive tool that allows us to gain insight into metabolism at the tissue level. In human investigations, it can be safely performed in the brain (neurosurgical patients), skeletal muscle and adipose tissue. Basically, the technique allows interstitial concentrations of small solutes to be evaluated. Several limitations of the method and possible ways to circumvent them are indicated. Recent technical developments are reviewed. At present, this method is rarely used in metabolic monitoring of critically ill patients, but its potential applications are highlighted.

Abstract: Raising plasma free fatty acid (FFA) levels reduces muscle glucose uptake, but the effect of FFAs on splanchnic glucose uptake, total glucose output, and glucose cycling may also be critical to producing lipid-induced glucose intolerance. In eight normal volunteers, we measured glucose turnover and cycling rates ([2H7]glucose infusion) during a moderately hyperglycemic (7.7 mmol/l) hyperinsulinemic clamp, before and after ingestion of a labeled (dideuterated) oral glucose load (700 mg/kg). Each test was performed twice, with either a lipid or a saline infusion; four subjects also had a third test with a glycerol infusion. As shown by similar rates of exogenous glucose appearance, the lipid infusion did not reduce first-pass splanchnic glucose uptake (saline 1.48+/-0.18, lipid 1.69+/-0.17, and glycerol 1.88+/-0.17 mmol/kg per 180 min; NS), but it reduced peripheral glucose uptake by 40% (P < 0.01 vs. both saline and glycerol infusions). Before oral ingestion of glucose, total glucose output was similarly increased by the lipid and glycerol infusions. Total glucose output was significantly increased by FFAs after oral ingestion of glucose (saline 3.68+/-1.15, glycerol 3.68+/-1.70, and lipid 7.92+/-0.88 micromol x kg(-1) x min(-1); P < 0.01 vs. saline and P < 0.05 vs. glycerol). The glucose cycling rate was approximately 2.7 micromol x kg(-1) x min(-1) with the three infusions and tended to decrease all along the lipid infusion, which argues against a stimulation of glucose-6-phosphatase by FFAs. It is concluded that in situations of moderate hyperinsulinemia-hyperglycemia, FFAs reduce peripheral but not splanchnic glucose uptake. Total glucose output is increased by FFAs, by a mechanism that does not seem to involve stimulation of glucose-6-phosphatase.

Abstract: Fatty acid transporter protein (FATP)-1 mRNA expression was investigated in skeletal muscle and in subcutaneous abdominal adipose tissue of 17 healthy lean, 13 nondiabetic obese, and 16 obese type 2 diabetic subjects. In muscle, FATP-1 mRNA levels were higher in lean women than in lean men (2.2 +/- 0.1 vs. 0.6 +/- 0.2 amol/microg total RNA, P < 0.01). FATP-1 mRNA expression was decreased in skeletal muscle in obese women both in nondiabetic and in type 2 diabetic patients (P < 0.02 vs. lean women in both groups), and in all women there was a negative correlation with basal FATP-1 mRNA level and body mass index (r = -0.74, P < 0.02). In men, FATP-1 mRNA was expressed at similar levels in the three groups both in skeletal muscle (0.6 +/- 0.2, 0.6 +/- 0.2, and 0.8 +/- 0.2 amol/microg total RNA in lean, obese, and type 2 diabetic male subjects) and in adipose tissue (0.9 +/- 0.2 amol/microg total RNA in the 3 groups). Insulin infusion (3 h) reduced FATP-1 mRNA levels in muscle in lean women but not in lean men. Insulin did not affect FATP-1 mRNA expression in skeletal muscle in obese nondiabetic or in type 2 diabetic subjects nor in subcutaneous adipose tissue in any of the three groups. These data show a gender-related difference in the expression of the fatty acid transporter FATP-1 in skeletal muscle of lean individuals and suggest that changes in FATP-1 expression may not contribute to a large extent to the alterations in fatty acid uptake in obesity and/or type 2 diabetes.

Abstract: The metabolic fate of an oral long-chain-triacylglycerol (LCT) load and of a mixed oral LCT and medium-chain-triacylglycerol (MCT) load was followed for 6 h in eight control and eight obese subjects with normal postabsorptive triacylglycerol concentrations. Labeled triacylglycerol and indirect calorimetry were used. Results showed that LCTs were less oxidized in obese than in control subjects (3.2+/-0.5 compared with 6.0+/-0.4 g, P < 0.01). Moreover, the amount of LCT oxidized was negatively correlated with fat mass (r = -0.77, P < 0.01). Appearance in plasma of dietary triacyglycerol-derived long-chain fatty acids was blunted in obese subjects and it was negatively related to fat mass (r = -0.84, P < 0.01) and positively to LCT oxidation (r = 0.70, P < 0.01). On the contrary, MCT oxidation was not altered in obese subjects compared with control subjects. Furthermore, the proportion of MCTs oxidized was higher in both groups compared with LCTs (x+/-SEM: 57.5+/-2.6% compared with 15.2+/-1.6%, P < 0.01, n = 16). Our conclusion is that obesity is associated with a defect in the oxidation of dietary LCTs probably related to an excessive uptake by the adipose tissue of meal-derived long-chain fatty acids. MCTs, the oxidation of which is not altered in obesity, could therefore be of interest in the dietary treatment of obesity.

Abstract: To determine the steps involved in the metabolism of ingested triglycerides (TG), 10 healthy women were studied during 6 h after ingestion of 30 g olive oil labeled with [1,1,1-13C3] triolein. The appearance of 13C was followed in chylomicron-TG (CM-TG), nonesterified fatty acid (NEFA), very low-density lipoprotein (VLDL)-TG, and in expired gas. Indirect calorimetry was used to determine total lipid oxidation. After 90 min, labeling was higher in CM-TG than in NEFA or VLDL. At 180 min, a plateau of enrichment was obtained for CM-TG and NEFA, demonstrating the entry of exogenous lipids in the NEFA pool. After 300 min, a plateau was observed for VLDL-TG with levels of enrichment (0.38 +/- 0.04%) similar to those observed for NEFA (0.36 +/- 0.03%), suggesting a precursor-product relationship. Only 19 +/- 2% of the load was oxidized. From 300 to 360 min, 70% of total lipid oxidation was from exogenous TG. We conclude that, after ingestion of a lipid load, a cycle of fatty acids-TG occurs from CM to NEFA and from NEFA to VLDL. Furthermore, this lipid load has a sparing effect on endogenous lipid stores.

Abstract: The study of triglyceride (TG) metabolism using stable isotope tracers would be facilitated by being able to detect low 13C enrichment. To meet this goal, we developed a gas chromatography/isotope ratio-mass spectrometry technique to measure the enrichment of palmitate in nonesterified fatty acids (NEFA) and TG as its methyl derivative. This method allows accurate and reproducible measurements of enrichment as low as 0.009 mole percent excess (MPE), in a range between 0-0.65 MPE. The usefulness of this method is shown by two studies of lipid metabolism in human beings. First, we studied the metabolic fate of an oral TG load labeled with [1,1,1-13C3]tripalmitin. Labeled palmitate appeared concurrently in plasma NEFA and TG, and four hours after the load, the labeling was higher in NEFA than in TG (MPE NEFA: 1.53 +/- 0.31 vs. MPE TG: 0.78 +/- 0.06, P < 0.05). In a second study, the hepatic reesterification of NEFA was estimated by measuring the appearance of infused [1-13C]palmitate in circulating TG. The estimated contribution of plasma NEFA to circulating TG increased to a maximum of 22%. Thus, gas chromatography/isotope ratio-mass spectrometry appears to be a useful tool for future studies of lipid metabolism in humans.