Genetic variants in the FTO gene have been associated with obesity and type 2 diabetes in European populations. We aimed to test the role of FTO genetic variants in obesity and type 2 diabetes in the Chinese population.
We genotyped 19 single nucleotide polymorphisms (SNPs) spanning from the 3' end of the neighboring RPGRIP1L gene to the 5' flanking region of the FTO gene. We analyzed their associations with obesity (638 cases and 1,610 controls), type 2 diabetes (759 cases and 784 controls), and obesity-related traits in non-diabetic subjects.
Among the 19 SNPs, the rs9939609 A allele was strongly associated with obesity (p= 7.0x10–4) and body mass index (BMI) (p= 0.0024) in the Chinese population. The odds ratio for obesity was 2.60 (95% CI: 1.24-5.46; p=0.011) for the AA genotype and 1.32 (95% CI: 1.05-1.66; p= 0.018) for the AT genotype as compared to the TT genotype. Each additional copy of the rs9936609 A allele was associated with a BMI increase of ~ 0.37 kg/m2. The rs9939609 A allele was substantially less common in the Chinese population than in the European population (12.6% vs. 45%). We did not find significant associations of the 19 SNPs with type 2 diabetes or other obesity-related traits.
Genetic variation in the FTO gene is strongly associated with obesity and BMI in the Chinese population. The risk variant is less common in the Chinese population but its effect size on BMI is comparable to that in the European population.
Recent studies identified accumulation of reactive oxygen species (ROS) as a common pathway causing insulin resistance. However, whether and how the reduction of ROS levels improve insulin resistance remains to be elucidated. The present study was designed to define this mechanism.
We investigated the effect of overexpression of superoxide dismutase 1 (SOD1) in liver of obese diabetic model mice, db/db mice by adenoviral injection.
Db/db mice had high ROS levels in liver. Overexpression of SOD1 in liver of db/db mice reduced hepatic ROS and blood glucose level. These changes were accompanied by improvement in insulin resistance and reduction of hepatic gene expression of phosphoenol-pyruvate carboxykinase (PEPCK) and peroxisome proliferator-activated receptor gamma co-activator-1 (PGC-1), which is the main regulator of gluconeogenic genes. The inhibition of hepatic insulin resistance was accompanied by attenuation of phosphorylation of cAMP-responsive element-binding protein (CREB), which is a main regulator of PGC-1 expression, and attenuation of JNK phosphorylation. Simultaneously, overexpression of SOD1 in db/db mice enhanced the inactivation of Foxo1 that is another regulator of PGC-1 expression without the changes of insulin-induced Akt phosphorylation in liver. In hepatocyte cell lines, ROS induced phosphorylation of JNK and CREB, and the latter, together with PGC-1 expression, were inhibited by a JNK inhibitor.
Our results indicate that the reduction of ROS is a potential therapeutic target of liver insulin resistance at least partly by the reduced expression of PGC-1.
Accumulation of intracellular lipid droplets (LD) in non-adipose tissues is recognized as a strong prognostic factor for the development of insulin resistance in obesity. LD are coated with PAT proteins that are thought to regulate LD turnover by modulating lipolysis. Our hypothesis is that PAT proteins modulate LD metabolism and therefore insulin resistance.
We used a cell culture model (murine AML12 loaded with oleic acid) and siRNA to directly assess the impact of PAT proteins on LD accumulation, lipid metabolism and insulin action. PAT proteins associated with excess fat deposited in livers of DIO mice were also measured.
Cells lacking PAT proteins exhibited a dramatic increase in LD size and a decrease in LD number. Further, the lipolytic rate increased by ~2-2.5- fold in association with increased adipose triglyceride lipase (ATGL) at the LD surface. Down-regulation of PAT proteins also produced insulin resistance as indicated by decreased insulin stimulation of Akt phosphorylation (p<0.001). PDK-1 and PI3 kinase decreased; IRS-1 307 phosphorylation increased. Increased lipid in DIO mice livers was accompanied by changes in PAT composition, but also increased ATGL, suggesting a relative PAT deficiency.
These data establish an important role for PAT proteins as surfactant at the LD surface packaging lipids in smaller units and restricting access of lipases, preventing insulin resistance. We suggest that a deficiency of PAT proteins relative to the quantity of ectopic fat could contribute to cellular dysfunction in obesity and T2D.
Glucagon-like peptide 1 (GLP-1) promotes glucose homeostasis through regulation of islet hormone secretion, as well as hepatic and gastric function. Because GLP-1 is also synthesized in the brain, where it regulates food intake, we hypothesized that the CNS GLP-1 system regulates glucose tolerance as well.
We found that central, but not peripheral, administration of low doses of a GLP-1 receptor (GLP-1r) antagonist caused relative hyperglycemia during a glucose tolerance test suggesting that activation of CNS GLP-1r regulates key processes involved in the maintenance of glucose homeostasis. Central administration of GLP-1 augmented glucose-stimulated insulin secretion, and direct administration of GLP-1 into the arcuate, but not the paraventricular nucleus of the hypothalamus reduced hepatic glucose production. Consistent with a role for GLP-1r in the arcuate, GLP-1r mRNA was found to be expressed in 68.1% of arcuate neurons that expressed POMC mRNA but was not significantly co-expressed with NPY.
These data suggest that the arcuate GLP-1r are a key component of the GLP-1 system to improve glucose homeostasis by both regulating insulin secretion and glucose production.
Pathogenic mechanisms underlying diabetes-induced retinal dysfunction are not fully understood. The aim of the present study was to show the relationship of the renin-angiotensin system (RAS) with the synaptic vesicle protein synaptophysin and neuronal activity in the diabetic retina.
C57BL/6 mice with streptozotocin-induced diabetes were treated with the angiotensin II type 1 receptor (AT1R) blocker telimsartan or valsartan, and retinal function was analyzed by electroretinography (ERG). Retinal production of the RAS components and phosphorylation of ERK (extracellular-signal regulated kinase) were examined by immunoblotting. Retinal mRNA and protein levels of synaptophysin were measured by quantitative RT-PCR and immunoblot analyses, respectively. In vitro, synaptophysin levels were also evaluated using angiotensin II-stimulated PC12D neuronal cells cultured with or without the inhibition of ERK signaling or ubiquitin-proteasome system (UPS).
Induction of diabetes led to a significant increase in retinal production of angiotensin II and AT1R together with ERK activation in the downstream of AT1R. AT1R blockade significantly reversed diabetes-induced ERG changes and reduction of synaptophysin protein, but not mRNA, levels in the diabetic retina. In agreement with the AT1R-mediated post-transcriptional downregulation of synaptophysin in vivo, in vitro application of angiotensin II to PC12D neuronal cells caused the UPS-mediated degradation of synaptophysin protein via AT1R, which proved to be induced by ERK activation.
These data indicate the first molecular evidence of the RAS-induced synaptophysin degradation and neuronal dysfunction in the diabetic retina, suggesting the possibility of AT1R blockade as a novel, neuroprotective treatment for diabetic retinopathy.
Autoimmune diabetes in the Non Obese Diabetic (NOD) mouse model results from a breakdown of T cell tolerance caused by impaired tolerogenic dendritic cells development and regulatory T cells (Tregs) differentiation. Re-establishment of the Tregs pool has been shown to confer T cell tolerance and protection against diabetes. Here, we have investigated whether murine Thymic Stromal Lymphopoietin (TSLP) re-established tolerogenic function of DCs and induced differentiation and/or expansion of Tregs in NOD mice and protection against diabetes.
We examined the phenotype of TSLP-conditioned bone marrow DCs (TSLP-DCs) of NOD mice and their functions to induce non-inflammatory Th2 response and differentiation of Treg. The functional relevance of TSLP and TSLP-DCs to development of diabetes was also tested.
Our results showed that bone marrow DCs of NOD mice cultured in the presence of TSLP acquired signatures of tolerogenic DCs such as an absence of production of proinflammatory cytokines and a decreased expression of DCs costimulatory molecules (CD80, CD86, MHC class II) as compared to LPS-treated DCs. Furthermore, TSLP-DCs promoted non-inflammatory Th2 response and induced the conversion of naïve T cells into functional CD4+CD25+Foxp3+ Tregs. We further showed that subcutaneous injections of TSLP for 6 days or a single intravenous injection of TSLP-DCs protected NOD mice against diabetes.
Our study demonstrates that TSLP re-established a tolerogenic immune response in NOD mice and protects from diabetes suggesting that TSLP may have a therapeutic potential for the treatment of type 1 diabetes.
Acute activation of G protein-coupled Receptor 40 (GPR40) by free fatty acids (FFAs) or synthetic GPR40 agonists enhances insulin secretion. However, it is still a matter of debate if activation of GPR40 would be beneficial for the treatment of type 2 diabetes since chronic exposure to FFAs impairs islet function. We sought to evaluate the specific role of GPR40 in islets and its potential as a therapeutic target using compounds that specifically activate GPR40.
We developed a series of GPR40 selective small molecule agonists and studied their acute and chronic effects on glucose-dependent insulin secretion (GDIS) in isolated islets, as well as effects on blood glucose levels during intraperitoneal glucose tolerance tests (IPGTT) in wild-type (WT) and in GPR40 knock-out mice (GPR40-/-).
Small molecule GPR40 agonists significantly enhanced GDIS in isolated islets and improve glucose tolerance in WT mice, but not in GPR40-/- mice. While a 72-hour exposure to FFA in tissue culture significantly impaired GDIS in islets from both WT and GPR40-/- mice, similar exposure to the GPR40 agonist did not impair GDIS in islets from WT mice. Furthermore, the GPR40 agonist enhanced insulin secretion in perfused pancreata from neonatal streptozotocin-induced diabetic rats, and improved glucose levels in high fat diet-induced obese mice acutely and chronically.
GPR40 does not mediate the chronic toxic effects of FFAs on islet function. Pharmacological activation of GPR40 may potentiate GDIS in humans and be beneficial on overall glucose control in patients with type 2 diabetes mellitus.
Cannabinoid type 1 (CB1) receptor blockade decreases body weight and adiposity in obese subjects, however the underlying mechanism is not yet fully understood. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) induces mitochondrial biogenesis and function in adipocytes. This study was undertaken to test the hypothesis whether CB1 receptor blockade increases the espression of eNOS and mitochondrial biogenesis in white adipocytes.
We examined the effects on eNOS and mitochondrial biogenesis of selective pharmacological blockade of CB1 receptors by SR141716 (rimonabant) in mouse primary white adipocytes. We also examined eNOS expression and mitochondrial biogenesis in white adipose tissue (WAT) and isolated mature white adipocytes of CB1 receptor deficient (CB1–/–) and chronically SR141716-treated mice on either standard (STD) or high-fat diet (HFD).
SR141716 treatment increased eNOS expression in cultured white adipocytes. Moreover, SR141716 increased mitochondrial DNA amount, mRNA levels of genes involved in mitochondrial biogenesis, and mitochondrial mass and function through eNOS induction, as demonstrated by reversal of SR141716 effects by small interfering RNA-mediated decrease in eNOS. While HFD-fed wild-type mice showed reduced eNOS expression and mitochondrial biogenesis in WAT and isolated mature white adipocytes, genetic CB1 receptor deletion or chronic treatment with SR141716 restored these parameters to the levels observed in wild-type mice on STD, an effect linked to the prevention of adiposity and body weight increase.
CB1 receptor blockade increases mitochondrial biogenesis in white adipocytes by inducing the expression of eNOS. This is linked to the prevention of HFD-induced fat accumulation, without concomitant changes in food intake.
The implication of innate immunity in T1D development has long been proposed. HMGB1, an evolutionarily conserved chromosomal protein, was recently recognized to be a potent innate inflammatory mediator when released extracellularly. We sought to test the hypothessis that HMGB1 acts as an innate immune-mediator implicated in T1D pathogenesis.
8wk- and 12wk-old NOD mice were treated with an HMGB1 neutralizing antibody once a week till 25wk-old and monitored for insulitis progression and diabetes onset. The underlying mechanisms of HMGB1 regulation of autoimmune response were further explored.
During autoimmunity, HMGB1 can be passively released from damaged pancreatic β cells and actively secreted by islet infiltrated immune cells. Extracellular HMGB1 is potent in inducing NOD DC maturation and stimulating macrophage activation. Blockade of HMGB1 significantly inhibited insulitis progression and diabetes development both in 8wk- and 12wk-old NOD mice. HMGB1 antibody treatment decreased the number and maturation of pancreatic lymph node (PLN) CD11c++CD11b+ DCs, a subset of DCs probably associated with auto-antigen presentation to naïve T cells, but increased the number for PLN CD4+Foxp3+ regulatory T cells. Blockade of HMGB1 also decreased splenic DC allo-stimulatory capability associated with increased tolergenic CD11c+CD8a+ DCs. Interestingly, the number of CD8+IFN+ (Tc1) T cells was increased in the PLN and spleen after blockade of HMGB1, which could be associated with retarded migration of activated autoreactive T cells into the pancreatic islets.
Extracellular HMGB1 functions as a potent innate immune-mediator contributing to insulitis progression and diabetes onset.
To identify, map, clone and functionally validate a novel mouse model for impaired glucose tolerance and insulin secretion.
Haploinsufficiency of the insulin receptor (IR) and associated mild insulin resistance has been utilised to sensitise an ENU screen to identify novel mutations resulting in impaired glucose tolerance and diabetes. The new IGT4 model was selected using an intraperitoneal glucose tolerance test and inheritance of the phenotype confirmed by generation of backcross progeny. Segregation of the phenotype was correlated with genotype information to map the location of the gene and candidates sequenced for mutations. The function of the Sox4 gene in insulin secretion was tested using another ENU allele and by siRNA silencing in insulinoma cells.
We describe two allelic autosomal dominant mutations in the highly conserved HMG box of the transcription factor Sox4. Previously associated with pancreas development, Sox4 mutations in the adult mouse result in an insulin secretory defect, which in association with IR+/– induced insulin resistance exhibits impaired glucose tolerance. Elimination of the Sox4 transcript in INS1 and Min6 cells resulted in the abolition of glucose-stimulated insulin release similar to that observed for silencing of the key metabolic enzyme glucokinase. Intracellular calcium measurements in treated cells indicates that this defect lies downstream of the KATP channel and calcium influx.
IGT4 represents a novel di-genic model of insulin resistance coupled with an insulin secretory defect. The Sox4 gene has a role in insulin secretion in the adult beta cell downstream of the KATP channel.
Fluxes through mitochondrial pathways are defective in insulin resistant skeletal muscle, but it is unclear whether similar mitochondrial defects play a role in the liver during insulin resistance and/or diabetes. The purpose of this study is to determine if abnormal mitochondrial metabolism might play a role in the dysregulation of both hepatic fat and glucose metabolism during diabetes.
Mitochondrial fluxes were measured using 2H/13C tracers and nuclear magnetic resonance (NMR) spectroscopy in Zucker Diabetic Fatty (ZDF) rats during early and advanced diabetes. To determine whether defects in hepatic fat oxidation can be corrected by PPAR activation, rats were treated with WY14,643 for 3-weeks prior to tracer administration.
Hepatic mitochondrial fat oxidation in the diabetic liver was impaired 2-fold secondary to decreased ketogenesis, but TCA cycle activity and pyruvate carboxylase flux were normal in newly diabetic rats and elevated in older rats. Treatment of diabetic rats with a PPAR agonist induced hepatic fat oxidation via ketogenesis and hepatic TCA cycle activity, but failed to lower fasting glycemia or endogenous glucose production. In fact, PPAR agonism over-stimulated mitochondrial TCA cycle flux and induced pyruvate carboxylase flux and gluconeogenesis in lean rats.
The impairment of certain mitochondrial fluxes, but preservation or induction of others, suggests a complex defect in mitochondrial metabolism in the diabetic liver. These data indicate an important co-dependence between hepatic fat oxidation and gluconeogenesis in the normal and diabetic state and potentially explain the sometimes equivocal effect of PPAR agonists on glycemia.
Insulin resistance and type 2 diabetes (DM2) are associated with an atherogenic lipoprotein profile. We examined the role of visceral and subcutaneous fat depots, independent of BMI, on the dyslipidemia associated with DM2.
Three hundred eighty-two subjects with DM2 underwent abdominal computed tomography to evaluate subcutaneous (SAT) and visceral (VAT) adipose tissue distribution, and had anthropometric measurements to determine BMI, waist and hip circumference. Fasting blood was obtained for lipoprotein particle number and size using nuclear magnetic resonance spectroscopy. The relationship of lipoprotein particle number and size to BMI, SAT and VAT was examined using multivariable regression models adjusted for age, sex, diabetes therapy, duration of diabetes, smoking, statin use and hemoglobin A1c levels. The relation of VAT to lipoprotein particle number and size was further evaluated after the addition of BMI, BMI plus SAT, or BMI plus HOMAIR to the model.
VAT was positively related to VLDL particle number (P<0.0001), LDL particle number (P<0.01), VLDL size (P<0.0001) and negatively related to LDL size (P<0.0001) and HDL size (P<0.0001). These relationships remained unchanged after addition of BMI and SAT to the model. After Addition of HOMAIR, VAT remained positively related to VLDL particle number (P<0.0001) and size (P<0.01) and negatively related to LDL and HDL particle size (P<0.0001 for both comparisons). Neither BMI nor SAT were independently related to lipoprotein parameters.
In patients with DM2, higher VAT independent of BMI, was associated with higher VLDL and LDL particle number, larger VLDL particles and smaller LDL and HDL particles. This lipoprotein pattern has been associated with increased risk for atherosclerosis and cardiovascular disease.
Islet transplantations have been performed clinically, but the practical applications are limited. An extensive effort has been made towards the identification of pancreatic β cell stem cells which has yielded many insights to date, yet targeted reconstitution of β cell mass remains elusive. Here, we present a mouse model for inducible and reversible ablation of pancreatic β cells named the PANIC-ATTAC mouse (pancreatic islet β cell - apoptosis through targeted activation of caspase 8).
We efficiently induce β cell death through apoptosis and concomitant hyperglycemia by administration of a chemical dimerizer to the transgenic mice. In contrast to streptozotocin-treated animals, the diabetes phenotype and β cell loss are fully reversible in the PANIC-ATTAC mice and we find significant β cell recovery with normalization of glucose levels after two months.
The rate of recovery can be enhanced by various pharmacological interventions with agents acting on the GLP-1 axis as well as agonists of peroxisome proliferator-activated receptor-. During recovery, we find an increased population of Glut2 positive, insulin negative cells in the islets of PANIC-ATTAC mice, which may represent a novel pool of potential β cell precursors.
The PANIC-ATTAC mouse may be used as an animal model of inducible and reversible β cell ablation and therefore has applications in many areas of diabetes research that include identification of β cell precursors, evaluation of glucotoxicity effects in diabetes and examination of pharmacological interventions.
Recent genome-wide association studies have identified six novel genes for type 2 diabetes (T2D) and obesity and confirmed TCF7L2 as the major T2D gene up to date in Europeans. However, the implications of these genes in Asians are unclear.
We studied 13 associated single nucleotide polymorphisms (SNPs) from these genes in 3041 patients with T2D and 3678 controls of Asian ancestry from Hong Kong and Korea.
We confirmed the associations of TCF7L2, SLC30A8, HHEX, CDKAL1, CDKN2A/CDKN2B, IGF2BP2 and FTO with risk for T2D, with odds ratios ranged from 1.13 to 1.35 (1.3x 10–12 < Punadjusted <0.016). In addition, the A allele of rs8050136 at FTO was associated with increased body mass index in the control subjects (Punadjusted=0.008). However, we did not observe significant association of any genetic variants with surrogate measures of insulin secretion or insulin sensitivity indices in a subset of 2662 control subjects. Compared to subjects carrying 0, 1 or 2 risk alleles, each additional risk allele was associated with 17% increased risk, and up to 3.3-fold increased risk for T2D in those carrying 8 or more risk alleles. Despite most of the effect sizes being similar between Asians and Europeans in the meta-analyses, the ethnic differences in risk allele frequencies in most of these genes lead to variable attributable risks in these two populations.
Our findings support the important but differential contribution of these genetic variants for T2D and obesity in Asians as compared to Europeans.
Synthetic ligands for peroxisome proliferator-activated receptor (PPAR) improve insulin sensitivity in obesity but it is still unclear if inflammatory signals modulate their metabolic actions. In this study we tested whether targeted disruption of iNOS, a key inflammatory mediator in obesity, modulates the metabolic effects of rosiglitazone (RSG) in obese mice.
iNOS–/– and iNOS+/+ were subjected to a high-fat diet or standard diet for 18 weeks and were then treated with RSG for 2 weeks. Whole-body insulin sensitivity and glucose tolerance were determined and metabolic tissues harvested to assess activation of insulin and AMPK signaling pathways and the levels of inflammatory mediators.
RSG was found to similarly improve whole-body insulin sensitivity and insulin signaling to Akt/PKB in skeletal muscle of obese iNOS–/– and obese iNOS+/+ mice. However, RSG further improved glucose tolerance and liver insulin signaling only in obese mice lacking iNOS. This genotype-specific effect of RSG on glucose tolerance was linked to a markedly increased ability of the drug to raise plasma adiponectin levels. Accordingly, RSG increased AMPK activation in muscle and liver only in obese iNOS–/– mice. PPAR transcriptional activity was increased in adipose tissue of iNOS–/– mice. Conversely, treatment of 3T3-L1 adipocytes with a nitric oxide donor blunted PPAR activity.
Our results identify the iNOS/NO pathway as a critical modulator of PPAR activation and circulating adiponectin levels, and show that invalidation of this key inflammatory mediator improves the efficacy of PPAR agonism in an animal model of obesity and insulin resistance.
Berardinelli-Seip Congenital Lipodystrophy type 2 (BSCL2) is a recessive disorder featuring near-complete absence of adipose tissue. Remarkably, whilst the causative gene, BSCL2, has been known for several years, its molecular function and its role in adipose tissue development have not been elucidated. Therefore, we examined whether BSCL2 is involved in the regulation of adipocyte differentiation, and the mechanism whereby pathogenic mutations in BSCL2 cause lipodystrophy.
Following the characterisation of BSCL2 expression in developing adipocytes, C3H10T1/2 mesenchymal stem cells were generated in which BSCL2 expression was knocked down using shRNA. These cells were used to investigate whether BSCL2 is required for adipogenesis. BSCL2 constructs harbouring pathogenic mutations known to cause lipodystrophy were also generated and characterised.
BSCL2 expression was strongly induced during adipocyte differentiation and the induction of BSCL2 expression was essential for adipogenesis to occur. The initial induction of key adipogenic transcription factors including PPAR and C/EBP was preserved in cells lacking BSCL2. However, the expression of these critical factors was not sustained suggesting that the activity of PPAR was impaired. Moreover, expression of key genes mediating triglyceride synthesis including AGPAT2, lipin-1 and DGAT2 was persistently reduced and lipid accumulation was inhibited. Analysis of pathogenic missense mutants of BSCL2 revealed that the amino acid substitution A212P causes aberrant targeting of BSCL2 within the cell, suggesting that sub-cellular localisation of BSCL2 may be critical to its function.
This study demonstrates that BSCL2 is an essential, cell autonomous regulator of adipogenesis.
Congenital hyperinsulinism, usually associated with severe neonatal hypoglycemia, may progress to diabetes, typically during the 4th decade of life in non-pancreatectomized patients.
A 10.5y old female presented with new onset, antibody-negative diabetes (HbA1C-10.6%). She was born large for gestational age (5Kg) to a non-diabetic mother, and developed frequent hypoglycemic episodes, which persisted until age 3y, responding initially to intravenous glucose and later to oral sweets. Currently, she is fully pubertal, obese (BMI-30.2kg/m2), with a partially controlled convulsive disorder (since age 1y) and poor school performance. Glucose levels were >11.1mmol/l throughout 72 hours of continuous glucose monitoring, with low insulin secretion during IVGTT. KCNJ11 and ABCC8 mutation analysis was performed and the mutation identified was characterized in COSm6 cells.
A novel, de novo heterozygous ABCC8 (SUR1) mutation (R370S) was identified in the patient's DNA but not in that of either parent. Co-transfection of Kir6.2 and mutant SUR1 demonstrated that the mutated protein is expressed efficiently at the cell surface but fails to respond to MgADP, resulting in minimal channel activity. Interestingly, the heterozygous channel (WT:R370S) responded well to glibenclamide, a finding that lead to the successful initiation of sulfonylurea therapy.
This new ABCC8 mutation is associated with neonatal hyperinsulinism progressing within 10 years to insulinopenic diabetes. Consistent with in-vitro findings, the patient responded to sulfonylurea treatment. The mechanism causing the relatively rapid loss in β-cell function is not clear, but it may be due to mutation-induced increased β-cell apoptosis related to increased metabolic demand.
Chronic exposure to fatty acids causes β-cell failure, often referred to as lipotoxicity. We investigated its mechanisms focusing on contribution of SREBP-1c, a key transcription factor for lipogenesis.
We studied in vitro and in vivo effects of saturated and polyunsaturated acids on insulin secretion, insulin-signaling and expression of genes involved in β-cell functions. Pancreatic islets isolated from C57BL/6 control and SREBP-1-null mice, and adenoviral gene-delivery or -knockdown systems of related genes were used.
Incubation of C57BL/6 islets with palmitate (PA) caused inhibition of both glucose- and potassium-stimulated insulin secretion, but addition of eicosapentaenoate (EPA) restored both inhibitions. Concomitantly, PA activated, and EPA abolished both mRNA and nuclear protein of SREBP-1c, accompanied by reciprocal changes of SREBP-1c-target genes such as IRS-2 and granuphilin. These PA-EPA effects on insulin secretion were abolished in SREBP-1-null islets. Suppression of IRS-2/Akt pathway could be a part of the downstream mechanism for the SREBP-1c-mediated insulin secretion defect because adenoviral constitutive-active Akt compensated it. UCP-2 also plays a crucial role in the PA inhibition of insulin secretion as confirmed by knockdown experiments, but SREBP-1c-contribution to UCP-2-regulation was partial. The PA-EPA regulation of insulin secretion was similarly observed in islets from C57BL/6 mice pretreated with dietary manipulations. Furthermore, administration of EPA to diabetic KKAy mice ameliorated impairment of insulin secretion in their islets.
SREBP-1c plays a dominant role in PA-mediated insulin secretion defect, and EPA prevents it through SREBP-1c inhibition, implicating a therapeutic potential for diabetes related to lipotoxicity.
Chronic activation of the nuclear factor (NF)-B in white adipose tissue leads to increased production of pro-inflammatory cytokines, which are involved in the development of insulin resistance. It is presently unknown whether Peroxisome Proliferator-Activated Receptor (PPAR)β/ activation prevents inflammation in adipocytes.
Firstly, we examined whether the PPARβ/ agonist GW501516 prevents LPS-induced cytokine production in differentiated 3T3-L1 adipocytes. Treatment with GW501516 blocked LPS-induced IL-6 expression and secretion by adipocytes and the subsequent activation of the STAT3-SOCS3 pathway. This effect was associated with the capacity of GW501516 to impede LPS-induced NF-B activation. Secondly, in in vivo studies, white adipose tissue from Zucker Diabetic Fatty (ZDF) rats, compared to that of lean rats, showed reduced PPARβ/ expression and PPAR DNA-binding activity, which was accompanied by enhanced IL-6 expression and NF-B DNA-binding activity. Furthermore, IL-6 expression and NF-B DNA-binding activity was higher in white adipose tissue from PPARβ/-null mice than in wild-type mice. Since mitogen-activated protein kinase (MAPK)–extracellular signal–related kinase (ERK)1/2 (MEK1/2) is involved in LPS-induced NF-B activation in adipocytes, we explored whether PPARβ/ prevented NF-B activation by inhibiting this pathway. Interestingly, GW501516 prevented ERK1/2-phosphorylation by LPS. Further, white adipose tissue from animal showing constitutively increased NF-B activity, such as ZDF rats and PPARβ/-null mice, also showed enhanced phospho-ERK1/2 levels.
These findings indicate that activation of PPARβ/ inhibits enhanced cytokine production in adipocytes by preventing NF-B activation via ERK1/2, an effect that may contribute to prevent insulin resistance.
Insulin-like growth factor-1 (IGF-1) and the IGF-1 receptor (IGF-1R) have been implicated in the regulation of adipocyte differentiation and lipid accumulation in vitro.
To investigate the role of IGF-1 receptor in vivo, we have inactivated the IGF-1 receptor gene in adipose tissue (IGF-1RaP2Cre mice) using conditional gene targeting strategies.
Conditional IGF-1R inactivation resulted in increased adipose tissue mass with a predominantly increased lipid accumulation in epigonadal fat pads. However, insulin-stimulated glucose uptake into adipocytes was unaffected by the deletion of the IGF-1R. Surprisingly, IGF-1RaP2Cre mice exhibited markedly increased somatic growth in the presence of elevated IGF-1 serum concentrations and IGF-1 mRNA expression was significantly increased in liver and adipose tissue. IGF-1 stimulation of wild type adipocytes significantly decreased IGF-1 mRNA expression, while the opposite effect was obeserved in IGF-1R deficient adipocytes.
IGF-1 receptor signaling in adipocytes does not appear to be crucial for the development and differentiation of adipose tissue in vivo, but we identified a negative IGF-1 receptor-mediated feedback mechanism of IGF-1 on its own gene expression in adipocytes, indicating an unexpected role for adipose tissue IGF-1 signaling in the regulation of IGF-1 serum concentrations in control of somatic growth.
Diabetic nephropathy clusters in families suggesting that genetic factors play a role in its pathogenesis. We investigated whether similar clustering exists for proliferative retinopathy in families with two or more siblings with type 1 diabetes.
The FinnDiane Study has characterized 20% (4800 patients) of adults with type 1 diabetes in Finland. In 188 families there were at least two siblings with type 1 diabetes. Ophthalmic records were obtained for 369/396 (93%) and fundus photographs for 251/369 (68%) patients. Retinopathy was graded based on photographs and/or repeated ophthalmic examinations using the ETDRS-grading scale.
Mean age at onset of diabetes was 14.3 (±10.2) years and mean duration 25.9 (±11.8) years. Proliferative retinopathy was found in 115/369 patients (31%). The familial risk of proliferative retinopathy was estimated in 168/188 sibships, adjusted for HbA1c, duration and mean blood pressure. Proliferative retinopathy in the probands (48/168) was associated with an increased risk (Odds Ratio [OR] 2.76 [95% CI 1.25- 6.11], P=0.01) of proliferative retinopathy in the siblings of probands (61/182). The heritability of proliferative retinopathy was h2=0.52 (± 0.31, P<0.05).
We found a familial clustering of proliferative retinopathy in patients with type 1 diabetes. The observation cannot be accounted for by conventional risk factors, suggesting a genetic component in the pathogenesis of proliferative retinopathy in type 1 diabetes.
Protein kinase C (PKC) , an upstream regulator of the Akt survival pathway, contributes to cellular dysfunction in the pathogenesis of diabetes. Herein we examined the role of PKC in neuronal apoptosis through Akt in the retinas of diabetic rats.
We used retinas from 24- and 35-week-old male Otsuka Long-Evans Tokushima fatty (OLETF) diabetic and Long-Evans Tokushima Otsuka (LETO) non-diabetic rats. To assess whether PKC affects Akt signaling and cell death in OLETF rat retinas, we examined i) PKC activity and apoptosis, ii) protein levels of phosphatidylinositol 3-kinase (PI3K) p85, heat shock protein 90 (HSP90), and protein phosphatase 2A (PP2A), iii) Akt phosphorylation, and iv) Akt binding to HSP90 or PP2A in LETO and OLETF retinas in the presence or absence of rottlerin, a highly specific PKC inhibitor, or small interfering RNAs (siRNAs) for PKC and HSP90.
In 35-week OLETF retinas, ganglion cell death, PKC and PP2A activity, and Akt-PP2A binding were significantly increased, and Akt phosphorylation and Akt-HSP90 binding were decreased as compared with 24-week OLETF and LETO rats. Rottlerin and PKC siRNA abrogated these effects in 35-week OLETF retinas. HSP90 siRNA significantly increased ganglion cell death and Akt-PP2A complexes and markedly decreased HSP90-Akt binding and Akt phosphorylation in 35-week LETO retinas as compared to non-treated LETO rats.
PKC activation contributes to neuro-retinal apoptosis in diabetic rats by inhibiting Akt-mediated signaling pathways.
Several whole genome association (WGA) studies have reported identification of type 2 diabetes mellitus (T2DM) susceptibility genes in various European-derived populations. Little investigation of these loci has been reported in other ethnic groups, specifically African Americans (AAs). Striking differences exist between these populations, suggesting they may not share identical genetic risk factors. Our objective was to examine the influence of T2DM genes identified in WGA studies in a large AA case-control population.
SNPs in 12 loci (e.g. TCF7L2, IDE/KIF11/HHEX, SLC30A8, CDKAL1, PKN2, IGF2BP2, FLJ39370, and EXT2/ALX4) associated with T2DM in European-derived populations were genotyped in 993 T2DM AA cases and 1054 AA controls. Additionally, 68 ancestry-informative markers (AIMs) were genotyped to account for the impact of admixture on association results.
Except for TCF7L2, little evidence of association was observed between SNPs and T2DM in AAs. One TCF7L2 SNP (rs7903146) showed compelling evidence of association with T2DM (admixture adjusted Pa=1.59x10–6). Only the intragenic SNP on 11p12 (rs9300039, Pd=0.029) was also associated with T2DM after admixture adjustments. Interestingly, 4 of the SNPs are monomorphic in the Yoruba population of the HAPMAP project with only the "risk" allele from the populations of European descent present.
Results suggest these variants do not significantly contribute to inter-individual susceptibility to T2DM in AAs. Consequently, genes contributing to T2DM in AAs may, in part, be different from those in European-derived populations. High frequency of risk alleles in several of these genes may, however, contribute to the increased prevalence of T2DM in AAs.
Cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; encoded by Pck1) catalyzes the first committed step in gluconeogenesis. Extensive evidence demonstrates a direct correlation between PEPCK-C activity and glycemia control. Therefore, we aimed to evaluate the metabolic impact, and their underlying mechanisms, of knocking-down hepatic PEPCK-C in a type 2 diabetic model.
PEPCK-C gene targeting was achieved using adenovirus-transduced RNAi. The study assessed several clinical symptoms of diabetes and insulin signaling in peripheral tissues, in addition to changes in gene expression, protein and metabolites in the liver. Liver bioenergetics was also evaluated.
Treatment resulted in reduced PEPCK-C mRNA and protein. After treatment, improved glycemia and insulinemia, together with lower triglyceride and higher total and HDL cholesterol were measured. Unsterified fatty acid accumulation was observed in the liver, in the absence of de novo lipogenesis. In spite of hepatic lipidosis, treatment resulted in improved insulin signaling in the liver, muscle and adipose. O2 consumption measurements in isolated hepatocytes demonstrated unaltered mitochondrial function and a consequent increased cellular energy charge. Key regulatory factors (FOXO1, HNF4 and PGC-1) and enzymes (G6Pase) implicated in gluconeogenesis were down-regulated after treatment. Finally, the levels of Sirt1, a redox-state sensor that modulates gluconeogenesis through PGC1-, were diminished.
Our observations indicate that silencing PEPCK-C has direct impact on glycemia control and energy metabolism, and provides new insights into the potential significance of the enzyme as a therapeutic target for the treatment of diabetes.
Epidemiological and experimental studies have led to the hypothesis of fetal origin of adult diseases, suggesting that some adult diseases might be determined before birth by altered fetal development. We have previously demonstrated in the rat that in utero exposure to maternal diabetes impairs renal development leading to a reduction in nephron number. Little is known on the long term consequences of in utero exposure to maternal diabetes. The aim of the study was to assess, in the rat, long-term effects of in utero exposure to maternal diabetes on blood pressure and renal function in adulthood.
Diabetes was induced in Sprague-Dawley pregnant rats by streptozotocin on day 0 of gestation. Systolic blood pressure, plasma renin activity, renal function, were measured in the offsprings from 1 to 18 months of age. High salt diet experiments were performed at the pre-hypertensive stage and the abundance of tubular sodium transporters was elavuated by western-blot analysis. Kidney tissues were processed for histopathology and glomerular computer-assisted histomorphometry.
We demonstrated that in utero exposure to maternal diabetes induces a salt-sensitive hypertension in the offsprings associated with a decrease in renal function in adulthood. High salt diet experiments show an alteration of renal sodium handling that may be explained by a fetal re-programmation of tubular functions in association or as a result of the inborn nephron deficit induced by in utero exposure to maternal diabetes.
Pro-inflammatory cytokines contribute to systemic low-grade inflammation and insulin resistance. Tumor necrosis factor-alpha (TNF-) impedes insulin signaling in insulin target tissues. We determined the role of IKKβ in TNF--induced impairments in insulin signaling and glucose metabolism in skeletal muscle.
Small interfering RNA (siRNA) was used to silence IKKβ gene expression in primary human skeletal muscle myotubes from non-diabetic subjects. siRNA gene silencing reduced IKKβ protein expression 73% (p<0.05). Myotubes were incubated in the absence or presence of insulin and/or TNF- and effects of IKKβ silencing were determined on insulin signaling and glucose metabolism.
Insulin increased glucose uptake 1.7-fold (p<0.05), and glucose incorporation into glycogen 3.8-fold (p<0.05) in myotubes from non-diabetic subjects. TNF- exposure fully impaired insulin-mediated glucose uptake and metabolism. IKKβ siRNA protected against TNF--induced impairments in glucose metabolism, since insulin-induced increases in glucose uptake uptake (1.5-fold, p<0.05) and glycogen synthesis (3.5-fold, p<0.05) were restored. Conversely, TNF--induced increases in insulin receptor substrate-1 (IRS-1) serine phosphorylation (Ser312), JNK or ERK-1/2 MAPK phosphorylation were unaltered by siRNA-mediated IKKβ reduction. siRNA-mediated IKKβ reduction also prevented TNF--induced insulin resistance on Akt Ser473 and Thr308 phosphorylation and phosphorylation of the 160kDa Akt substrate AS160. IKKβ silencing was without effect on cell differentiation. Finally, mRNA expression of GLUT1, GLUT4, or protein expression of mitogenic-activated protein kinase kinase kinase kinase isoform 4 (MAP4K4) was unaltered by IKKβ siRNA.
IKKβ silencing prevents TNF--induced impairments in insulin action on Akt phosphorylation and glucose uptake and metabolism in human skeletal muscle.
We performed a comprehensive genetic association study of common variation spanning the IGF2BP2 locus, in order to replicate the association of the ‘confirmed’ type 2 diabetes susceptibility variants rs4402960 and rs1470579 in the French Caucasian population, and to further characterise the susceptibility variants at this novel locus.
We genotyped a total of 21 tagging SNPs spanning the IGF2BP2 locus in our type 2 diabetes case-control cohort comprising in 3,093 French Caucasian subjects.
IGF2BP2 variants rs4402960 and rs1470579 were not associated with type 2 diabetes in the present study (P = 0.632 and P = 0.896, respectively). Meta-analysis of genotype data from over 34,000 subjects demonstrated that our inability to replicate rs4402960/rs1470579 was consistent with the findings from several previous GWAS datasets that were under-powered to detect this modest association signal (OR 1.14). We obtained novel evidence that rs9826022, a borderline rare variant (5% MAF) in the 3' downstream region, was associated with type 2 diabetes (P = 0.0002; OR 1.53 [95% CI 1.22-1.91]). This result was corroborated by the meta-analysis of 10,542 genotypes from the current study and GWAS datasets using both fixed (P = 9.47 x 10–6; OR 1.30 [95% CI 1.16-1.46]) and random effects (P = 0.001; 1.30 [95 %CI 1.11-1.52)] calculations.
We were unable to replicate the confirmed rs4402960/rs1470579 susceptibility variants, but found novel evidence for a rare variant in the 3' downstream region of IGF2BP2. Further genetic and functional studies are required to identify the aetiological IGF2BP2 variants.
To search for a possible association of type 1 diabetes with ten validated type 2 diabetes (type 2 diabetes) loci, i.e. PPARG, KCNJ11, WFS1, HNF1B, IDE/HHEX, SLC30A8, CDKAL1, CDKN2A/B, IGF2BP2, and FTO/RPGRIP1L.
Two European population samples were studied: one case-control cohort of 514 type 1 diabetes cases and 2,027 controls, and one family cohort of 483 complete type 1 diabetes case-parent trios (total 997 affected). Thirteen tag SNPs from the ten type 2 diabetes loci were analyzed for type 1 diabetes association.
No association of type 1 diabetes was found with any of the ten type 2 diabetes loci and no age-of-onset effect was detected. By combined analysis using the Wellcome Trust Case-Control Consortium type 1 diabetes data, SNP rs1412829 in the CDKN2A/B locus show bordering significance (P=0.039), OR (95% CI)=0.929 (0.867, 0.995), which did not reach the statistical significance threshold adjusted for 13 tests (=0.00385).
This study suggests that the type 2 diabetes loci do not play any obvious role in type 1 diabetes genetic susceptibility. The distinct molecular mechanisms of the two diseases highlighted the importance of differentiation diagnosis and different treatment principles.
A recent meta-analysis demonstrated a nominal association of the ectonucleotide pyrophosphate phosphodiesterase 1 (ENPP1) K121Q polymorphism with type 2 diabetes. We set out to confirm the association of ENPP1 K121Q with hyperglycemia, expand this association to insulin resistance traits, and determine whether the association stems from K121Q or another variant in linkage disequilibrium with it.
We characterized the haplotype structure of ENPP1 and selected 39 tag SNPs that captured 96% of common variation in the region (minor allele frequency ≥5%) with an r2≥0.80. We genotyped the SNPs in 2,511 Framingham Heart Study participants and used age-sex-adjusted linear mixed effects models to test for association with quantitative metabolic traits. We also examined whether interaction between K121Q and BMI affected glycemic trait levels.
The Q allele of K121Q (rs1044498) was associated with increased fasting plasma glucose (FPG), HbA1c, fasting insulin, and insulin resistance by homeostasis model assessment (HOMA-IR; all P=0.01-0.006). Two non-coding SNPs (rs7775386, rs7773477) demonstrated similar associations, but linear mixed effect models indicated that their effects were not independent from K121Q. We found no association of K121Q with obesity, but interaction models suggested that the effect of the Q allele on FPG and HOMA-IR was stronger in those with a higher BMI (P=0.008 and 0.01 for interaction, respectively).
The Q allele of ENPP1 K121Q is associated with hyperglycemia and insulin resistance in whites. We found an adiposity-SNP interaction, with a stronger association of K121Q with diabetes-related quantitative traits in people with a higher BMI.
The mechanisms behind the association between retinol binding protein 4 (RBP4) and insulin resistance are not well understood. An interaction between iron and vitamin A status, of which RBP4 is a surrogate, has long been recognized. We hypothesized that iron-associated insulin resistance could be behind the impaired insulin action caused by RBP4.
Serum ferritin and RBP4 concentration, and insulin resistance were evaluated in a sample of middle-aged men (n=132), and in a replication, independent study. Serum RBP4 was also studied before and after iron depletion in patients with type 2 diabetes. Finally, the effect of iron on RBP4 release was evaluated in vitro in adipose tissue.
A positive correlation between circulating RBP4 and log serum ferritin (r=0.35 and r=0.61, p<0.0001) was observed in both independent studies. Serum RBP4 concentration was higher in men than women in parallel to increased ferritin levels. On multiple regression analyses to predict serum RBP4, log serum ferritin contributed significantly to RBP4 variance after controlling for BMI, age and HOMA value. Serum RBP4 concentration decreased after iron depletion type 2 diabetic patients (percent mean differences –13.7 (–25.4 to –2.04, p=0.024).
The iron donor lactoferrin led to increased dose-dependent adipose tissue release of RBP4 (2.4 fold, p=0.005) and increased RBP4 expression while apotransferrin and deferoxamine led to decreased RBP4 release.
The relationship between circulating RBP4 and iron stores, both cross-sectional and after iron depletion, and in vitro findings suggest that iron could play a role in the RBP4-insulin resistance relationship.
We have developed a novel platform for display and delivery of bioactive peptides that links the biological properties of the peptide to the pharmacokinetic properties of an antibody. Peptides engineered in the MIMETIBODYTM platform have improved biochemical and biophysical properties quite distinct from those of Fc-fusion proteins. CNTO736 is a GLP-1 receptor agonist engineered in our MIMETIBODYTM platform. It retains many activities of native GLP-1 yet has a significantly enhanced pharmacokinetic profile. Our goal was to develop a long acting GLP-1 receptor agonist with sustained efficacy.
In vitro and in vivo activity of CNTO736 was evaluated using a variety of rodent cell lines and diabetic animal models.
Acute pharmacodynamic studies in diabetic rodents demonstrate that CNTO736 reduces fasting and postprandial glucose, decreases gastric emptying, and inhibits food intake in a GLP-1R-specific manner. Reduction of food intake following CNTO736 dosing is coincident with detection of the molecule in the circumventricular organs of the brain and activation of c-fos in regions protected by the blood brain barrier. Diabetic rodents dosed chronically with CNTO736 have lower fasting and postprandial glucose and reduced body weight.
Taken together, our data demonstrate that CNTO736 produces a spectrum of GLP-1R-dependent actions while exhibiting significantly improved pharmacokinetics relative to the native GLP-1 peptide.
Many prevalent diseases of advanced societies, such as obesity-induced Type 2 diabetes, are linked to indolent mononuclear cell-dependent inflammation. We previously proposed that blockade of 4 integrin signaling can inhibit inflammation, while limiting mechanism-based toxicities of loss of 4 function. Thus, we hypothesized that mice bearing an 4(Y991A) mutation, which blocks signaling, would be protected from development of high fat diet (HFD)-induced insulin resistance.
6-8 weeks old wild-type (WT) and 4(Y991A) C57Bl/6 male mice were placed on either a HFD that derived 60% calories from lipids, or a chow diet. Metabolic testing was performed after 16 to 22 weeks of diet.
4(Y991A) mice were protected from development of HFD-induced insulin resistance. This protection was conferred on WT mice by 4(Y991A) bone marrow transplantation. In the reverse experiment, WT bone marrow renders HFD-fed 4(Y991A) acceptor animals insulin-resistant. Furthermore, fat-fed 4(Y991A) mice showed a dramatic reduction of monocyte/macrophages in adipose tissue. This reduction was due to reduced monocyte/macrophage migration rather than reduced monocyte chemoattractant protein-1 (MCP-1) production.
4 integrins contribute to the development of HFD-induced insulin resistance by mediating the trafficking of monocytes into adipose tissue; hence, blockade of 4 integrin signaling can prevent the development of obesity-induced insulin resistance.
Estradiol (E2) is known to modulate insulin sensitivity and consequently, glucose homeostasis. Resveratrol (RSV), an agonist of estrogen receptor (ER), has exerted antihyperglycemic effect in streptozotocin-induced type 1 diabetic rats in our previous study and also shown to improve insulin resistance in other reports. However, it remains unknown whether activation of ER is involved in the metabolic effects of RSV via insulin-dependent and independent mechanisms.
Here we show that RSV shifts the metabolic characteristics of rats on high cholesterol-fructose (HCF) diet towards those of rats on standard diet. RSV treatment increased insulin-stimulated whole body glucose uptake and steady-state glucose uptake of soleus muscle and liver in HCF-fed rats as well as enhanced membrane trafficking activity of glucose transporter 4 (GLUT 4) and increased phosphorylation of insulin receptor in insulin resistant soleus muscles. Interestingly, the phosphorylated ER level in insulin-resistant soleus muscle was significantly elevated in rats with RSV treatment in both basal and euglycemic hyperinsulinemic conditions. RSV exerted insulin-like stimulatory effect on isolated soleus muscle, epididymal fat and hepatic tissue and C2C12 myotubes. The RSV-stimulated glucose uptake in C2C12 myotubes was depended on Erk/p38 (early phase, 1 hr) and p38/PI3k (late phase, 14 hrs) activation respectively. Inhibition of ER abrogated RSV-induced glucose uptake in both early and late phases.
Collectively, these results indicate that ER is a key regulator in RSV-stimulating insulin-dependent and independent glucose uptake which might account for the protective effects of RSV on diet-induced insulin resistance syndrome.
The underlying genetic component of obesity-related traits is not well understood, and there is limited evidence to support genetic association shared across multiple studies, populations, and environmental contexts. The present study investigated the association between candidate variants and obesity-related traits in a sample of 1,886 adult Filipino women from the Cebu Longitudinal Health and Nutrition Survey (CLHNS) cohort.
We selected and genotyped 19 single nucleotide polymorphisms (SNPs) in 10 genes (ADRB2, ADRB3, FTO, GNB3, INSIG2, LEPR, PPARG, TNF, UCP2, UCP3), that had been reported previously to be associated with an obesity-related quantitative trait.
We observed evidence for association of the A-allele of rs9939609 (FTO intron 1) with increased body mass index (BMI) (p=.0072, before multiple test correction), baseline BMI (p=.0015), longitudinal BMI based on eight surveys from 1983 to 2005 (p=.000029), waist circumference (p=.0094), and weight (p=.021). The increase in average BMI was approximately .4 for each additional A-allele. We also observed association of the ADRB3 Trp64Arg variant with BMI, waist circumference, percent body fat, weight, fat mass, arm fat area, and arm muscle area (all p<.05), although the direction of effect is inconsistent with the majority of previous reports.
Our study confirms that FTO is a common obesity susceptibility gene in Filipinos with an effect size similar to that seen in samples of European origin.
Measurement of plasma C2 glucose enrichment is cumbersome. Therefore the plasma C5 glucose/2H2O rather than the plasma C5/C2 glucose ratio commonly has been used to measure gluconeogenesis and glycogenolysis during hyperinsulinemic euglycemic clamps. The validity of this approach is unknown.
10 non-diabetic and 10 diabetic subjects ingested 2H2O the evening before study. The following morning insulin was infused at a rate of 0.6 mU/kg/min and glucose was clamped at ~5.3 mmol/l for five hours. Plasma C5 glucose, C2 glucose and 2H2O enrichments were measured hourly from two hours onward.
Plasma C2 glucose and plasma 2H2O enrichment were equal in both groups before the clamp resulting in equivalent estimates of gluconeogenesis and glycogenolysis. In contrast, plasma C2 glucose and plasma C5 glucose enrichments fell throughout the clamp whereas plasma 2H2O enrichment remained unchanged. Since the C5 glucose concentration and hence the C5glucose/2H2O ratio is influenced by both gluconeogenesis and glucose clearance whereas the C5/C2 glucose ratio only is influenced by gluconeogenesis, the C5glucose/2H2O ratio overestimated (p<0.01) gluconeogenesis during the clamp. This resulted in biologically implausible "negative" (i.e. calculated rates of gluconeogenesis exceeding total endogenous glucose production) rates of glycogenolysis in both the non-diabetic and diabetic subjects.
The plasma C5 glucose/2H2O ratio does not provide an accurate assessment of gluconeogenesis in non-diabetic or diabetic subjects during a traditional (i.e. 2-3 hour) hyperinsulinemic euglycemic clamp. The conclusions of studies that have used this approach need to be reevaluated.
The aim of our study was to explore the immunomodulatory activity of soluble ILT3-Fc in pancreatic islet transplantation and determine its mechanism of action.
NOD/SCID mice in which diabetes was induced by streptozotocin injection were transplanted with human pancreatic islet cells. Mice in which the transplant restored euglycemia were humanized with allogeneic PBMC and treated with ILT3-Fc, control human IgG, or left untreated. The blood glucose level was monitored twice a week and rejection was diagnosed after two consecutive readings >350mg/dl. Tolerated and rejected grafts were studied histologically and by immunostaining for human T cells and insulin production. CD4 and CD8 T cells from the spleen were studied for suppressor activity, expression of cytokines and CD40L.
Although human T cell engraftment was similar in all groups, ILT3-Fc treated mice tolerated the islets for the entire period of observation (91 days), while control mice rejected the graft within 7 weeks (p<0.0001). ILT3-Fc treatment suppressed the expression of cytokines and CD40L and induced the differentiation of human CD8+ Ts that inhibited Th alloreactivity against graft HLA antigens. T cells allostimulated in vitro in the presence of ILT3-Fc inhibited CD40L-induced upregulation of CD40 in human pancreatic islet cells. Histochemical studies showed dramatic differences between human pancreatic islets from tolerant, ILT3-Fc treated mice and control recipients rejecting the grafts.
The data indicated that ILT3-Fc is a potent immunoregulatory agent which suppressed islet allograft rejection in humanized NOD/SCID mice.
The transcription factor NF-B and the MAP kinases JNK1/2 are known to play decisive roles in cytokine-induced damage of rodent β-cells. The upstream events by which these factors are activated in response to cytokines are, however, uncharacterized. The aim of the present investigation was to elucidate a putative role of the MAP kinase kinase kinase-1 (MEKK-1) in cytokine-induced signaling.
To establish a functional role of MEKK-1, the effects of transient MEKK-1 overexpression in βTC-6 cells, achieved by lipofection and cell sorting, and MEKK-1 downregulation in βTC-6 cells and human islet cells, achieved by diced-siRNA treatment, were studied.
We observed that overexpression of wild type MEKK-1, but not of a kinase dead MEKK-1 mutant, resulted in potentiation of cytokine-induced JNK activation, IB degradation, NF-B translocation and cell death. Downregulation of MEKK-1 in human islet cell provoked opposite effects, i.e. attenuation of cytokine-induced JNK and MKK4 activation, IB stability and a less pronounced NF-B translocation. βTC-6 cells with a downregulated MEKK-1 expression displayed also a weaker cytokine-induced iNOS expression and lower cell death rates. Also primary mouse islet cells with downregulated MEKK-1 expression were protected against cytokine-induced cell death.
MEKK-1 mediates cytokine-induced JNK- and NF-B activation and this event is necessary for iNOS expression and cell death.
This study determined the effects of high glucose exposure and cytokine treatment on generation of reactive oxygen species (ROS) and activation of inflammatory and apoptotic pathways in human retinal endothelial cells (HREC).
Glucose consumption of HREC, human retinal pigment epithelial cells (HRPE), and human Müller cells (HMC) under elevated glucose conditions was measured and compared to cytokine treatment. Production of reactive oxygen species (ROS) in HREC was examined using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), spin trap-EPR, and MitoTracker Red staining following high glucose and cytokine treatment. The activation of different signaling cascades including the MAPK pathways, tyrosine phosphorylation pathways, and apoptosis by high glucose and cytokines in HRECs was determined.
HREC, in contrast to HRPE and HMC, did not increase glucose consumption in response to increasing glucose concentrations. Exposure of HREC to 25mM glucose did not stimulate endogenous ROS production, activation of nuclear factor kappa B (NFB), ERK, p38 and JNK, tyrosine phosphorylation, IL-1β or TNF production, and only slightly affected apoptotic cell death pathways compared to normal glucose (5mM). In marked contrast, exposure of HRECs to pro-inflammatory cytokines IL-1β or TNF increased glucose consumption, mitochondrial superoxide production, ERK and JNK phosphorylation, tyrosine phosphorylation, NFB activation, and caspase activation.
Our in vitro results indicate that HREC respond to cytokines rather than high glucose suggesting that in vivo diabetes-related endothelial injury in the retina may be due to glucose-induced cytokine release by other retinal cells and not a direct effect of high glucose.
Human adenovirus type 36 (Ad-36) increases adiposity, but improves insulin sensitivity in experimentally infected animals. We determined the ability of Ad-36 to increase glucose uptake by human skeletal muscle cells.
The effect of Ad-36 on glucose uptake and cell signaling was determined in human primary skeletal muscle (HSKM) cells obtained from type 2 diabetic and healthy lean subjects. Ad-2, another human adenovirus, was used as a negative control. Gene expression and proteins of GLUT1 and GLUT4 were measured by real time PCR and Western Blotting. Role of Insulin and Ras signaling pathways was determined in Ad-36 infected HSKM cells.
Ad-36 and Ad-2 infections were confirmed by the presence of respective viral mRNA and protein expressions. In a dose dependent manner, Ad-36 significantly increased glucose uptake in diabetic and non-diabetic HSKM cells. Ad-36 increased gene expression and protein abundance of GLUT1 and GLUT4, GLUT4 translocation to plasma membrane, and phosphotidyl inositol-3-kinase (PI3K) activity, in an insulin independent manner. In fact, Ad-36 decreased IRS-1 tyrosine phosphorylation, and IRS-1 and IRS-2 associated PI3K activities. On the other hand, Ad-36 increased Ras gene expression and protein abundance, and Ras RNAi abrogated Ad-36 induced PI3K activation, GLUT4 protein abundance and glucose uptake. These effects were not observed with Ad-2 infection.
Ad-36 infection increases glucose uptake in HSKM cells via Ras activated PI3K pathway, in an insulin independent manner. These findings may provide impetus to exploit the role of Ad-36 proteins as novel therapeutic targets for improving glucose handling.
Coinhibitory signals mediated via programmed death 1 (PD-1) receptor play a critical role in downregulating immune responses and in maintaining peripheral tolerance. Programmed death 1 ligand 1 (PD-L1), the interacting ligand for PD-1, widely expressed in many cell types, acts as a tissue-specific negative regulator of pathogenic T cell responses. We investigated the protective potential of PD-L1 on autoimmune diabetes by transgenically overexpressing PD-L1 in pancreatic β cells in nonobese diabetic (NOD) mice.
We established an insulin promoter-driven murine PD-L1 transgenic NOD mouse model to directly evaluate the protective effect of an organ-specific PD-L1 transgene against autoimmune diabetes. Transgene expression, insulitis, and diabetic incidence were characterized in these transgenic NOD mice. Lymphocyte development, Th1 cells and regulatory T cells were analyzed in these transgenic mice; and T cell proliferation, adoptive transfer and islet transplantation were performed to evaluate the PD-L1 transgene-mediated immune protective mechanisms.
The severity of insulitis in these transgenic mice is significantly decreased, disease onset is delayed and the incidence of diabetes is markedly decreased compared with littermate controls. NOD/SCID mice that received lymphocytes from transgenic mice became diabetic at a slower rate than mice receiving control lymphocytes. Moreover, lymphocytes collected from recipients transferred by lymphocytes from transgenic mice revealed less proliferative potential than lymphocytes obtained from control recipients. Transgenic islets transplanted in diabetic recipients survived moderately longer than control islets.
Our results demonstrate the protective potential of transgenic PD-L1 in autoimmune diabetes as well as illustrating its role in downregulating diabetogenic T cells in NOD mice.
Insufficient development of a new intra-islet capillary network following transplantation may be one contributing factor to the failure of islet grafts in clinical transplantation. The present study tested the hypothesis that the angiostatic factor thrombospondin-1 (TSP-1), which is normally present in islets, restricts intra-islet vascular expansion posttransplantation.
Pancreatic islets of TSP-1 deficient (TSP-1 (-/-)) mice, or wild-type islets transfected with siRNA for TSP-1, were transplanted beneath the renal capsule of syngeneic or immunocompromised recipient mice.
Both genetically TSP-1 (-/-) islets and TSP-1 siRNA-transfected islet cells demonstrated an increased vascular density when compared to control islets one month following transplantation. This was also reflected in a markedly increased blood perfusion and oxygenation of the grafts. The functional importance of the improved vascular engraftment was analyzed by comparing glucose-stimulated insulin release from islet cells transfected with either TSP-1 siRNA or scramble siRNA prior to implantation. These experiments showed that the increased revascularization of grafts composed of TSP-1 siRNA-transfected islet cells correlated to increments in both their first and second phase of glucose-stimulated insulin secretion.
Our findings demonstrate that inhibition of TSP-1 in islets intended for transplantation may be a feasible strategy to improve islet graft revascularization and function.
Subjects with diabetes experience an increased risk of myocardial infarction and cardiac failure compared to non-diabetic age-matched individuals. The Receptor for Advanced Glycation Endproducts (RAGE) is upregulated in diabetic tissues. In this study, we tested the hypothesis that RAGE impacted on ischemia/reperfusion (I/R) injury in the diabetic myocardium. In diabetic rat hearts, expression of RAGE and its ligands was enhanced, and localized particularly to both endothelial cells (EC) and mononuclear phagocytes (MP).
To specifically dissect the impact of RAGE, homozygous RAGE null mice and transgenic mice expressing cytoplasmic domain-deleted RAGE (or DN RAGE), in which RAGE-dependent signal transduction was deficient in EC or MP, were rendered diabetic with streptozotocin. Isolated perfused hearts were subjected to I/R.
Diabetic RAGE null mice were significantly protected from the adverse impact of I/R injury in the heart, as indicated by decreased release of LDH and lower glycoxidation products CML and pentosidine, improved functional recovery, and increased ATP. In diabetic transgenic mice expressing DN RAGE in EC or MP, markers of ischemic injury and CML were significantly reduced, and levels of ATP were increased in heart tissue compared to littermate diabetic controls. Further, key markers of apoptosis, caspase-3 activity and cytochrome c release, were reduced in the hearts of diabetic RAGE-modified mice compared to wild-type diabetic littermates in I/R.
These findings demonstrate novel and key roles for RAGE in I/R injury in the diabetic heart.
Amino acids (AA) stimulate glucagon responses to hypoglycaemia, and may be utilized by the brain. Aim of study was to assess the responses to hypoglycemia in nondiabetic and type 1 diabetic subjects after ingestion of AA mixture.
10 nondiabetic and 10 diabetic type 1 subjects were studied on three different occasions during intravenous insulin (2 mU/kg/min) + variable glucose for 160 minutes. In two studies, clamped hypoglycemia (plasma glucose 47 mg/dl for 40 min) was induced and either oral placebo (P) or AA mixture (42g) was given at +30 min. In the third study AA were given, but euglycemia was maintained.
Plasma glucose and insulin were no different in the hypoglycemia studies both with P and AA (p>0.2). After AA, plasma AA concentration increased to levels observed after mixed meal (2.4±0.13 vs P study 1.7±0.1 mmol/l, p=0.02). During clamped euglycemia, AA resulted in transient increase in glucagon concentrations, which returned to basal by the end of study. During clamped hypoglycemia, glucagon response was sustained and increased more in AA studies vs P in nondiabetics and diabetics (p<0.05), but other counterregulatory hormones and total symptom score were not different. Beta-OH-butyrate was less suppressed after AA (200±15 vs 93±9 µmol/L, p=0.01). Among the cognitive tests administered, the following indicated less deterioration after AA than P: Trail-making B, PASAT (2 sec), Digit span forward, Stroop colored words and verbal memory tests (nondiabetics), and Trail-Making B, Digit span backwards and Stroop color tests (diabetics).
Oral amino acids improve cognitive function in response to hypoglycemia, and enhance the response of glucagon in nondiabetic and in diabetic subjects.
Polycystic ovary syndrome (PCOS) is associated with insulin resistance and obesity. Vaspin levels increase with hyperinsulinemia and obesity. Currently, no data exists on vaspin in PCOS women.
To assess mRNA and protein levels of vaspin from subcutaneous (sc) and omental (om) adipose tissue (AT) of PCOS women and matched controls, including circulating vaspin. Ex vivo regulation of AT vaspin and the effects of metformin treatment on circulating vaspin levels in PCOS subjects were also studied.
Real-time RT-PCR and western blotting were used to assess mRNA and protein expression of vaspin. Serum vaspin was quantified by ELISA. The effects of D-glucose, insulin, gonadal and adrenal steroids on AT vaspin were analysed ex vivo.
There were significantly higher levels of circulating vaspin (P < 0.05), vaspin mRNA (P < 0.05) and protein (P < 0.05) in om AT of PCOS women. Interestingly, in om AT explants, glucose significantly