Abstract: Introduction: Statins reduce cardiovascular risk in populations targeted for both primary and secondary
prevention. The potential of statin use to impact the risk of developing new onset diabetes has been suggested.
However, there have been a few large-scale RCTs of pitavastatin which is the statin, but adverse effects on glucose
metabolism and diabetes onset were not documented by meta analysis.
Case reports: We report two patients with type 2 diabetes and hypercholesterolemia showing amelioration
of insulin resistance with pitavastatin. The improvements were attributed to effects of pitavastatin on glucose and
insulin metabolism. A 75-year-old man and a 70-year-old woman with type 2 diabetes, hypercholesterolemia and
hypertension were given pitavastatin (2 mg/day) for 6 months, without changes in other medication. Before and after
treatment, both received euglycemic hyperinsulinemic clamp studies to assess insulin sensitivity. After 6 months,
glucose infusion rates were increased in both subjects.
Conclusion: These cases suggest that pitavastatin, unlike other statins, has beneficial effects on insulin
sensitivity in an insulin-resistant state.
Abstract: We investigated the effect between resistin and adiponectin on the proliferation of vascular smooth muscle cells (VSMCs). We confirmed that resistin significantly increases the number of rat VSMCs as well as thymidine incorporation with them, whereas adiponectin diminishes resistin-induced cell proliferation. Resistin significantly increased p42/44 mitogen-activated protein kinase (MAPK) phosphorylation within rat VSMCs, whereas adiponectin inhibited resistin-induced MAPK phosphorylation. Moreover, resistin significantly increased c-fos expression, whereas adiponectin suppressed resistin-induced c-fos expression. Cell cycle progression is a tightly controlled event that is negatively regulated by cyclin-dependent kinases inhibitors (CDKIs) such as p53, p21, and p27. Resistin significantly decreased the expression of these CDKIs, whereas adiponectin restored the resistin-induced decrease in CDKIs expression. These effects were abolished in the MAPK inhibitors. In conclusion, resistin plays a role in the development of atherosclerosis, whereas adiponectin may be an important in its prevention in insulin-resistant patients.
Abstract: Objective:Yacon is a perennial plant forming a clump of >20 big, edible underground tubers. Yacon, which originates from South America, has become increasingly popular in the Japanese diet for tubers have a lower caloric value and a high fiber content. Recent studies have suggested that yacon feeding ameliorates diabetes as indicated by reduced blood glucose.Methods:We fed male Zucker fa/fa rats for 5 weeks with isocaloric normal chow diet containing from 6.5% control aroid or 6.5% yacon. Insulin sensitivity was evaluated by euglycemic-hyperinsulinemic clamp study.Results:Body weight was comparable between yacon- and aroid-fed rats. In the basal state, yacon feeding had an effect to lower fasting glucose levels from 184.1±4.1 to 167.8±2.7 mg dl(-1) (P<0.01), as well as basal hepatic glucose output (HGO) from 9.9±0.4 to 7.4 ± 0.2 mg kg(-1) per min (P<0.01). During the clamp studies, the glucose infusion rate required to maintain euglycemia was increased by 12.3% in yacon-fed rat. The insulin suppression of HGO was also increased in yacon-fed rats compared with control rats (85.3±2.4% vs 77.0±3.0%; P<0.05), whereas the glucose disposal rate was not different between the two groups. Consistent with the clamp data, the insulin-stimulated phosphorylation of Akt was significantly enhanced in liver but not in skeletal muscle. Furthermore, tribbles 3 (Trb3) expression, which is a negative regulator of Akt activity, was markedly reduced in the liver of yacon-fed rats compared with control rats.Conclusion:These results indicate that the effect of yacon feeding to reduce blood glucose is likely due to its beneficial effects on hepatic insulin sensitivity in the insulin resistant state.
Abstract: Adipocytokines such as resistin, TNF-α, and adiponectin, which are adipocyte-derived peptides, play important
roles in glucose metabolism. Resistin and TNF-α have been implicated as factors associated with the development
of insulin resistance in obesity. In contrast, adiponectin has been shown to improve insulin sensitivity in insulin
resistance. However, the interaction among these adipocytokines is still unknown. In this study, we investigated
the mechanism of the effects of these 3 adipocytokines (resistin, adiponectin, and TNF-α) on glucose transport in
3T3-L1 adipocytes.
Glucose uptake was evaluated by 2-[3H] deoxy-glucose (DOG) uptake assay in 3T3-L1 adipocytes. Resistin and
adiponectin secretion were analyzed by western blotting.
Adenovirus-mediated overexpression of resistin inhibited insulin-stimulated 2-DOG uptake by only 15% compared
with control cells. In contrast, pretreatment of cells with 10 ng/mL TNF-α for 3 hrs did not inhibit insulin stimulated
2-DOG uptake compared with control cells, whereas overexpression of resistin led to a ~ 40% decrease in insulin
stimulated 2-DOG uptake following pretreatment with TNF-α. TNF-α has been shown to suppress the expression
and secretion of adiponectin from adipocytes. Therefore, we speculated that this potentiating effect of resistin might
be caused by the reduction in adiponectin secretion. We confirmed that the secretion of adiponectin was decreased
by ~ 50% in TNF-α treated cells compared to control cells. Furthermore, overexpression of adiponectin prevented
this additive effect of resistin and TNF-α.
In conclusion, these results suggest that: (1) TNF-α enhances the action of resistin via the reduction of
adiponectin, 2) Resistin may cause severe insulin resistance under low adiponectin levels.
Abstract: AIMS:
Pioglitazone, a full peroxisome proliferator-activated receptor (PPAR)-γ agonist, improves insulin sensitivity by increasing circulating adiponectin levels. However, the molecular mechanisms by which pioglitazone induces insulin sensitization are not fully understood. In this study, we investigated whether pioglitazone improves insulin resistance via upregulation of either 2 distinct receptors for adiponectin (AdipoR1 or AdipoR2) expression in 3T3-L1 adipocytes.
MAIN METHODS:
Glucose uptake was evaluated by 2-[(3)H] deoxy-glucose uptake assay in 3T3-L1 adipocytes with pioglitazone treatment. AdipoR1 and AdipoR2 mRNA expressions were analyzed by qRT-PCR.
KEY FINDINGS:
We first confirmed that pioglitazone significantly increased insulin-induced 2-deoxyglucose (2-DOG) uptake in 3T3-L1 adipocytes. Next, we investigated the mRNA expression and regulation of AdipoR1 and AdipoR2 after treatment with pioglitazone. Interestingly, pioglitazone significantly induced AdipoR2 expression but it did not affect AdipoR1 expression. In addition, adenovirus-mediated PPARγ expression significantly enhanced the effects of pioglitazone on insulin-stimulated 2-DOG uptake and AdipoR2 expression in 3T3-L1 adipocytes. These data suggest that pioglitazone enhances adiponectin's autocrine and paracrine actions in 3T3-L1 adipocytes via upregulation of PPARγ-mediated AdipoR2 expression. Furthermore, we found that pioglitazone significantly increased AMP-activated protein kinase (AMPK) phosphorylation in insulin-stimulated 3T3-L1 adipocytes, but it did not lead to the phosphorylation of IRS-1, Akt, or protein kinase Cλ/ζ.
Abstract: Apolipoprotein (apo) E plays a key role in lipoprotein metabolism and has been proposed to modulate triglyceride (TG) lipolysis. However, no systematic investigation on lipolysis using all 3 isoforms of apoE has been performed. To clarify the role of common human apoE isoforms in the lipolysis of very low-density lipoprotein (VLDL) TGs, we overexpressed human apoE isoforms in apoE and low-density lipoprotein receptor-deficient mice using adenoviral-mediated gene transfer and used VLDL particles obtained from these mice for in vitro lipolysis assay. Overexpression of apoE, regardless of its isoforms, increased the TG content of VLDL in mice in vivo. In vitro analysis of the effect of apoE on lipolysis revealed that irrespective of its isoforms, apoE did inhibit TG lipolysis at every concentration of apoE examined, and this inhibitory effect became more pronounced as the apoE content of VLDL increased. No difference was observed in TG lipolysis activity among isoforms at low apoE/TG ratio; however, intermediate ratios of apoE/TG, which reflect physiologic VLDL apoE/TG ratios, demonstrated a significantly greater level of lipolysis inhibition in apoE2, but less so in apoE4 compared with other isoforms. This differential effect by apoE isoforms on lipolysis was attenuated at higher apoE/TG ratios; nevertheless, apoE2 still inhibited lipolysis significantly more than did apoE4. Enrichment of VLDL with apoE decreased both the apoC contents and apoC-II/C-III ratios of VLDL, contributing, at least in part, to the inhibitory function of apoE on lipolysis. The present study clarifies the differential lipolysis-modulating effect of apoE isoforms, which would help explain the difference in pre- and postprandial TG levels among humans carrying different apoE isoforms.
Abstract: Lipid infusion and high fat feeding are established causes of systemic and adipose tissue insulin resistance. In this study, we treated 3T3-L1 adipocytes with a mixture of free fatty acids (FFAs) to investigate the molecular mechanisms underlying fat-induced insulin resistance. FFA treatment impaired insulin receptor-mediated signal transduction and decreased insulin-stimulated GLUT4 translocation and glucose transport. FFAs activated the stress/inflammatory kinases c-Jun N-terminal kinase (JNK) and IKK{beta}, and the suppressor of cytokine signaling protein 3, increased secretion of the inflammatory cytokine tumor necrosis factor (TNF)-{alpha}, and decreased secretion of adiponectin into the medium. RNA interference-mediated down-regulation of JNK blocked JNK activation and prevented most of the FFA-induced defects in insulin action. Blockade of TNF-{alpha} signaling with neutralizing antibodies to TNF-{alpha} or its receptors or with a dominant negative TNF-{alpha} peptide had a partial effect to inhibit FFA-induced cellular insulin resistance. We found that JNK activation by FFAs was not inhibited by blocking TNF-{alpha} signaling, whereas the FFA-induced increase in TNF-{alpha} secretion was inhibited by RNA interference-mediated JNK knockdown. Together, these results indicate that 1) JNK can be activated by FFAs through TNF-{alpha}-independent mechanisms, 2) activated JNK is a major contributor to FFA-induced cellular insulin resistance, and 3) TNF-{alpha} is an autocrine/paracrine downstream effector of activated JNK that can also mediate insulin resistance.
Abstract: In this study, we investigated the chronic in vivo effect of adiponectin on insulin sensitivity and glucose metabolism by overexpressing the adiponectin protein in male Wistar rats using intravenous administration of an adenovirus (Adv-Adipo). Virally infected liver secreted adiponectin as high and low molecular weight complexes. After 7 days of physiological or supraphysiological hyperadiponectinemia, the animals displayed enhanced insulin sensitivity during the glucose tolerance and insulin tolerance tests. Glucose clamp studies performed at submaximal and maximal insulin infusion rates (4 and 25 mU {middle dot} kg-1 {middle dot} min-1, respectively) also demonstrated increased insulin sensitivity in Adv-Adipo animals, with the insulin-stimulated glucose disposal rate being increased by 20-67%. In contrast, insulin's effect on the suppression of hepatic glucose output and plasma free fatty acid levels was not enhanced in Adv-Adipo rats compared with controls, suggesting that high levels of adiponectin expression in the liver may lead to a local desensitization. Consistent with the clamp data, the activation of AMP-activated protein kinase was significantly enhanced in skeletal muscle (by 50%) but not in liver. One interesting finding was that in male Wistar rats, both AdipoR1 and AdipoR2 expression levels were higher in skeletal muscle than in liver, as it is the case in humans. These results indicate that chronic adiponectin treatment enhances insulin sensitivity and could serve as a therapy for human insulin resistance.
Abstract: G protein-coupled receptor kinases (GRKs) regulate seven-transmembrane receptors (7TMRs) by phosphorylating agonist-activated 7TMRs. Recently, we have reported that GRK2 can function as a negative regulator of insulin action by interfering with G protein-q/11 {alpha}-subunit (G{alpha}q/11) signaling, causing decreased glucose transporter 4 (GLUT4) translocation. We have also reported that chronic endothelin-1 (ET-1) treatment leads to heterologous desensitization of insulin signaling with decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and G{alpha}q/11, and decreased insulin-stimulated glucose transport in 3T3-L1 adipocytes. In the current study, we have investigated the role of GRK2 in chronic ET-1-induced insulin resistance. Insulin-induced GLUT4 translocation was inhibited by pretreatment with ET-1 for 24 h, and we found that this inhibitory effect was rescued by microinjection of anti-GRK2 antibody or GRK2 short interfering RNA. We further found that GRK2 mediates the inhibitory effects of ET-1 by two distinct mechanisms. Firstly, adenovirus-mediated overexpression of either wild-type (WT)- or kinase-deficient (KD)-GRK2 inhibited G{alpha}q/11 signaling, including tyrosine phosphorylation of G{alpha}q/11 and cdc42-associated phosphatidylinositol 3-kinase activity. Secondly, ET-1 treatment caused Ser/Thr phosphorylation of IRS-1 and IRS-1 protein degradation. Overexpression of KD-GRK2, but not WT-GRK2, inhibited ET-1-induced serine 612 phosphorylation of IRS-1 and restored activation of this pathway. Taken together, these results suggest that GRK2 mediates ET-1-induced insulin resistance by 1) inhibition of G{alpha}q/11 activation, and this effect is independent of GRK2 kinase activity, and 2) GRK2 kinase activity-mediated IRS-1 serine phosphorylation and degradation.
Abstract: We investigated the chronic in vivo effect of resistin on insulin sensitivity and glucose metabolism by overexpressing resistin protein in male Wistar rats using intravenous administration of an adenovirus encoding mouse resistin. After 7 days of elevated resistin levels at a supraphysiological concentration, the animals displayed glucose intolerance and hyperinsulinemia during glucose tolerance tests, and insulin tolerance tests demonstrated an impaired glucose-lowering effect of insulin. The glucose clamp studies were performed at submaximal (4 mU/kg/min) and maximal (25 mU/kg/min) insulin infusion rates and demonstrated the presence of insulin resistance induced by elevated resistin levels. Indeed, the insulin-stimulated glucose infusion rate was decreased by 12-31%; suppression of hepatic glucose output was attenuated by 28-55%; and insulin suppression of circulating FFA levels was inhibited by 7%. Insulin receptor substrate-1 and -2 phosphorylation and Akt activation were impaired in muscle and adipose tissue. Interestingly, activation of AMP-activated protein kinase in skeletal muscle, liver, and adipose tissue was also significantly downregulated. Together, these results indicate that chronic "hyper-resistinemia" leads to whole-body insulin resistance involving impaired insulin signaling in skeletal muscle, liver, and adipose tissue, resulting in glucose intolerance, hyperinsulinemia, and hypertriglyceridemia. Thus elevated resistin levels in normal rats fed a regular chow diet produce many of the features of human syndrome X.
Abstract: {beta}-Arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether {beta}-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) {beta}-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous {beta}-arrestin-1 was knocked down by transfection of {beta}-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT {beta}-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both {beta}-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) {beta}-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) {beta}-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on {beta}-arrestin and the amino terminus of {beta}-arrestin-1 are required for this effect of {beta}-arrestin on IRS-1 degradation; and (iv) inhibition of {beta}-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.
Abstract: Insulin resistance is a major hallmark in the development of type 2 diabetes, which is characterized by an impaired ability of insulin to inhibit glucose output from the liver and to promote glucose uptake in muscle. The nuclear hormone receptor coactivator PGC-1 (peroxisome proliferator-activated (PPAR)-gamma coactivator-1) has been implicated in the onset of type 2 diabetes. Hepatic PGC-1 expression is elevated in mouse models of this disease, where it promotes constitutive activation of gluconeogenesis and fatty acid oxidation through its association with the nuclear hormone receptors HNF-4 and PPAR-alpha, respectively. Here we show that PGC-1-deficient mice, generated by adenoviral delivery of PGC-1 RNA interference (RNAi) to the liver, experience fasting hypoglycemia. Hepatic insulin sensitivity was enhanced in PGC-1-deficient mice, reflecting in part the reduced expression of the mammalian tribbles homolog TRB-3, a fasting-inducible inhibitor of the serine-threonine kinase Akt/PKB (ref. 6). We show here that, in the liver, TRB-3 is a target for PPAR-alpha. Knockdown of hepatic TRB-3 expression improved glucose tolerance, whereas hepatic overexpression of TRB-3 reversed the insulin-sensitive phenotype of PGC-1-deficient mice. These results indicate a link between nuclear hormone receptor and insulin signaling pathways, and suggest a potential role for TRB-3 inhibitors in the treatment of type 2 diabetes.
Notes: Using Smart Source Parsing xD;May; Epub 2004 Apr 25
Abstract: G protein-coupled receptor kinases (GRKs) represent a class of proteins that classically phosphorylate agonist-activated G protein-coupled receptors, leading to uncoupling of the receptor from further G protein activation. Recently, we have reported that the heterotrimeric G protein {alpha}-subunit, G{alpha}q/11, can mediate insulin-stimulated glucose transport. GRK2 contains a regulator of G protein signaling (RGS) domain with specificity for G{alpha}q/11. Therefore, we postulated that GRK2 could be an inhibitor of the insulin signaling cascade leading to glucose transport in 3T3-L1 adipocytes. In this study, we demonstrate that microinjection of anti-GRK2 antibody or siRNA against GRK2 increased insulin-stimulated insulin-responsive glucose transporter 4 (GLUT4) translocation, while adenovirus-mediated overexpression of wild-type or kinase-deficient GRK2 inhibited insulin-stimulated GLUT4 translocation as well as 2-deoxyglucose uptake. Importantly, a mutant GRK2 lacking the RGS domain was without effect. Taken together, these results indicate that through its RGS domain endogenous GRK2 functions as a negative regulator of insulin-stimulated glucose transport by interfering with G{alpha}q/11 signaling to GLUT4 translocation. Furthermore, inhibitors of GRK2 can lead to enhanced insulin sensitivity.
Abstract: Objective-- Plasma platelet-activating factor (PAF) acetylhydrolase (AH) is an enzyme bound with lipoproteins that degrades not only PAF but also PAF-like oxidized phospholipids that are proposed to promote atherosclerosis. In this study, we investigated the distribution of PAF-AH protein among lipoprotein classes by using adenovirus-mediated gene transfer in mice, and we examined its effects on lipoprotein oxidation and foam cell formation of macrophages. Methods and Results-- Adenovirus-mediated overexpression of PAF-AH in mice resulted in a 76- to 140-fold increase in plasma PAF-AH activity. Contrary to the previous report, overexpressed human PAF-AH protein was bound to very low density lipoprotein, intermediate density lipoprotein, low density lipoprotein, and high density lipoprotein (HDL). All the lipoproteins with overexpressed human PAF-AH revealed more resistance against oxidative stress, which was associated with lower levels in autoantibody against oxidized low density lipoprotein in the plasma. In addition, HDL with human PAF-AH inhibited foam cell formation and facilitated cholesterol efflux in macrophages. Conclusions-- These results suggest that human plasma PAF-AH exerts an antiatherogenic effect by binding to all the lipoproteins and thereby protecting them from oxidation, producing less proatherogenic lipoproteins and preserving HDL functions.
Abstract: Insulin stimulates glucose transport by promoting translocation of GLUT4 proteins from the perinuclear compartment to the cell surface. It has been previously suggested that the microtubule-associated motor protein kinesin, which transports cargo toward the plus end of microtubules, plays a role in translocating GLUT4 vesicles to the cell surface. In this study, we investigated the role of Rab4, a small GTPase-binding protein, and the motor protein KIF3 (kinesin II in mice) in insulin-induced GLUT4 exocytosis in 3T3-L1 adipocytes. Photoaffinity labeling of Rab4 with [{gamma}-32P]GTP-azidoanilide showed that insulin stimulated Rab4 GTP loading and that this insulin effect was inhibited by pretreatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 or expression of dominant-negative protein kinase C-{lambda} (PKC-{lambda}). Consistent with previous reports, expression of dominant-negative Rab4 (N121I) decreased insulin-induced GLUT4 translocation by 45%. Microinjection of an anti-KIF3 antibody into 3T3-L1 adipocytes decreased insulin-induced GLUT4 exocytosis by 65% but had no effect on endocytosis. Coimmunoprecipitation experiments showed that Rab4, but not Rab5, physically associated with KIF3, and this was confirmed by showing in vitro association using glutathione S-transferase-Rab4. A microtubule capture assay demonstrated that insulin stimulation increased the activity for the binding of KIF3 to microtubules and that this activation was inhibited by pretreatment with the PI3-kinase inhibitor LY294002 or expression of dominant-negative PKC-{lambda}. Taken together, these data indicate that (i) insulin signaling stimulates Rab4 activity, the association of Rab4 with kinesin, and the interaction of KIF3 with microtubules and (ii) this process is mediated by insulin-induced PI3-kinase-dependent PKC-{lambda} activation and participates in GLUT4 exocytosis in 3T3-L1 adipocytes.
Abstract: Probucol, an antioxidative and hypolipidemic agent, has been postulated to increase reverse cholesterol transport by enhancing cholesteryl ester transfer protein (CETP) activity. However, its clinical implication in CETP deficient patients has not been fully defined. To characterize the effects of probucol in the absence of CETP, we evaluated the changes in lipid profile, lipid peroxidation, and paraoxonase 1 (PON1) activity in two complete CETP deficient patients, caused by treatment with probucol.When the patients were not receiving probucol, low-density lipoprotein (LDL) particles were smaller and high-density lipoprotein (HDL) particles were larger in these patients than in controls. Treatment with probucol (500mg/day) resulted in the decrease in the levels of HDL-C and apolipoprotein (apo) A-I up to 22%. The size of HDL particles became smaller. LDL cholesterol concentration did not change in one patient, while it decreased by 47% in the other. PON1 activity/HDL-C, which was about 40% lower in the patients before treatment than in controls with the matching PON1 genotype, increased by 30% during the treatment. Lag time for LDL and HDL in both cases became prolonged more than 1.8 times after administration of probucol.This study demonstrated for the first time that probucol reduces HDL-C even in humans with complete CETP deficiency. Probucol treatment in these patients was also associated with protection of lipoproteins against oxidative stress, suggesting a clinical benefit of this drug even in such a state.
Abstract: Mutations in Na+-HCO[IMG]img002.gif" ALT="<UP><SUB>3</SUB><SUP></SUP></UP>"> cotransporter (NBC-1) cause proximal renal tubular acidosis (pRTA) associated with ocular abnormalities. One pRTA patient had increased serum amylase, suggesting possible evidence of pancreatitis. To further delineate a link between NBC-1 inactivation and pancreatic dysfunction, immunohistochemical analysis was performed on rat and human pancreas using antibodies against kidney-type (kNBC-1) and pancreatic-type (pNBC-1) transporters. In rat pancreas, the anti-pNBC-1 antibody labeled acinar cells and both apical and basolateral membranes of medium and large duct cells. In human pancreas, on the other hand, the anti-pNBC-1 antibody did not label acinar cells, although it did label the basolateral membranes of the entire duct system. The labeling by anti-kNBC-1 antibody was detected in only a limited number of rat pancreatic duct cells. To examine the effects of pRTA-related mutations, R342S and R554H, on pNBC-1 function, we performed functional analysis and found that both mutants had reduced transport activities compared with the wild-type pNBC-1. These results indicate that pNBC-1 is the predominant variant that mediates basolateral HCO[IMG]img002.gif" ALT="<UP><SUB>3</SUB><SUP></SUP></UP>"> uptake into duct cells in both rat and human pancreas. The loss of pNBC-1 function is predicted to have significant impact on overall ductal HCO[IMG]img002.gif" ALT="<UP><SUB>3</SUB><SUP></SUP></UP>"> secretion, which could potentially lead to pancreatic dysfunction.
Abstract: BACKGROUND & AIMS: Sphingosine 1-phosphate (S1P), a ligand for G protein-coupled endothelial differentiation gene-1 (Edg-1), Edg-3, Edg-5, Edg-6, and Edg-8, elicits a variety of responses by cells. Prominent among these is cell proliferation. S1P is abundantly stored in platelets and released upon their activation, suggesting that S1P plays a pathophysiologic role in vivo. Because the major part of injected S1P was distributed into the liver in mice, we wondered whether the liver would be one of its targets. The effects of S1P on hepatocytes, the major constituent cells in the liver, were examined. METHODS & RESULTS: Northern blot analysis revealed the expression of Edg-1 and Edg-5 messenger RNA (mRNA) in cultured rat hepatocytes, in which S1P decreased DNA synthesis induced by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) without affecting total protein synthesis. This inhibitory effect was attenuated by inactivation of small GTPase Rho with C3 exotoxin but not by inactivation of G(i) with pertussis toxin. Moreover, in the presence of JTE-013, a newly developed and specific binding antagonist for Edg-5, the inhibitory effect was also cancelled. Finally, the administration of S1P after 70% partial hepatectomy in rats reduced the peak of DNA synthesis in hepatocytes with increased Rho activity. Furthermore, Edg-5 but not Edg-1 mRNA expression was enhanced in hepatocytes 24-72 hours after partial hepatectomy, which coincides with decreasing hepatocyte proliferation. CONCLUSIONS: S1P has an antiproliferative property in rat hepatocytes by activating Rho via Edg-5. Our results raise the possibility that S1P is a negative regulator in liver regeneration.
Abstract: We investigated the role of cdc42, a Rho GTPase family member, in insulin-induced glucose transport in 3T3-L1 adipocytes. Microinjection of anti-cdc42 antibody or cdc42 siRNA led to decreased insulin-induced and constitutively active Gq (CA-Gq; Q209L)-induced GLUT4 translocation. Adenovirus-mediated expression of constitutively active cdc42 (CA-cdc42; V12) stimulated 2-deoxyglucose uptake to 56% of the maximal insulin response, and this was blocked by treatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin, or LY294002. Both insulin and CA-Gq expression caused an increase in cdc42 activity, showing that cdc42 is activated by insulin and is downstream of G[alpha]q/11 in this activation pathway. Immunoprecipitation experiments showed that insulin enhanced a direct association of cdc42 and p85, and both insulin treatment and CA-cdc42 expression stimulated PI3-kinase activity in immunoprecipitates with anti-cdc42 antibody. Furthermore, the effects of insulin, CA-Gq, and CA-cdc42 on GLUT4 translocation or 2-deoxyglucose uptake were inhibited by microinjection of anti-protein kinase C[lambda] (PKC[lambda]) antibody or overexpression of a kinase-deficient PKC[lambda] construct. In summary, activated cdc42 can mediate 1) insulin-stimulated GLUT4 translocation and 2) glucose transport in a PI3-kinase-dependent manner. 3) Insulin treatment and constitutively active Gq expression can enhance the cdc42 activity state as well as the association of cdc42 with activated PI3-kinase. 4) PKC[lambda] inhibition blocks CA-cdc42, CA-Gq, and insulin-stimulated GLUT4 translocation. Taken together, these data indicate that cdc42 can mediate insulin signaling to GLUT4 translocation and lies downstream of G[alpha]q/11 and upstream of PI3-kinase and PKC[lambda] in this stimulatory pathway.
Abstract: Objective-- Apolipoprotein E (apoE) mediates cellular cholesterol efflux and plays a crucial role in the inhibition of atherogenesis. We investigated whether there is an isoform-specific difference in its function for cholesterol efflux from cholesterol-loaded RAW264.7 cells, a murine macrophage cell line that lacks endogenous apoE expression. Methods and Results-- When human apoE was expressed in RAW264.7 cells, apoE2 reduced cellular total cholesterol (TC) and esterified cholesterol (EC) levels significantly, whereas apoE3 and apoE4 had no effect. However, treatment of cells with 4-methylumbelliferyl-7-{beta}-D-xyloside ({beta}-DX) resulted in all 3 isoforms' reducing cellular TC and EC contents significantly. We also investigated the effect of exogenously derived apoE on cholesterol efflux by utilizing the medium harvested from HeLa cells expressing apoE. ApoE2 and E3 reduced both cellular TC and EC contents significantly, whereas apoE4 did not. However, treatment of the cells with {beta}-DX resulted in all 3 exogenously derived apoE isoforms' reducing TC and EC contents significantly. The binding ability of apoE to heparan sulfate proteoglycans examined by heparinase I treatment revealed less binding ability of apoE2 compared with that of apoE3 or apoE4. Conclusions-- The present study clarified the differential cellular cholesterol-modulating effect of apoE isoforms in macrophages, which would be due to the difference in their binding to proteoglycans.
Abstract: A 52-year-old woman with early-onset diabetes mellitus, severe diabetic retinopathy, and a family history of a dominant inheritance pattern was referred to our hospital. Direct sequencing revealed a novel mutation in the HNF-4alpha gene (R244Q). The position of the mutation in the amino-acid sequence of the gene is well conserved among species and transcriptional activity of the mutant gene was significantly reduced. Therefore, a diagnosis of maturity-onset diabetes of the young (MODY)-1 was made. Genetic testing enabled early diagnosis of diabetes in the patient's 20-year-old daughter, which we consider to be important for the prevention of diabetic complications.
Abstract: Nitric oxide (NO) derived from endothelial cells is profoundly related to the maintenance of physiological vascular tone. Impairment of endothelial NO generation brought about by gene polymorphism is considered the major deterioration factor for progressive renal disease, including diabetic nephropathy. The present study aimed to elucidate the Glu298Asp polymorphism of endothelial NO synthase (eNOS) in patients with end-stage renal disease (ESRD) and its role as a predisposing factor for cardiovascular complications. Glu298Asp in exon 7 of the eNOS gene was determined by polymerase chain reaction, followed by restriction fragment length polymorphism analysis, in ESRD patients (n=185) and compared with that of unrelated healthy individuals (n=304). The occurrence of 298Asp was significantly higher in the ESRD group (P=0.0020; odds ratio [OR] 1.65; 95% confidential interval [CI]: 1.21 to 2.25). In this group, 72 patients had type 2 diabetes mellitus (DM). Although 298Asp did not reach a significant level in the non-DM ESRD subgroup, the occurrence of 298Asp was significantly higher in DM-derived ESRD patients (P=0.0010; OR 2.02; 95% CI: 1.37 to 3.07). The functional effect of the Glu298Asp was examined using Chinese hamster ovary (CHO) cells stably overexpressing either 1917G or 1917T. NO-selective electrode measurements and fluorometric nitrite assay revealed a statistically significant difference in NO production or nitrite accumulation between CHO 1917G and 1917T (P<0.01). These data indicated that Glu298Asp is the predisposing factor in ESRD, especially DM-derived ESRD. The functional difference in NO generation depending on eNOS with either glutamate or aspartate at position 298 was also confirmed in vitro.
Abstract: Paraoxonase1 (PON1) is a high-density lipoprotein (HDL)-associated protein which removes peroxidized lipids from lipoproteins. It has been proposed that apolipoprotein A-I (apoA-I) is an important determinant for its stabilization on HDL. However, little is known about its existence and activity in an apoA-I-deficient state in humans. To characterize the nature of PON1 in apoA-I deficiency, we investigated PON1 in an apoA-I-deficient patient. When serum was analyzed on fast protein liquid chromatography, PON1 protein was distributed almost exclusively on HDL despite the absence of apoA-I; on the other hand, 38.5% of PON1 protein was found in the lipoprotein-free fraction when the lipoproteins were fractionated through ultracentrifugation. The stability of PON1 activity in the patient serum was almost the same as in the normal control sera throughout incubation at 14 degrees C for 7 days. However, when the sera were incubated at 37 degrees C for 24 h, its activity declined more than those in the normal controls (19% versus 4% reduction of the initial values). Our results demonstrated that PON1 protein possesses a preferential association with HDL even in the absence of apoA-I, although apoA-I is a crucial factor for the maximal activity and stabilization of PON1. (c)2001 Elsevier Science.
Abstract: Proximal renal tubular acidosis associated with ocular abnormalities such as band keratopathy, glaucoma, and cataracts is caused by mutations in the Na+-HCO3- cotransporter (NBC-1). However, the mechanism by which NBC-1 inactivation leads to such ocular abnormalities remains to be elucidated. By immunological analysis of human and rat eyes, we demonstrate that both kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters are present in the corneal endothelium, trabecular meshwork, ciliary epithelium, and lens epithelium. In the human lens epithelial (HLE) cells, RT-PCR detected mRNAs of both kNBC-1 and pNBC-1. Although a Na+-HCO3- cotransport activity has not been detected in mammalian lens epithelia, cell pH (pHi) measurements revealed the presence of Cl--independent, electrogenic Na+-HCO3- cotransport activity in HLE cells. In addition, up to 80% of amiloride-insensitive pHi recovery from acid load in the presence of HCO3-/CO2 was inhibited by adenovirus-mediated transfer of a specific hammerhead ribozyme against NBC-1, consistent with a major role of NBC-1 in overall HCO3- transport by the lens epithelium. These results indicate that the normal transport activity of NBC-1 is indispensable not only for the maintenance of corneal and lenticular transparency but also for the regulation of aqueous humor outflow.
Abstract: Two women with multiple xanthomas, intermittent arthritis and thrombocytopenia were diagnosed as phytosterolaemia, an autosomal-recessive lipid storage disease, based on their increased serum concentrations of beta-sitosterol, campesterol and sitostanol. The gene responsible for this disease is located within a distance of 18 cM between microsatellite markers of D2S 1788 and D2S1352 at chromosome 2p21. We genotyped the patients and their family members with 16 microsatellite markers around this locus. The results from the homozygosity mapping of one family suggested that the gene was located within the distance of 12.6 cM between D2S2328 and D2S1352. We have shortened the genetic distance by 5.4 cM.
Abstract: In the present study we investigated whether an immortalized human renal proximal cell line, HKC-8, expresses a recently cloned Na+-HCO3- cotransporter (NBC-1) and, if so, which isoform (kNBC-1 from kidney or pNBC-1 from pancreas) is expressed in this cell line. Cell pH (pHi) measurements using a pH-sensitive fluorescence probe in the absence of HCO3-/CO2 revealed the presence of a Na+/H+ exchanger that required high concentrations of amiloride for full inhibition. In the presence of HCO3-/CO2 another pHi recovery process, dependent on Na+ but independent of Cl-, was identified. This process was electrogenic and was inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid (DIDS), being consistent with the Na+-HCO3- cotransporter. In addition, the pHi responses to Cl- removal were compatible with the presence of a Na+-independent Cl-/HCO3- exchanger that was also inhibited by DIDS. Reverse transcriptase polymerase chain reaction (RT-PCR) using primers designed for specific and common regions detected mRNAs of both kNBC-1 and pNBC-1 and Western blot analysis confirmed the expression of NBC-1 protein. These results indicate that HKC-8 has transport activities similar to intact proximal tubules and also suggest that both kNBC-1 and pNBC-1 may contribute to the Na+-HCO3- cotransport activity in this cell line.
Abstract: The rate of thrombin inhibition by heparin cofactor II (HCII) is facilitated by heparin or dermatan sulfate in vitro. The distributions of these glycosaminoglycans (GAGs) in vivo are not the same; heparin-like substance is rich on the surface of endothelial cells and dermatan sulfate is relatively dominant in the extravascular region. When inflammation takes place, at least two other possible existent forms of HCII, the complexed form with thrombin and the cleaved form by leukocyte elastase, are assumed to be present at relatively high concentrations in a local circumstance. We examined the interactions of HCII with the two forms of HCII on thrombin inhibition in the presence of the GAGs. By HCII in complex with thrombin or cleaved by leukocyte elastase, the affinity of HCII moiety for heparin increases and that for dermatan sulfate decreases. The two forms possibly occur at relatively high concentrations in a local pathological situation, although the heparin cofactor activity for thrombin inhibition by HCII decreases and dermatan sulfate determines the cofactor activity. These results indicate efficient thrombin inhibitory activity of HCII in the extravascular region.
Abstract: Sphingosine 1-phosphate (S-1-P), a lipid mediator shown to be a ligand for G protein-coupled receptors (AGRs), endothelial differentiation gene (EDG)1, EDG3, and AGR16/EDG5, is stored in platelets and released on their activation. Platelet consumption occurs in acute liver injury. Hepatic stellate cells (HSCs) play an important role in wound healing. Effects of S-1-P on HSCs were investigated. S-1-P enhanced proliferation of culture-activated HSCs. The mitogenic effect was pertussis toxin sensitive, mitogen-activated protein kinase dependent, and more prominent at lower cell density. S-1-P increased contraction of collagen lattices containing HSCs, irrespective of activation state, in a C3 exotoxin-sensitive manner. mRNAs of EDG1 and AGR16, but not of EDG3, were detected in HSCs. In HSC activation, EDG1 mRNA levels were downregulated, whereas AGR16 mRNA levels were unchanged. Considering that HSCs are capable of production of extracellular matrices and modulation of blood flow in sinusoids, our results suggest that S-1-P may play a role in wound healing process in the liver.
Abstract: AIMS/METHODS: To investigate whether smoking affects the serum level of leptin, 708 male workers aged 25-65 years old were cross-sectionally surveyed. RESULTS: Multiple regression analysis indicated that among the various parameters examined, the level of leptin was positively associated with the body mass index and the levels of insulin, total cholesterol and uric acid, and was inversely associated with physical activity and the level of creatinine. The partial correlation coefficient of leptin was highest against the body mass index (r = 0.40), followed by insulin (0.29) and physical activity (-0.14), after adjustment for other leptin-related variables. However, no association was observed between the level of leptin and smoking (0.05), alcohol consumption (0.09) or age (0.09). CONCLUSIONS: The findings suggest that among life-style habits, physical activity, but not smoking or alcohol consumption, significantly affects the serum level of leptin in Japanese men. Copyright 2001 S. Karger AG, Basel
Abstract: A total of 5,174 Japanese men were included in a cross-sectional study to examine the relationship between the glycated hemoglobin (HbA1C) level and the prevalence of proteinuria as determined using a reagent strip. The prevalence of proteinuria rose significantly at HbA1C levels above 5.9%, whereas no relationship was observed at HbA1C levels below 5.9%. Multiple logistic regression analysis showed that blood pressure and a family history of diabetes were independent factors associated with proteinuria in subjects with a HbA1C below 5.9% who were not under medication for diabetes. In contrast, HbA1C, obesity and smoking were associated with proteinuria in subjects who were under medication for diabetes and/or have a HbA1C above 5.9%. These findings suggest that maintaining a HbA1C level below 5.9%, non-smoking and a standard body weight may reduce the prevalence of proteinuria in Japanese men. Healthy life-style and standard body weight are especially important for subjects with a family history of diabetes.
Abstract: The effect of peroxisome proliferator-activated receptor (PPAR) gamma activators, thiazolidinediones, on plasminogen activator type 1 (PAI-1) was examined in cultured human umbilical vein endothelial cells (HUVEC). Tumor necrosis factor alpha (TNF-alpha) enhanced PAI-1 secretion and mRNA expression by approximately 2-fold. The thiazolidinediones, troglitazone and pioglitazone, decreased basal and TNF-alpha-stimulated PAI-1 secretion and mRNA expression in HUVEC in a dose-dependent fashion. PPARgamma mRNA in HUVEC could be detected by reverse transcriptase-polymerase chain reaction using specific primers. These results suggest that PPARgamma may regulate PAI-1 expression in HUVEC and that thiazolidinediones have a therapeutic potential for improving endothelial dysfunction observed in insulin resistance.
Abstract: We examined the effect of troglitazone on immunoreactive endothelin-1 (ET-1) secretion from cultured bovine vascular endothelial cells (bVECs). Insulin (10(-9)-10(-7) M) stimulated ET-1 secretion in a dose-dependent fashion without any kinetic change. Troglitazone (1-20 microM) dose-dependently inhibited both spontaneous and insulin-stimulated ET-1 secretion. This inhibitory effect of troglitazone was associated with reduced ET-1 mRNA levels. Addition of indomethacin (100 microM) or Nw-nitro-l-arginine methyl ester (1 mM) and downregulation of protein kinase C by prolonged pretreatment of the cells with a phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, did not affect the inhibitory effect of troglitazone at concentrations up to 10 microM. Troglitazone did not change the intracellular Ca2+ concentration stimulated by angiotensin II (10 microM). Other PPARgamma ligands, pioglitazone (1-10 microM) and 15-deoxy-delta 12, 14-prostaglandin J2 (1-10 microM), but not a PPARalpha ligand, bezafibrate (1-10 microM), dose-dependently suppressed spontaneous ET-1 secretion from bVECs. These results, taken together, suggest that troglitazone inhibits ET-1 mRNA expression and secretion in bVECs possibly through activation of PPARgamma. This inhibition may contribute to the hypotensive effect of troglitazone in insulin-resistant subjects.
Abstract: Lysophosphatidic acid (LPA) is a growth factor-like mediator for fibroblasts or smooth muscle cells produced and released by activated platelets. Platelet activation occurs with hepatic necrosis and subsequent liver regeneration and fibrosis. In the fibrosis, hepatic stellate cells proliferate with phenotypic transformation to myofibroblasts. Thus, effects of LPA on proliferation of hepatocytes and stellate cells were investigated. In cultured rat stellate cells, LPA increased DNA synthesis with enhanced MAP kinase activity. Pertussis toxin (PTX) attenuated this mitogenic action. In contrast, LPA decreased DNA synthesis by cultured rat hepatocytes induced by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) without affecting protein synthesis. Enhanced MAP kinase activity by HGF or EGF was not changed by LPA. This anti-mitogenic action was attenuated by PTX. TGFbeta level in the medium was less than the level effective for inhibiting the DNA synthesis in the presence of LPA. Our results suggest that LPA might affect proliferation of hepatocytes and stellate cells in liver diseases complicating platelet activation.
Abstract: We evaluated the characteristics of the protein kinase C (PKC)-independent mechanism for ATP release in platelet-rich plasma. When ADP (10 microM) and U46619 (1 microM) were both added as agonists, a significant release was observed immediately after stimulation. The PKC inhibitor, Ro-31-7549 (10 microM), or a cyclooxygenase inhibitor, aspirin (400 microM) or indomethacin (20 microM), partially inhibited ATP release with little effect on platelet aggregation. The ATP release observed in the presence of Ro-31-7549 was abolished by a cyclooxygenase inhibitor or by preventing aggregation without stirring. In the nonstirred condition, thromboxane B2 formation was reduced by 93%. When sodium arachidonate (1 mM) rather than U46619 was used with ADP, ATP release in the presence of Ro-31-7549 was abolished by stopping the stirring with no effect on thromboxane B2 formation. In contrast, ADP/U46619-induced ATP release observed in the presence of aspirin was only partially inhibited when the stirring was stopped. This release was also inhibited dose-dependently by Ro-31-7549 at concentrations between 1 and 10 microM. These results suggest that PKC-independent ATP-release in this system requires aggregation and is inhibited by a cyclooxygenase inhibitor, while PKC-dependent exocytosis is insensitive to aggregation and a cyclooxygenase inhibitor.
Abstract: Advanced glycation end products of bovine serum albumin (AGEs-BSA) exhibited biphasic effects on the proliferation of cultured rabbit vascular smooth muscle cells (VSMCs) in terms of [3H]thymidine incorporation and cell number count; a stimulatory effect was observed at 1-10 micrograms/ml and an inhibitory effect at more than 20 micrograms/ml, while it inhibited [3H]thymidine incorporation even at 1-10 micrograms/ml in cultured bovine vascular endothelial cells (VECs). Transient activation of p42 mitogen-activated protein kinase (MAPK) with a peak at around 5 min and a subsequent sustained phase was induced by AGEs-BSA in VSMCs, but not in VECs. The dependence of MAPK activation on AGEs-BSA dose was correlated with that of VSMCs proliferation.
Abstract: In a previous study, we characterized prostanoid and thrombin receptors expressed on a megakaryoblastic cell line, MEG-01s (Blood 78, 2328-2336, 1991). In this study, we examines the mechanism of cross-talk between intracellular Ca2+ ([Ca2+]i) and cAMP signalings through prostanoid and thrombin receptors. Addition of a thromboxane (TX)A2 mimetic (U46619 or STA2) or thrombin stimulated the formation of inositol phosphates and dose-dependently augmented a prostaglandin (PG)I2 mimetic (iloprost)- or forskolin-induced cAMP formation. 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin, to lesser extent, also augmented iloprost-induced cAMP formation. The enhancing effect of U46619 or TPA on cAMP formation was inhibited by prolonged pretreatment of the cells with TPA (2.5 microM, 24 h), but not with calmodulin-antagonists; W-7, W-5, or KN-62. The elevation of [Ca2+]i induced by thrombin, STA2 or PGE2 was significantly suppressed by pretreatment of the cells with TPA (100 nM) as well as cAMP mimetics such as dibutyryl cAMP (5 mM), forskolin (5 microM) and iloprost (1 microM). These results suggest the key role of PKC on the cross-talk between [Ca2+]i and cAMP signalings through prostanoid and thrombin receptors; PKC, which is activated with TXA2 or thrombin, concomitantly suppress further [Ca2+]i elevation and enhances the PGI2 receptor-mediated cAMP formation, which, in turn, suppress [Ca2+]i elevation.
Abstract: Among major eicosanoids and their analogs, prostaglandin (PG) F2 alpha > PGD2 > PGE1 > or = PGE2 > iloprost, a stable agonist of PGI2, dose-dependently stimulated DNA synthesis in quiescent NIH-3T3 cells. PGF2 alpha, PGD2, and PGE2, in that order, formed inositol phosphates and elevated intracellular Ca2+ ([Ca2+]i) but did not form cAMP nor inhibit forskolin-induced cAMP formation. Iloprost, PGI2, and PGE1 induced cAMP formation dose dependently with an ED50 of around 10(-7) M, and PGE2 at more than 10(-6) M did it. [3H]PGF2 alpha and [3H]PGD2 bindings membranes from NIH-3T3 cells were displaced in the order of PGF2 alpha > PGD2 > or = PGE2, while [3H]PGE2 binding was displaced by PGE2 > PGD2 > or = PGF2 alpha. Expression of mRNA encoding EP1 and EP4 (EP2) subtypes could be detected by reverse transcription- polymerase chain reaction using primers specific for EP1 and EP4 (EP2) cDNAs, but not that of EP3 subtype mRNA. The dose dependence of cAMP formation on iloprost and PGI2 and that of [Ca2+]i elevation on PGF2 alpha, D2, and E2 were similar to that of [3H]thymidine incorporation on the corresponding agonists. Fluprostenol (1 microM), a PGF2 alpha receptor agonist > 17-phenyl-trinor-PGE2 (1 microM), an EP1 receptor agonist stimulated [3H]thymidine incorporation, but an EP3 receptor agonist, ONO-AP-324 nor an EP4 (EP2) receptor agonist, 11-deoxy-PGE1 (1 microM) did not. Iloprost, dibutyryl cAMP, forskolin, or cholera toxin, when applied alone, enhanced [3H]thymidine incorporation, while they inhibited [3H]thymidine incorporation induced by submaximal concentrations of PGF2 alpha or epidermal growth factor (EGF), when applied within 12 hr after agonist stimulation. These results suggest that the proliferation of NIH-3T3 cells is stimulated by PGs via the PGF2 alpha receptor, EP1 subtype of PGE receptor, and the PGI2/PGE1 receptor through [Ca2+]i- and cAMP-dependent pathways, and that cAMP pathway negatively cross-talks with [Ca2+]i-or receptor tyrosine kinase-mediated DNA synthesis in a cell cycle-dependent manner.
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