Reduced expression of SIRT1 is associated with diminished glucose-induced insulin secretion in islets from calorie-restricted rats

Alterations in food intake such as caloric restriction modulate the expression of SIRT1 and SIRT4 proteins that are involved in pancreatic β-cell function. Here, we search for a possible relationship between insulin secretion and the expression of SIRT1, SIRT4, PKC and PKA in islets from adult rats submitted to CR for 21 days. Rats were fed with an isocaloric diet (CTL) or received 60% (CR) of the food ingested by CTL. The dose-response curve of insulin secretion to glucose was shifted to the right in the CR compared with CTL islets (EC₅₀ of 15.1±0.17 and 10.5±0.11 mmol/L glucose). Insulin release by the depolarizing agents arginine and KCl was reduced in CR compared with CTL islets. Total islet insulin content and glucose oxidation were also reduced in CR islets. Leucine-stimulated secretion was similar in both groups, slightly reduced in CR islets stimulated by leucine plus glutamine but higher in CR islets stimulated by ketoisocaproate (KIC). Insulin secretion was also higher in CR islets stimulated by carbachol, compared with CTL islets. No differences in the rise of cytosolic Ca²⁺ concentrations stimulated by either glucose or KCl were observed between groups of islets. Finally, SIRT1, but not SIRT4, protein expression was lower in CR compared with CTL islets, whereas no differences in the expression of PKC and PKA proteins were observed. In conclusion, the lower insulin secretion in islets from CR rats was, at least in part, due to an imbalance between the expression of SIRT1 and SIRT4.     

ENPP1 affects insulin action and secretion: evidences from in vitro studies

The aim of this study was to deeper investigate the mechanisms through which ENPP1, a negative modulator of insulin receptor (IR) activation, plays a role on insulin signaling, insulin secretion and eventually glucose metabolism. ENPP1 cDNA (carrying either K121 or Q121 variant) was transfected in HepG2 liver-, L6 skeletal muscle- and INS1E beta-cells. Insulin-induced IR-autophosphorylation (HepG2, L6, INS1E), Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) phosphorylation (HepG2, L6), PEPCK mRNA levels (HepG2) and 2-deoxy-D-glucose uptake (L6) was studied. GLUT 4 mRNA (L6), insulin secretion and caspase-3 activation (INS1E) were also investigated. Insulin-induced IR-autophosphorylation was decreased in HepG2-K, L6-K, INS1E-K (20%, 52% and 11% reduction vs. untransfected cells) and twice as much in HepG2-Q, L6-Q, INS1E-Q (44%, 92% and 30%). Similar data were obtained with Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) in HepG2 and L6. Insulin-induced reduction of PEPCK mRNA was progressively lower in untransfected, HepG2-K and HepG2-Q cells (65%, 54%, 23%). Insulin-induced glucose uptake in untransfected L6 (60% increase over basal), was totally abolished in L6-K and L6-Q cells. GLUT 4 mRNA was slightly reduced in L6-K and twice as much in L6-Q (13% and 25% reduction vs. untransfected cells). Glucose-induced insulin secretion was 60% reduced in INS1E-K and almost abolished in INS1E-Q. Serum deficiency activated caspase-3 by two, three and four folds in untransfected INS1E, INS1E-K and INS1E-Q. Glyburide-induced insulin secretion was reduced by 50% in isolated human islets from homozygous QQ donors as compared to those from KK and KQ individuals. Our data clearly indicate that ENPP1, especially when the Q121 variant is operating, affects insulin signaling and glucose metabolism in skeletal muscle- and liver-cells and both function and survival of insulin secreting beta-cells, thus representing a strong pathogenic factor predisposing to insulin resistance, defective insulin secretion and glucose metabolism abnormalities.     

Common variants in the type 2 diabetes KCNQ1 gene are associated with impairments in insulin secretion during hyperglycaemic glucose clamp

Background

Genome-wide association studies in Japanese populations recently identified common variants in the KCNQ1 gene to be associated with type 2 diabetes. We examined the association of these variants within KCNQ1 with type 2 diabetes in a Dutch population, investigated their effects on insulin secretion and metabolic traits and on the risk of developing complications in type 2 diabetes patients.

Methodology

The KCNQ1 variants rs151290, rs2237892, and rs2237895 were genotyped in a total of 4620 type 2 diabetes patients and 5285 healthy controls from the Netherlands. Data on macrovascular complications, nephropathy and retinopathy were available in a subset of diabetic patients. Association between genotype and insulin secretion/action was assessed in the additional sample of 335 individuals who underwent a hyperglycaemic clamp.

Principal Findings

We found that all the genotyped KCNQ1 variants were significantly associated with type 2 diabetes in our Dutch population, and the association of rs151290 was the strongest (OR 1.20, 95% CI 1.07–1.35, p = 0.002). The risk C-allele of rs151290 was nominally associated with reduced first-phase glucose-stimulated insulin secretion, while the non-risk T-allele of rs2237892 was significantly correlated with increased second-phase glucose-stimulated insulin secretion (p = 0.025 and 0.0016, respectively). In addition, the risk C-allele of rs2237892 was associated with higher LDL and total cholesterol levels (p = 0.015 and 0.003, respectively). We found no evidence for an association of KCNQ1 with diabetic complications.

Conclusions

Common variants in the KCNQ1 gene are associated with type 2 diabetes in a Dutch population, which can be explained at least in part by an effect on insulin secretion. Furthermore, our data suggest that KCNQ1 is also associated with lipid metabolism.     

Patterns of deletions of the dystrophin gene in different European populations

The distribution of deletion breakpoints in the dystrophin gene was studied in a series of subjects belonging to different European populations. The data, obtained from the literature or directly from the present study, refer to population samples from France, Finland, Germany, Italy, Netherland, Switzerland, and U.K. (England, Scotland, Wales). In total, 1516 breakpoints were assigned to different introns, 359 in the region encompassing the first 40 exons and 1157 (76%) in the distal part of the gene. Intron 7 appears to be equally involved as the starting or ending breakpoint, whereas intron 44 is involved mostly as a starting breakpoint. Breakpoint distribution by intron seems to differ in different populations, reaching statistical significance in the case of introns 44, 49, and 53. This finding suggests that some intronic sequences might contain preferential breakpoints that might vary in different populations, possibly as a consequence of genetic drift.     

Genetic variants in the RELN gene are associated with otosclerosis in multiple European populations

Otosclerosis is a common form of hearing loss characterized by abnormal bone remodeling in the otic capsule. It is considered a complex disease caused by both genetic and environmental factors. In a previous study, we identified a region on chr7q22.1 located in the RELN gene that is associated with otosclerosis in Belgian–Dutch and French populations. Evidence for allelic heterogeneity was found in this chromosomal region in the form of two independent signals. To confirm this finding, we have completed a replication study that includes four additional populations from Europe (1,141 total samples). Several SNPs in this region replicated in these populations separately. While the power to detect significant association in each population is small, when all four populations are combined, six of seven SNPs replicate and show an effect in the same direction as in the previous populations. We also confirmed the presence of allelic heterogeneity in this region. These data further implicate RELN in the pathogenesis of otosclerosis. Functional research is warranted to determine the pathways through which RELN acts in the pathogenesis of otosclerosis.     

Variant in sulfonylurea receptor-1 gene is associated with high insulin concentrations in non-diabetic Mexican Americans: SUR-1 gene variant and hyperinsulinemia

The high-affinity sulfonylurea receptor (SUR1) gene regulates insulin secretion and may play a role in type 2 diabetes. A silent variant in exon 31 of SUR1 (AGG→AGA) was detected by single-strand conformational polymorphism and genotypes were determined for 396 Mexican American subjects (289 non-diabetic). The normal and mutant alleles were designated G and A, respectively. Among non-diabetics, those with the AA genotype had higher fasting insulin values than those with the AG and GG genotypes (113.4 pmol/l for AA vs 82.8 pmol/l for AG/GG, P=0.043). Similar results were observed for 2-h insulin (849.6 pmοl/l for AA vs 498.6 pmol/l for AG/GG, P=0.0003) and for the proinsulin to specific insulin ratio (0.068 for AA vs 0.056 for AG/GG, P=0.030). Specific insulin levels also differed significantly across the three genotypic classes (P=0.021). No differences in fasting glucose, body mass index, or waist circumference according to genotype were noted. Two-hour glucose was modestly higher in individuals with the AA genotype. Since we have previously reported linkage between SUR1 and hyperglycemia, the present association between a SUR1 variant and hyperinsulinemia in normal individuals from a high diabetes risk ethnic group raises the possibility of primary insulin hypersecretion as an antecedent of type 2 diabetes in at least some individuals from this population. Received: 3 April 1998 / Accepted: 5 June 1998     

LRRK2 A419V is not associated with Parkinson's disease in different Chinese populations

It has been suggested that a common LRRK2 polymorphic variant (A419V (rs34594498 C >T)) may be a risk factor among Asians (especially in Taiwan). In this study, we examined this variant in a larger and independent Taiwan cohort. We found the frequency of the variant (A419V) to be very rare in our Taiwan PD and controls (?0.6%). Further studies were conducted in two other Chinese populations (Singapore and China), comprising of a total of 3004 subjects including 1517 PD patients and 1487 control subjects. However, our multi-center Chinese study revealed that the frequency of the variant was rare (?0.4%) and was not associated with risk of PD, suggesting that the variant is not a major risk factor for PD among Chinese, at least in our study population.     

Molecular and histological evaluation of pancreata from patients with a mitochondrial gene mutation associated with impaired insulin secretion.

A mutation in mitochondrial DNA, which was originally identified in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), can be associated with a subtype of diabetes mellitus. To determine the molecular and histological basis of impaired insulin secretion in the subjects with this mutation, we studied autopsy pancreata specimens from eight subjects diagnosed as having MELAS. The 3243 bp mutation was identified in seven out of eight pancreata examined. Immunohistochemical studies demonstrated a reduction in total islet mass, and in the numbers of both B and A cells. No evidence of insulitis or apoptosis was found. These data suggested that the 3243 bp mutation may cause the reduction of islet cells, mainly through mechanisms other than autoimmune destruction.     

Inhibition of apoB secretion from HepG2 cells by insulin is amplified by naringenin, independent of the insulin receptor

Hepatic overproduction of apolipoprotein B (apoB)-containing lipoproteins is characteristic of the dyslipidemia associated with insulin resistance. Recently, we demonstrated that the flavonoid naringenin, like insulin, decreased apoB secretion from HepG2 cells by activation of both the phosphoinositide-3-kinase (PI3-K) pathway and the mitogen-activated protein kinase/extracellular-regulated kinase (MAPK(erk)) pathway. In the present study, we determined whether naringenin-induced signaling required the insulin receptor (IR) and sensitized the cell to the effects of insulin, and whether the kinetics of apoB assembly and secretion in cells exposed to naringenin were similar to those of insulin. Immunoblot analysis revealed that insulin stimulated maximal phosphorylation of IR and IR substrate-1 after 10 min, whereas naringenin did not affect either at any time point up to 60 min. The combination of naringenin and submaximal concentrations of insulin potentiated extracellular-regulated kinase 1/2 activation and enhanced upregulation of the LDL receptor, downregulation of microsomal triglyceride transfer protein expression, and inhibition of apoB-100 secretion. Multicompartmental modeling of apoB pulse-chase studies revealed that attenuation of secreted radiolabeled apoB in naringenin- or insulin-treated cells was similar under lipoprotein-deficient or oleate-stimulated conditions. Naringenin and insulin both stimulated intracellular apoB degradation via a kinetically defined rapid pathway. Therefore, naringenin, like insulin, inhibits apoB secretion through activation of both PI3-K and MAPK(erk) signaling, resulting in similar kinetics of apoB secretion. However, the mechanism for naringenin-induced signaling is independent of the IR. Naringenin represents a possible strategy for reduction of hepatic apoB secretion, particularly in the setting of insulin resistance.     

Protor-1 is required for efficient mTORC2-mediated activation of SGK1 in the kidney

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth and is a key target for therapeutic intervention in cancer. Two complexes of mTOR have been identified: complex 1 (mTORC1), consisting of mTOR, Raptor (regulatory associated protein of mTOR) and mLST8 (mammalian lethal with SEC13 protein 8) and complex 2 (mTORC2) consisting of mTOR, Rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated protein kinase-interacting protein 1), mLST8 and Protor-1 or Protor-2. Both complexes phosphorylate the hydrophobic motifs of AGC kinase family members: mTORC1 phosphorylates S6K (S6 kinase), whereas mTORC2 regulates phosphorylation of Akt, PKCα (protein kinase Cα) and SGK1 (serum- and glucocorticoid-induced protein kinase 1). To investigate the roles of the Protor isoforms, we generated single as well as double Protor-1- and Protor-2-knockout mice and studied how activation of known mTORC2 substrates was affected. We observed that loss of Protor-1 and/or Protor-2 did not affect the expression of the other mTORC2 components, nor their ability to assemble into an active complex. Moreover, Protor knockout mice display no defects in the phosphorylation of Akt and PKCα at their hydrophobic or turn motifs. Strikingly, we observed that Protor-1 knockout mice displayed markedly reduced hydrophobic motif phosphorylation of SGK1 and its physiological substrate NDRG1 (N-Myc downregulated gene 1) in the kidney. Taken together, these results suggest that Protor-1 may play a role in enabling mTORC2 to efficiently activate SGK1, at least in the kidney.     

Glucose-stimulated insulin secretion of insulinoma INS-1E cells is associated with elevation of both respiration and mitochondrial membrane potential

Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represents a plausible mechanism controlling the glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. Although specific bioenergetics might be involved, parallel studies of cell respiration and mitochondrial membrane potential (ΔΨ_m) during GSIS are lacking. Using high resolution respirometry and parallel ΔΨ_m monitoring by two distinct fluorescence probes we have quantified bioenergetics in rat insulinoma INS-1E cells representing a suitable model to study in vitro insulin secretion. Upon glucose addition to glucose-depleted cells we demonstrated a simultaneous increase in respiration and ΔΨ_m during GSIS and showed that the endogenous state 3/state 4 respiratory ratio hyperbolically increased with glucose, approaching the maximum oxidative phosphorylation rate at maximum GSIS. Attempting to assess the basis of the “toxic” effect of fatty acids on insulin secretion, GSIS was studied after linoleic acid addition, which diminished respiration increase, ΔΨ_m jump, and magnitude of insulin release, and reduced state 3/state 4 dependencies on glucose. Its effects were due to protonophoric function, i.e. uncoupling, since without glucose, linoleic acid accelerated both state 3 and state 4 respiration by similar extent. In turn, state 3 respiration increased marginally with linoleic acid at 10–20 mM glucose. We conclude that upon glucose addition in physiological range, the INS-1E cells are able to regulate the oxidative phosphorylation rate from nearly zero to maximum and that the impairment of GSIS by linoleic acid is caused by mitochondrial uncoupling. These findings may be relevant to the pathogenesis of type 2 diabetes.     

The CAPN10 gene is associated with insulin resistance phenotypes in the Spanish population

Cardiovascular disease is the leading cause of morbidity and mortality in the industrialized world. Familial aggregation of cardiovascular risk factors is a frequent finding, but genetic factors affecting its presentation are still poorly understood. The calpain 10 gene (CAPN10) has been associated with type 2 diabetes (T2DM), a complex metabolic disorder with increased risk of cardiovascular disease. Moreover, the CAPN10 gene has been associated with the presence of metabolic syndrome (MS) in T2DM and in polycystic ovary syndrome (PCOS). In this work, we have analysed whether the polymorphisms UCSNP44, -43, -19 and -63 are related to several cardiovascular risk factors in the context of MS. Molecular analysis of CAPN10 gene was performed in 899 individuals randomly chosen from a cross-sectional population-based epidemiological survey. We have found that CAPN10 gene in our population is mainly associated with two indicators of the presence of insulin resistance: glucose levels two hours after a 75-g oral glucose tolerance test (OGTT) and HOMA values, although cholesterol levels and blood pressure values are also influenced by CAPN10 variants. In addition, the 1221/1121 haplogenotype is under-represented in individuals that fulfil the International Diabetes Federation (IDF) diagnostic criteria for MS. Our results suggest that CAPN10 gene is associated with insulin resistance phenotypes in the Spanish population.     

Impaired glucose-stimulated insulin secretion in the GK rat is associated with abnormalities in islet nitric oxide production

We investigated implications of nitric oxide (NO) derived from islet neuronal constitutive NO synthase (ncNOS) and inducible NOS (iNOS) on insulin secretory mechanisms in the mildly diabetic GK rat. Islets from GK rats and Wistar controls were analysed for ncNOS and iNOS by HPLC, immunoblotting and immunocytochemistry in relation to insulin secretion stimulated by glucose or l-arginine in vitro and in vivo. No obvious difference in ncNOS fluorescence in GK vs control islets was seen but freshly isolated GK islets displayed a marked iNOS expression and activity. After incubation at low glucose GK islets showed an abnormal increase in both iNOS and ncNOS activities. At high glucose the impaired glucose-stimulated insulin release was associated with an increased iNOS expression and activity and NOS inhibition dose-dependently amplified insulin secretion in both GK and control islets. This effect by NOS inhibition was also evident in depolarized islets at low glucose, where forskolin had a further amplifying effect in GK but not in control islets. NOS inhibition increased basal insulin release in perfused GK pancreata and amplified insulin release after glucose stimulation in both GK and control pancreata, almost abrogating the nadir separating first and second phase in controls. A defective insulin response to l-arginine was seen in GK rats in vitro and in vivo, being partially restored by NOS inhibition. The results suggest that increased islet NOS activities might contribute to the defective insulin response to glucose and l-arginine in the GK rat. Excessive iNOS expression and activity might be deleterious for the beta-cells over time.     

The uncoupling protein 1 gene, UCP1, is expressed in mammalian islet cells and associated with acute insulin response to glucose in African American families from the IRAS Family Study

Background

Variants of uncoupling protein genes UCP1 and UCP2 have been associated with a range of traits. We wished to evaluate contributions of known UCP1 and UCP2 variants to metabolic traits in the Insulin Resistance and Atherosclerosis (IRAS) Family Study.

Methods

We genotyped five promoter or coding single nucleotide polymorphisms (SNPs) in 239 African American (AA) participants and 583 Hispanic participants from San Antonio (SA) and San Luis Valley. Generalized estimating equations using a sandwich estimator of the variance and exchangeable correlation to account for familial correlation were computed for the test of genotypic association, and dominant, additive and recessive models. Tests were adjusted for age, gender and BMI (glucose homeostasis and lipid traits), or age and gender (obesity traits), and empirical P-values estimated using a gene dropping approach.

Results

UCP1 A-3826G was associated with AIR_g in AA (P = 0.006) and approached significance in Hispanic families (P = 0.054); and with HDL-C levels in SA families (P = 0.0004). Although UCP1 expression is reported to be restricted to adipose tissue, RT-PCR indicated that UCP1 is expressed in human pancreas and MIN-6 cells, and immunohistochemistry demonstrated co-localization of UCP1 protein with insulin in human islets. UCP2 A55V was associated with waist circumference (P = 0.045) in AA, and BMI in SA (P = 0.018); and UCP2 G-866A with waist-to-hip ratio in AA (P = 0.016).

Conclusion

This study suggests a functional variant of UCP1 contributes to the variance of AIR_g in an AA population; the plausibility of this unexpected association is supported by the novel finding that UCP1 is expressed in islets.     

Increased intrahepatic triglyceride is associated with peripheral insulin resistance: in vivo MR imaging and spectroscopy studies

Recent studies have indicated that the mass/content of intramyocellular lipid (IMCL), intrahepatic triglyceride (IHTG), visceral fat (VF), and even deep abdominal subcutaneous fat (SF) may all be correlated with insulin resistance. Since simultaneous measurements of these parameters have not been reported, the relative strength of their associations with insulin action is not known. Therefore, the goals of this study were 1) to simultaneously measure IMCL, IHTG, VF, and abdominal SF in the same nondiabetic individuals using noninvasive (1)H-magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) and 2) to examine how these fat stores are correlated with systemic insulin sensitivity as measured by whole body glucose disposal (R(d)) during euglycemic-hyperinsulinemic clamp studies. Positive correlations were observed among IMCL, IHTG, and VF. There were significant inverse correlations between whole body R(d) and both IMCL and VF. Notably, there was a particularly tight inverse correlation between IHTG and whole body R(d) (r = -0.86, P < 0.001), consistent with an association between liver fat and peripheral insulin sensitivity. This novel finding suggests that hepatic triglyceride accumulation has important systemic consequences that may adversely affect insulin sensitivity in other tissues.