The results of the DCCT/EDIC revealed a strong association between dyslipidemia and the development of diabetic retinopathy. However, there are no experimental data on retinal fatty acid (FA) metabolism in diabetes. This study determined retinal specific FA metabolism in control and diabetic animals.

Tissue gene and protein expression profiles were determined by qRT-PCR and western blot in control and STZ diabetic rats at 3-6 weeks of diabetes. Fatty acid profiles were assessed by RP-HPLC, and phospholipid analysis was performed by nESI-MS/MS.

We found a dramatic difference between retinal and liver elongase and desaturase profiles with high elongase and low desaturases gene expression in the retina compared to liver. Elovl4, an elongase expressed in the retina, but not in the liver, showed the greatest expression level among retinal elongases, followed by Elovl2, Elovl1 and Elovl6. Importantly, early stage diabetes induced a marked decrease in retinal expression levels of Elovl4, Elovl2 and Elovl6.

Diabetes-induced downregulation of retinal elongases translated into a significant decrease in total retinal docosahexaenoic acid, as well as decreased incorporation of very long chain polyunsaturated fatty acids, particularly 32:6n3, into retinal phosphatidylcholine. This decrease in n3 PUFA was coupled with inflammatory status in diabetic retina, reflected by an increase in gene expression of pro-inflammatory markers IL-6, VEGF, and ICAM-1.

This is the first comprehensive study demonstrating diabetes-induced changes in retinal FA metabolism. Normalization of retinal FA levels by dietary means or/and modulating expression of elongases could represent a potential therapeutic target for diabetes-induced retinal inflammation.

In humans, multiple genes in the IL-2/IL-2R pathway are associated with T1D. However, no link between IL-2 responsiveness and CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Treg) has been demonstrated in T1D subjects despite the role of these IL-2-dependent cells in controlling autoimmunity. Here, we address whether altered IL-2 responsiveness impacts persistence of FOXP3 expression in Treg of T1D subjects.

Persistence of Treg was assessed by culturing sorted CD4⁺CD25^hi nTreg with IL-2 and measuring FOXP3 expression over-time by flow cytometry for control and T1D populations. The effects of IL-2 on FOXP3 induction were assessed 48 hours following activation of CD4⁺CD25^– T cells with anti-CD3 antibody. Cytokine receptor expression and signaling upon exposure to IL-2, IL-7 and IL-15 was determined by flow cytometry and western blot analysis.

Maintenance of FOXP3 expression in CD4⁺CD25⁺ Treg of T1D was diminished in the presence of IL-2, but not IL-7. Impaired responsiveness was not linked to altered expression of the IL-2R complex. Instead, IL-2R signaling was reduced in Treg and total CD4⁺ T cells of T1D. In some individuals, decreased STAT5 phosphorylation correlated with significantly higher expression of protein tyrosine phosphatase N2 (PTPN2), a negative regulator of IL-2R signaling.

Aberrant IL-2R signaling in CD4⁺ T cells of T1D subjects contributes to decreased persistence of FOXP3 expression that may impact establishment of tolerance. These findings suggest novel targets for treatment of T1D within the IL-2R pathway and suggest that an altered IL-2R signaling signature may be a biomarker for T1D.

Glycemia is a major risk factor for the development of long-term complications in type 1 diabetes, however no specific genetic loci have been identified for glycemic control in persons with type 1 diabetes. To identify such loci in type 1 diabetes, we analyzed longitudinal repeated measures of HbA1c from the Diabetes Control and Complications Trial (DCCT).

We performed a genome-wide association study using the mean of quarterly HbA1c values measured over 6.5 years, separately in the conventional (n=667) and intensive (n=637) treatment groups of the DCCT. At loci of interest, linear mixed models were used to take advantage of all the repeated measures. We then assessed the association of these loci with capillary glucose, and repeated measures of multiple complications of diabetes.

We identified a major locus for HbA1c levels in the conventional treatment group near SORCS1 (10q25.1, p=7x10^–10) which was also associated with mean glucose (p=2x10^–5). This was confirmed using HbA1c in the intensive treatment group (p=0.01). Other loci achieved evidence close to genome-wide significance: 14q32.13 (GSC) and 9p22 (BNC2) in the combined treatment groups; 15q21.3 (WDR72) in the intensive group. Further, these loci gave evidence for association with diabetic complications, specifically SORCS1 with hypoglycemia, and BNC2 with renal and retinal complications. We replicated the SORCS1 association in GoKinD controls (p=0.01), and the BNC2 association with HbA1c in non-diabetic individuals.

A major locus for HbA1c and glucose in individuals with diabetes is near SORCS1. This may influence the design and analysis of genetic studies attempting to identify risk factors for long-term diabetic complications.

We have previously shown that lack of thioredoxin-interacting protein (TXNIP) protects against diabetes and glucotoxicity-induced beta-cell apoptosis. Since the role of TXNIP in lipotoxicity is unknown, the goal of the present study was to determine whether TXNIP expression is regulated by fatty acids and whether TXNIP-deficiency also protects beta-cells against lipoapoptosis.

To determine the effects of fatty acids on beta-cell TXNIP expression, INS-1 beta-cell and isolated islets were incubated with/without palmitate and rats underwent cyclic infusions of glucose and/or intralipid prior to islet isolation and analysis by quantitative real-time RT-PCR and immunoblotting. Using primary wild-type and TXNIP-deficient islets, we then assessed the effects of palmitate on apoptosis (TUNEL), mitochondrial death pathway (cytochrome C release) and endoplasmic reticulum (ER) stress (BiP, CHOP). Effects of TXNIP-deficiency were also tested in the context of staurosporine (mitochondrial damage) or thapsigargin (ER-stress).

Glucose elicited a dramatic increase in islet TXNIP expression both in vitro and in vivo, whereas fatty acids had no such effect and, when combined with glucose, even abolished the glucose effect. We also found that TXNIP-deficiency does not effectively protect against palmitate or thapsigargin-induced beta-cell apoptosis, but specifically prevents staurosporine or glucose-induced toxicity.

Our results demonstrate that unlike glucose, fatty acids do not induce beta-cell expression of pro-apoptotic TXNIP. They further reveal that TXNIP deficiency specifically inhibits the mitochondrial death pathway underlying beta-cell glucotoxicity, whereas it has very little protective effects against ER-stress-mediated lipoapoptosis.

To investigate whether children born to older mothers have an increased risk of type 1 diabetes by performing a pooled analysis of previous studies using individual patient data to adjust for recognised confounders.

Relevant studies published before June 2009 were identified from MEDLINE, Web of Science and EMBASE. Authors of studies were contacted and asked to provide individual patient data or conduct pre-specified analyses. Risk estimates of type 1 diabetes by maternal age were calculated for each study, before and after adjustment for potential confounders. Meta-analysis techniques were used to derive combined odds ratios, and investigate heterogeneity between studies.

Data were available for 5 cohort and 25 case-control studies, including 14,724 cases of type 1 diabetes. Overall, there was, on average, a 5% (95% CI 2%, 9%) increase in childhood type 1 diabetes odds per 5 year increase in maternal age (P=0.006), but there was heterogeneity between studies (heterogeneity I²= 70%). In studies with a low risk of bias there was a more marked increase in diabetes odds of 10% per 5 year increase in maternal age. Adjustments for potential confounders little altered these estimates.

There was evidence of a weak but significant linear increase in the risk of childhood type 1 diabetes across the range of maternal ages, but the magnitude of association varied between studies. A very small percentage of the increase in the incidence of childhood type 1 diabetes in recent years could be explained by increases in maternal age.

Nephrin, an immunoglobulin-like-protein essential for the function of the glomerular podocyte and regulated in diabetic nephropathy, is also expressed in pancreatic β-cells, where its function remains unknown. The aim of this study was to investigate whether diabetes modulates nephrin expression in human pancreatic islets and to explore the role of nephrin in β-cell function.

Nephrin expression in human pancreas and in MIN6 insulinoma cells was studied by Western-Blot, PCR, confocal microscopy, subcellular fractionation and immunogold-labeling. Islets from diabetic (n=5) and non-diabetic (n=7) patients were compared. Stable transfection and siRNA knockdown in MIN-6 cells/human islets were utilized to study nephrin function in vitro and in vivo after transplantation in diabetic immunodeficient mice. Live imaging of GFP-nephrin transfected cells was utilized to study nephrin endocytosis.

Nephrin was found at the plasma membrane and on insulin vesicles. Nephrin expression was decreased in islets from diabetic patients when compared to non-diabetic controls. Nephrin transfection in MIN-6 cells/pseudoislets resulted in higher glucose-stimulated insulin release in vitro and in vivo after transplantation into immunodeficient diabetic mice. Nephrin gene silencing abolished stimulated insulin release. Confocal imaging of GFP-nephrin transfected cells revealed nephrin endocytosis upon glucose stimulation. Actin stabilization prevented nephrin trafficking as well as nephrin positive effect on insulin release.

Our data suggest that nephrin is an active component of insulin vesicle machinery that may affect vesicle-actin interaction and mobilization to the plasma membrane. Development of drugs targeting nephrin may represent a novel approach to treat diabetes.

To evaluate the significance of GAD antibodies (GADA) and family history for type 1 or type 2 diabetes (FH_T1) in non-diabetic subjects.

GADA were analysed in 4976 non-diabetic relatives of type 2 diabetic patients or control subjects from Finland. Altogether 289 (5.9%) were GADA+. 253 GADA+ and 2511 GADA- participated in repeated oral glucose tolerance tests during a median time of 8.1 years. The risk of progression to diabetes was assessed using Cox regression analysis.

Those within highest quartile of GADA+ (GADA+_high;) had more often 1^st degree FH_T1 (29.2% vs. 7.9%, P<0.00001) and GADA+ type 2 diabetic (21.3% vs. 13.7%, P=0.002) or non-diabetic (26.4% vs. 13.3%, P=0.010) relatives than GADA- subjects. During the follow-up, the GADA+ subjects developed diabetes significantly more often than the GADA- subjects [36/253 (14.2%) vs. 134/2511 (5.3%), P<0.00001]. GADA+_high conferred a 4.9-fold increased risk of diabetes (95% CI 2.8 – 8.5) as compared with GADA-. Seroconversion to positive during the follow–up was associated with 6.5-fold (2.8 – 15.2) and 1^st degree FH_T1 with 2.2-fold (1.2-4.1) risk of diabetes. Only three subjects developed type 1 diabetes, others had a non-insulin dependent phenotype one year after diagnosis. GADA+ and GADA- subjects did not clinically differ at baseline, but they were leaner and less insulin resistant after the diagnosis of diabetes.

GADA positivity clusters in families with type 1 diabetes or LADA. GADA positivity predicts diabetes independently of family history of diabetes and this risk was further increased with high GADA concentrations.

Temperature and nutrient homeostasis are two interdependent components of energy balance regulated by distinct sets of hypothalamic neurons. The objective is to examine the role of the metabolic signal insulin in the control of core body temperature (CBT).

The effect of preoptic area administration of insulin on CBT in mice was measured by radiotelemetry and respiratory exchange ratio. In vivo 2-[¹⁸F]fluoro-2-deoxyglucose (¹⁸F-FDG) uptake into brown adipose tissue was measured in rats after insulin treatment by positron emission tomography (PET) combined with X-ray computed tomography (CT) imaging. Insulin receptor-positive neurons were identified by retrograde tracing from the Raphe Pallidus. Insulin was locally applied on hypothalamic slices to determine the direct effects of insulin on intrinsically warm-sensitive neurons by inducing hyperpolarization and reducing firing rates.

Injection of insulin into the preoptic area of the hypothalamus induced a specific and dose dependent elevation of CBT mediated by stimulation of brown adipose tissue thermogenesis as shown by imaging and respiratory ratio measurements. Retrograde tracing indicates that insulin receptor expressing warm sensitive neurons activate brown adipose tissue through projection via the Raphe Pallidus. Insulin applied on hypothalamic slices, acted directly on intrinsically warm-sensitive neurons by inducing hyperpolarization and reducing firing rates. The hyperthermic effects of insulin were blocked by pretreatment with antibodies to insulin or with a PI3kinase inhibitor.

Our findings demonstrate that insulin can directly modulate hypothalamic neurons that regulate thermogenesis and CBT and indicate that insulin plays an important role in coupling metabolism and thermoregulation at the level of anterior hypothalamus.

Adipose tissue may contain few large adipocytes (hypertrophy) or many small adipocytes (hyperplasia). We investigated factors of putative importance for adipose tissue morphology.

Subcutaneous adipocyte size and total fat mass were compared in 764 subjects with body mass index 18-60 kg/m². A morphology value was defined as the difference between the measured adipocyte volume and the expected volume given by a curve-line fit, for a given body fat mass and was related to insulin values. In 35 subjects, in vivo adipocyte turnover was measured by exploiting incorporation of atmospheric¹⁴C into DNA.

Occurrence of hyperplasia (negative morphology value) or hypertrophy (positive morphology value) was independent of gender and body weight but correlated with fasting plasma insulin levels and insulin sensitivity, independently from adipocyte volume (beta coefficient=0.3, p<0.0001). Total adipocyte number and morphology were negatively related (r=-0.66); i.e. the total adipocyte number was greatest in pronounced hyperplasia and smallest in pronounced hypertrophy. The absolute number of new adipocytes generated each year was 70% lower (p<0.001) in hypertrophy than in hyperplasia and individual values for adipocyte generation and morphology were strongly related (r=0.7; p<0.001). The relative death rate (about 10% per year) or mean age of adipocytes (about 10 years) was not correlated with morphology.

Adipose tissue morphology correlates with insulin measures and is linked to the total adipocyte number independently of gender and the body fat level. Low generation rates of adipocytes associate with adipose tissue hypertrophy whereas high rates associate with adipose hyperplasia.

We demonstrated earlier that chronic maternal micronutrient restriction altered the body composition in rat offspring and may predispose them to adult onset diseases. Chromium regulates glucose and fat metabolism. The aim of this study is to determine the long term effects of maternal Cr restriction on adipose tissue development and function in a rat model.

Female, weanling WNIN rats received ad libitum, a control diet or the same with 65% restriction of Cr (CrR) for three months and mated with control males. Some pregnant CrR mothers were rehabilitated from conception or parturition and their pups weaned to control diet. While some CrR offspring were weaned to control diet, others continued on CrR diet. Various parameters were monitored in the offspring at three monthly intervals up to 15-18 months of age.

Maternal Cr restriction significantly increased body weight and fat %, specially the central adiposity in both male and female offspring. Further the expression of leptin and 11β-HSD1 genes were significantly increased in CrR offspring of both the genders. Adipocytokine levels were altered in plasma and adipose tissue, circulating triglyceride and FFA levels were increased albeit in female offspring only. Rehabilitation regimes did not correct body adiposity but restored the circulating levels of lipids and adipocytokines.

Chronic maternal chromium restriction increased body adiposity probably due to increased stress and altered lipid metabolism in WNIN rat offspring, which may predispose them to obesity and associated diseases in their later life.

Recently results from a meta-analysis of genome wide association studies have yielded a number of novel type 2 diabetes loci. However, conflicting results have been published regarding their effects on insulin secretion and insulin sensitivity. In this study we used hyperglycemic clamps with three different stimuli to test associations between these novel loci and various measures of beta cell function.

336 participants, 180 normal glucose tolerant and 156 impaired glucose tolerant, underwent a two hour hyperglycemic clamp. In a subset we also assessed the response to GLP-1 and arginine during an extended clamp (n=123). All subjects were genotyped for gene variants in JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, THADA, ADAMTS9, NOTCH2/ADAMS30, DCD, VEGFA, BCL11A, HNF1B, WFS1 and MTNR1B.

Gene variants in CDC123/CAMK1D, ADAMTS9, BCL11A and MTNR1B affected various aspects of the insulin response to glucose (all p<6.9*10^–3). The THADA gene variant was associated with lower beta cell response to GLP-1 and arginine (both p<1.6*10^–3) suggesting lower beta cell mass as a possible pathogenic mechanism. Remarkably we also noted a trend towards an increased insulin response to GLP-1 in carriers of MTNR1B (p=0.03) which may offer new therapeutic possibilities. The other seven loci were not detectably associated with beta cell function.

Diabetes risk alleles in CDC123/CAMK1D, THADA, ADAMTS9, BCL11A and MTNR1B are associated with various specific aspects of beta cell function. These findings point to a clear diversity in the impact that these different gene variants may have on (dys-)function of pancreatic beta cells.

Previous studies have indicated that the LADA phenotype is heterogeneous and that LADA patients share features of type 1 and type 2 diabetes in varying proportion. We tested for association of known type 1 and type 2 diabetes susceptibility genes in LADA subjects and analysed relationships to a marker of autoimmune activity (titres of antiGAD) and a phenotypic risk factor of type 2 diabetes (BMI).

Data were assembled from the HUNT study, which comprises the adult population of an entire county in Norway. We genotyped 60 SNPs known to be associated with type 1 or type 2 diabetes, including 14 tag SNPs used for HLA-haplotyping in 120 type 1 diabetic, 126 LADA, 1090 type 2 diabetic patients and 1503 age and gender matched non-diabetic subjects.

The majority of the strongly associated HLA-haplotypes for type 1 diabetes, were significantly associated with LADA in general, but mainly with high antiGAD LADA patients. Two distinct HLA-haplotypes were only associated with LADA and mainly in low antiGAD LADA patients. There were no associations of non-HLA type 1 diabetes loci with LADA. Of type 2 diabetes associated genes, the CC/CT genotypes of rs7961581 (TSPAN8) and the obesity-linked AA/AC genotypes of rs8050136 (FTO) were associated with LADA in general, but mainly in low antiGAD LADA patients (p=0.004 and p=0.004).

Genetic heterogeneity in LADA is linked to a varying degree of autoimmune activity and may be partly distinct from both type 1 and type 2 diabetes.

White adipose tissue (WAT) and brown adipose tissue (BAT) play distinct roles in adaptation to changes in nutrient availability, with WAT serving as an energy store and BAT regulating thermogenesis. We previously showed that mice maintained on a leucine-deficient diet unexpectedly experienced a dramatic reduction in abdominal fat mass (1). The cellular mechanisms responsible for this loss, however, are unclear. The goal of current study is to investigate possible mechanisms.

Male C57BL/6J mice were fed either control, leucine-deficient, or pair-fed diets for 7 days. Changes in metabolic parameters and expression of genes and proteins related to lipid metabolism were analyzed in WAT and BAT.

We found that leucine deprivation for 7 days increases oxygen consumption, suggesting increased energy expenditure. We also observed increases in lipolysis and expression of β-oxidation genes, and decreases in expression of lipogenic genes and activity of fatty acid synthase (FAS) in WAT, consistent with increased utilization and decreased synthesis of fatty acids, respectively. Furthermore, we observed that leucine deprivation increases expression of uncoupling protein (UCP)1 in BAT, suggesting increased thermogenesis.

We show for the first time that elimination of dietary leucine produces significant metabolic changes in WAT and BAT. The effect of leucine deprivation on UCP1 expression is a novel and unexpected observation and suggests that the observed increase in energy expenditure may reflect an increase in thermogenesis in BAT. Further investigation will be required to determine the relative contribution of UCP1 upregulation and thermogenesis in BAT to leucine deprivation-stimulated fat loss.

We tested the primary hypotheses that sphingolipid and diacylglycerol (DAG) content is higher within insulin resistant muscle and that the association between intramyocellular triglycerides (IMTG) and insulin resistance is muscle fiber type-specific.

A nested case-control analysis was conducted in 22 obese (BMI >30 kg/m²) women who were classified as insulin resistant (IR; n = 12) or insulin sensitive (IS; n = 10), determined by hyperinsulinemic euglycemic clamp (>30% greater in IS compared to IR, P <0.01). Sphinoglipid and DAG content was determined by HPLC/MS/MS. Fiber type-specific IMTG content was histologically determined. Gene expression was determined by qPCR.

Total (555±53 vs. 293±54 pMol/mg protein, P =0.004), saturated (361±29 vs. 179±34 pMol/mg protein, P =0.001) and unsaturated (198±29 vs. 113±66 pMol/mg protein, P =0.034) ceramides were higher in IR compared to IS. DAG concentrations, however, were similar. IMTG content within type I myocytes, but not type II myocytes, was higher in IR compared to IS (P =0.005). Insulin sensitivity was negatively correlated with IMTG within type I myocytes (R = –0.51, P =0.026), but not with IMTG within type II myocytes. The proportion of type I myocytes was lower (41% vs. 59%, P <0.01) in IR. Several genes involved in lipid droplet and fatty acid metabolism were differentially expressed in IR compared to IS.

Human skeletal muscle insulin resistance is related to greater IMTG content in type I, but not type II myocytes, to greater ceramide content, and to alterations in gene expression associated with lipid metabolism.

SNPs in intron 1 of fat mass and obesity associated gene (FTO) are strongly associated with human adiposity, while Fto–/– mice are lean and Fto+/– mice are resistant to diet-induced obesity. We aimed to determine whether FTO mutations are disproportionately represented in lean or obese humans and to use these mutations to understand structure-function relationships within FTO.

We sequenced all coding exons of FTO in 1433 severely obese and 1433 lean individuals. We studied the enzymatic activity of selected non-synonymous variants.

We identified 33 heterozygous non-synonymous variants in lean (2.3%) and 35 in obese (2.4%) individuals, with 8 mutations unique to the obese and 11 unique to the lean. Two novel mutations replace absolutely conserved residues; R322Q in the catalytic domain and R96H in the predicted substrate recognition lid. R322Q was unable to catalyse the conversion of 2-OG to succinate in the presence or absence of 3-methylthymidine. R96H retained some basal activity, which was not enhanced by 3-methylthymidine. However, both were found in lean and obese individuals.

Heterozygous, loss-of-function mutations in FTO exist but are found in both lean and obese subjects. While intron 1 SNPs are unequivocally associated with obesity in multiple populations and murine studies strongly suggest that FTO has a role in energy balance, it appears that loss of one functional copy of FTO in humans is compatible with being either lean or obese. Functional analyses of FTO mutations have given novel insights into structure-function relationships in this enzyme.

Sympathetic nervous system (SNS) overactivity contributes to the pathogenesis and target organ complications of obesity. This study was conducted to examine the effects of lifestyle interventions (weight loss alone or together with exercise) on SNS function.

Untreated men and women (mean age 55 ± 1 yrs; BMI 32.3 ± 0.5 kg/m²) who fulfilled Adult Treatment Panel III MetS criteria were randomly allocated to either dietary weight loss (WL, n=20), dietary weight loss and moderate-intensity aerobic exercise (WL+EX, n=20) or no treatment (Control, n=19). Whole-body norepinephrine (NE) kinetics, muscle sympathetic nerve activity (MSNA) by microneurography, baroreflex sensitivity (BRS), fitness (maximal oxygen consumption), metabolic and anthropometric measurements were made at baseline and 12 weeks.

Body weight decreased by -7.1 ± 0.6 and -8.4 ± 1.0 kg in the WL and WL+EX groups respectively (both P<0.001). Fitness increased by 19 ± 4% (P<0.001) in the WL+EX group only. Resting SNS activity decreased similarly in the WL and WL+EX groups groups: NE spillover by -96 ± 30 and -101 ± 34 ng/min (both P<0.01) and MSNA by -12 ± 6 and -19 ± 4 bursts/100 heart beats, respectively (both P<0.01), but remained unchanged in Controls. Blood pressure, BRS and metabolic parameters improved significantly and similarly in the two lifestyle intervention groups.

The addition of moderate-intensity aerobic exercise training to a weight loss program does not confer additional benefits on resting SNS activity. This suggests that weight loss is the prime mover in sympathetic neural adaptation to a hypocaloric diet.

Because vitamin D deficiency is associated with a variety of chronic diseases, understanding the characteristics that promote vitamin D deficiency in otherwise healthy adults could have important clinical implications. Few studies relating vitamin D deficiency to obesity have included direct measures of adiposity. Furthermore, the degree to which vitamin D is associated with metabolic traits after adjusting for adiposity measures is unclear.

We investigated the relations of serum 25-hydroxyvitamin D (25[OH]D) concentrations with indices of cardiometabolic risk in 3,890 non-diabetic individuals; 1,882 had subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) volumes measured by multi-detector computed tomography (CT).

In multivariable-adjusted regression models, 25(OH)D was inversely associated with winter season, waist circumference, and serum insulin ( P<0.005 for all). In models further adjusted for CT measures, 25(OH)D was inversely related to SAT (–1.1 ng/mL per standard deviation [SD] increment in SAT, P=0.016) and VAT (–2.3 ng/mL per SD, P<0.0001). The association of 25(OH)D with insulin resistance measures became non-significant after adjustment for VAT. Higher adiposity volumes were correlated with lower 25(OH)D across different categories of body mass index (BMI), including in lean individuals (BMI <25 kg/m²). The prevalence of vitamin D deficiency (25(OH)D <20 ng/mL ) was 3-fold higher in those with high SAT and high VAT than in those with low SAT and low VAT (P<0.0001).

Vitamin D status is strongly associated with variation in subcutaneous and especially visceral adiposity. The mechanisms by which adiposity promotes vitamin D deficiency warrant further study.

Hippocampal neurons in adult animals and humans are vulnerable to severe hypoglycemia and hyperglycemia. Effects are hypothesized to be exacerbated during development, but existing studies on developing human brains are limited. We examined whether hypoglycemia or hyperglycemia experienced during brain development in humans affects hippocampal volumes.

We analyzed T1-weighted magnetic resonance images in 95 youth with type 1 diabetes and 49 sibling controls ages 7-17. Youth with diabetes were categorized as having either 0 (n=37), 1-2 (n=41) or 3 or more (3+; n=17) prior severe hypoglycemic episodes. Hyperglycemia exposure was estimated from median lifetime HbA1c, weighted for duration of diabetes. Stereologic measurements of hippocampal volumes were performed in atlas-registered space to correct for whole brain volume.

Greater exposure to severe hypoglycemia was associated with larger hippocampal volumes (F(3, 138)=3.6, p=.016; 3+ larger than all other groups, p<.05). Hyperglycemia exposure was not associated with hippocampal volumes (R² change =.003, F(1,89)=.31, p=.58, semi-partial r=.06; one outlier removed for high median HbA1c), and the 3+ severe hypoglycemia group still had larger hippocampal volumes after controlling for age of onset and hyperglycemia exposure (main effect of hypoglycemia category, F(2,88)=6.4,p=.002; 3+ larger than all other groups, p<.01).

Enlargement of the hippocampus may reflect a pathological reaction to hypoglycemia during brain development, such as gliosis, reactive neurogenesis or disruption of normal developmental pruning.

Test the hypothesis that FFA and muscle glycogen modify exercise-induced regulation of PDH in human skeletal muscle through regulation of PDK4 expression.

On two occasions, healthy male subjects lowered (by exercise) muscle glycogen in one leg (LOW) relative to the contra-lateral leg (CON) the day before the experiment day. On the experimental days, plasma FFA was ensured normal or remained elevated by consuming breakfast rich (low FFA) or poor (high FFA) in carbohydrate, 2 hours before performing 20 min of two-legged knee extensor exercise. Vastus lateralis biopsies were obtained before and after exercise.

PDK4 protein content was ~2.2 and ~1.5 fold higher in LOW than CON leg in high FFA and low FFA, respectively, and the PDK4 protein content in CON leg was ~2 fold higher in high FFA than in low FFA. In all conditions, exercise increased PDHa activity resulting in similar levels in LOW leg in both trials and CON leg in high FFA, but higher level in CON leg in low FFA. PDHa activity was closely associated with PDH-E1 phosphorylation.

Muscle glycogen and plasma FFA attenuate exercise-induced PDH regulation in human skeletal muscle in a non-additive manner. This might be through regulation of PDK4 expression. The activation of PDH by exercise independent of changes in muscle glycogen or plasma FFA suggests that exercise overrules FFA-mediated inhibition of PDH (i.e. carbohydrate oxidation), and this may thus be one mechanism behind health promoting effects of exercise.

RAGE interacts with the endogenous ligands S100 calgranulins and high mobility group box 1 (HMGB1) to induce inflammation. Since hyperglycemia-induced ROS activate many pathways of diabetic tissue damage, the effect of these ROS on RAGE and RAGE ligand expression was evaluated.

Expression of RAGE, S100A8, S100A12, and HMGB1 was evaluated in human aortic endothelial cells (HAECs) incubated in normal glucose, high glucose, and high glucose after overexpression of either uncoupling protein 1 (UCP1), superoxide dismutase 2 (SOD2), or glyoxalase 1 (GLO1). Expression was also evaluated in normal glucose after knockdown of GLO1. Expression was next evaluated in high glucose after knockdown of NFB p65 (RAGE) and after knockdown of AP-1 (S100A8, S100A12, and HMGB1), and chromatin immunoprecipitation (ChIP) was performed ± GLO1 overexpression for NFB p65 (RAGE promoter) and AP-1 (S100A8, S100A12, and HMGB1 promoters). Finally, endothelial cells from non-diabetic mice, STZ diabetic mice, and STZ diabetic mice treated with the superoxide dismutase mimetic MnTBAP were evaluated.

High glucose increased RAGE, S100A8, S100A12, and HMGB1 expression, which was normalized by overexpression of UCP1, SOD2, or GLO1. GLO1 knockdown mimicked the effect of high glucose, and in high glucose, overexpression of GLO1 normalized increased binding of NFB p65 and AP-1. Diabetes increased RAGE, S100A8 and HMGB1 expression, and MnTBAP treatment normalized this.

These results show that hyperglycemia-induced ROS production increases expression of RAGE and RAGE ligands. This effect is mediated by ROS-induced methylglyoxal, the major substrate of glyoxalase 1.

Insulin resistance in skeletal muscle is an early phenomenon in the pathogenesis of type 2 diabetes. Studies of insulin resistance usually are highly focused. However, approaches that give a more global picture of abnormalities in insulin resistance are useful in pointing out new directions for research. In previous studies, gene expression analyses show a coordinated pattern of reduction in nuclear-encoded mitochondrial gene expression in insulin resistance. However, changes in mRNA levels may not predict changes in protein abundance. An approach to identify global protein abundance changes involving the use of proteomics was used here.

Muscle biopsies were obtained basally from lean, obese, and type 2 diabetic volunteers (n=8 each); glucose clamps were used to assess insulin sensitivity. Muscle protein was subjected to mass spectrometry-based quantification using normalized spectral abundance factors.

Of 1218 proteins assigned, 400 were present in at least half of all subjects. Of these, 92 were altered by a factor of 2 in insulin resistance, and of those, 15 were significantly increased or decreased by analysis of variance (P < 0.05). Analysis of protein sets revealed patterns of decreased abundance in mitochondrial proteins, and altered abundance of proteins involved with cytoskeletal structure (desmin and alpha actinin-2 both decreased), chaperone function (TCP-1 subunits increased), and proteasome subunits (increased).

The results confirm the reduction in mitochondrial proteins in insulin resistant muscle and suggest that changes in muscle structure, protein degradation and folding also characterize insulin resistance.

So far it is unclear whether chronic peripheral hyperinsulinemia per se might contribute to ectopic lipid accumulation and consequently insulin resistance. We investigated the effects of systemic instead of portal insulin release in type-1 diabetic (T1DM) patients after successful pancreas-kidney-transplantation with systemic venous drainage (PKT) on the intracellular lipid content in liver (IHCL) and soleus muscle (IMCL), endogenous glucose production (EGP) and insulin-sensitivity.

In 9 PKT and 9 matching nondiabetic controls (CON), IHCL and IMCL were measured employing ¹H-nuclear-magnetic-resonance-spectroscopy. Fasting EGP was measured using D-[6,6-²H₂]-glucose-tracer dilution. A 3-hour 75g-oral glucose tolerance test (OGTT) allowed us to assess kinetics of glucose, free fatty acids, insulin and C-peptide concentrations in plasma and to calculate the clamp-like insulin sensitivity index (CLIX) and the hepatic insulin resistance index (HIR).

PKT displayed ~2fold increased fasting insulin (20±6 vs. 9±3µU/ml; p<0.0002 vs. CON) and ~10% increased fasting glucose (p<0.02) concentrations, but during the OGTT areas under the concentration curves of C-peptide and insulin were similar. IHCL (PKT:2.9±2.5%; CON:4.4±6.6%) and IMCL (PKT:1.0±0.4%; CON:1.0±0.5%), CLIX (PKT: 8±2, CON: 7±3), HIR [PKT:25.6±13.2; CON:35.6±20(mg·min^-1·kg^-1)x(µU/ml)] and EGP (PKT:1.6±0.2; CON:1.7±0.2 mg·min^-1·kg^-1) were comparable between PKT and CON. IHCL was negatively correlated with CLIX in all participants (r=-0.55, p<0.04).

Despite fasting peripheral hyperinsulinemia because of systemic venous drainage, T1DM after PKT show similar IHCL, IMCL, insulin sensitivity and fasting EGP in comparison to non-diabetic controls. These results suggest that systemic hyperinsulinemia per se does not cause ectopic lipid accumulation in liver and skeletal muscle.

Activation of the alternative pathway of the complement system, in which factor H (fH; CFH) is a key regulatory component, has been suggested as a link between obesity and metabolic disorders. To study the associations between circulating and adipose tissue gene expressions of CFH and complement factor B (fB; CFB) with obesity and insulin resistance.

Circulating fH and fB were determined by ELISA in 398 subjects. CFH and CFB gene expressions were evaluated in 76 adipose tissue samples, in isolated adipocytes and stromo-vascular cells (SVC) (n=13). The effects of weight loss and rosiglitazone were investigated in independent cohorts.

Both circulating fH and fB were positively associated with BMI, waist diameter, triglycerides and inflammatory parameters; and negatively with insulin sensitivity and HDL-cholesterol. For the first time, CFH gene expression was detected in human adipose tissue (significantly increased in subcutaneous compared with omental fat). CFH gene expression in omental fat was significantly associated with insulin resistance. In contrast, CFB gene expression was significantly increased in omental fat but also in association with fasting glucose and triglycerides. The SVC fraction was the responsible of these differences, although isolated adipocytes also expressed fB and fH at low levels. Both weight loss and rosiglitazone led to significantly decreased circulating fB and fH levels.

Increased circulating fH and fB concentrations in subjects with altered glucose tolerance could reflect increased SVC-induced activation of the alternative pathway of complement in omental adipose tissue linked to insulin resistance and metabolic disturbances.

Recently, several drugs have been shown to exert their beneficial effects for metabolic syndrome through mild regulation of mitochondrial function. Hence, we explored a strategy of targeting mitochondrial function to improve glucose and lipid metabolism.

Mitochondrial membrane potential (m) is a marker of mitochondrial function, therefore we set up a high-throughput screening (HTS) assay of m in L6 myotubes. The effects of a selected lead compound were investigated in vitro and in vivo in relation to metabolic syndrome.

A novel small-molecule compound, C1, was identified through this HTS. C1 depolarized m in L6 myotubes without cytotoxicity and led to increased cellular AMP/ATP ratio, activation of AMP-activated protein kinase (AMPK) and enhanced glucose uptake. It also stimulated the AMPK pathway in HepG2 cells, leading to decreased lipid content. Intriguingly, C1 inhibited respiration in L6 myotubes, but did not affect respiration in isolated muscle mitochondria, suggesting that it may depolarize m indirectly by affecting the supply of electron donors. Acute administration of C1 in C57BL/6J mice markedly increased fat oxidation and the phosphorylation of AMPK and ACC in the liver. In diabetic db/db mice, chronic administration of C1 significantly reduced hyperglycemia, plasma fatty acids, glucose intolerance and the mRNA levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in liver.

Our results demonstrate a novel small molecule which mildly depolarizes m, is able to improve glucose and lipid metabolism to exert beneficial effects for metabolic syndrome. These findings suggest that compounds regulating mitochondrial function may have therapeutic potential for type 2 diabetes.

Our aim was to study the recurrence risk of type 1 diabetes in the offspring of parents with adult onset (15-39 years) type 1 diabetes and to evaluate the transmission of diabetes within a continuum of parental age at onset of diabetes from childhood to adulthood.

Diabetes status of all offspring (n=9,715) of two Finnish cohorts of parents with type 1 diabetes was defined until the end of year 2007. Cumulative incidences of type 1 diabetes among the offspring were estimated and several factors contributing the risk were assessed.

During 137,455 person-years a total of 413 offspring were diagnosed with type 1 diabetes. The cumulative incidence by 20 years was 4.0% (95% CI 3.1-4.8) for the offspring of parents with adult onset diabetes. The risk was equal according to the sex of the parents. The cumulative incidence decreased in parallel with the increase in age at onset of diabetes in the fathers. In the offspring of diabetic mothers the risk was equal regardless of the age at onset of diabetes. However, the reduced risk in the maternal offspring was most pronounced in the daughters of the mothers with diagnosis age less than 10 years.

Type 1 diabetes transmission ratio distortion is strongly related to the sex and age at onset of diabetes in the diabetic parents.

Regulation of pancreatic β-cell mass is essential to preserve sufficient insulin levels for the maintenance of glucose homeostasis. Previously, we reported that DNA double strand breaks (DSBs) resulting from non-homologous end-joining (NHEJ) deficiency induce apoptosis, and when combined with p53 deficiency progressed rapidly into lymphomagenesis in mice. Combination of NHEJ-deficiency with a hypomorphic mutation, p53R172P, leads to the abrogation of apoptosis, up-regulation of p21 and senescence in precursor lymphocytes. This was sufficient to prevent tumorigenesis. However these mutant mice succumb to severe diabetes and die at an early age. The aim of this study is to determine the pathogenesis of diabetes in these mutant mice.

We analyzed the morphology of the pancreatic islets, the function, proliferation rate and senescence of β-cells. We also profiled DNA damage, p53 and p21 expression in the pancreas.

NHEJ-p53R172P mutant mice succumb to diabetes at 3-5 months of age. These mice show a progressive decrease in pancreatic islet mass that is independent of apoptosis and innate immunity. We observe an accumulation of DNA damage, accompanied with increased levels of p53 and p21, a significant decrease in β-cell proliferation, and cellular senescence in the mutant pancreatic islets.

Combined DSBs with an absence of p53-dependent apoptosis activates p53-dependent senescence, which leads to a diminished β-cell self-replication, massive depletion of the pancreatic islets and severe diabetes. This is a model that connects impaired DNA repair and accumulative DNA damage, a common phenotype in ageing individuals, to the onset of diabetes.

To determine whether the cell cycle transcription factor, FoxM1, is required for glucose homeostasis and β-cell mass expansion in maternal islets during pregnancy, and whether FoxM1 is essential for placental lactogen (PL)-induced β-cell proliferation.

β-cell mass, β-cell proliferation, and glucose homeostasis were assessed in virgin, pregnant, and postpartum mice with a pancreas-wide Foxm1 deletion (FoxM1^panc). Wild type islets were cultured with or without PL and examined for Foxm1 induction. Transgenic mice over-expressing PL in β-cells were bred with FoxM1^panc mice and β-cell proliferation was examined.

Foxm1 was up-regulated in maternal islets during pregnancy. In contrast to controls, β-cell proliferation did not increase in pregnant FoxM1^panc females. Mutant islets showed increased Menin and nuclear p27. FoxM1^panc females developed gestational diabetes as pregnancy progressed. After parturition, euglycemia was restored in FoxM1^panc females, but islet size was significantly reduced. Strikingly, β-cell mass was normal in postpartum FoxM1^panc pancreata, due to a combination of increased β-cell size and islet neogenesis. Evidence for neogenesis included increased number of endocrine clusters, increased proportion of smaller islets, and increased NGN3 or insulin expression in cells adjacent to ducts. PL induced Foxm1 expression in cultured islets and FoxM1 was essential for PL-mediated increases in β-cell proliferation in vivo.

FoxM1 is essential for β-cell compensation during pregnancy. In the absence of increased β-cell proliferation, neogenesis is induced in postpartum FoxM1^panc pancreata. Our results suggest that FoxM1 functions downstream of PL to mediate its effects on β-cell proliferation.

Reductions in insulin sensitivity in conjunction with muscle mitochondrial dysfunction have been reported to occur in many conditions including aging. The objective was to determine whether insulin-resistance and mitochondrial dysfunction are directly related to chronological age, or are related to age-related changes in body composition.

Twelve young lean, 12 young obese, 12 elderly lean and 12 elderly obese sedentary adults were studied. Insulin sensitivity was measured by a hyperinsulinemic-euglycemic clamp and skeletal muscle mitochondrial ATP production rates (MAPR) were measured in freshly isolated mitochondria obtained from vastus lateralis biopsy samples using the luciferase reaction.

Obese participants, independent of age, had reduced insulin sensitivity based on lower rates of glucose infusion during hyperinsulinemic-euglycemic clamp. In contrast, age had no independent effect on insulin sensitivity. However, the elderly participants had lower muscle MAPR than the young participants, independent of obesity. Elderly participants also had higher levels inflammatory cytokines and total adiponectin. In addition, higher muscle MAPR were also noted in men than in women whereas glucose infusion rates were higher in women.

The results demonstrate that age-related reductions in insulin sensitivity are likely due to an age-related increase in adiposity rather than a consequence of advanced chronological age. The results also indicate that age-related decrease in muscle mitochondrial function is neither related to adiposity or insulin sensitivity. Of interest, a higher mitochondrial ATP production capacity was noted in the men, whereas the women were more insulin sensitive demonstrating further dissociation between insulin sensitivity and muscle mitochondrial function.

The ~45 cM Insulin-dependent diabetes 9 (Idd9) region on mouse chromosome 4 harbors several different type 1 diabetes (T1D)-associated loci. Nonobese diabetic (NOD) mice congenic for the Idd9 region of C57BL/10 (B10) mice, carrying anti-diabetogenic alleles in three different Idd9 sub-regions (Idd9.1, Idd9.2 and Idd9.3), are strongly resistant to T1D. However, the mechanisms remain unclear. This study aimed to define mechanisms underlying the T1D resistance afforded by B10 Idd9.1, Idd9.2 and/or Idd9.3.

We used a reductionist approach that involves comparing the fate of a T1D-relevant autoreactive CD8+ T-cell population, specific for residues 206–214 of islet-specific glucose 6 phosphatase catalytic subunit-related protein (IGRP_206-214), in non-congenic versus B10-Idd9-congenic (Idd9.1+Idd9.2+Idd9.3, Idd9.2+Idd9.3, Idd9.1, Idd9.2 and Idd9.3) TCR-transgenic (8.3) NOD mice.

Most of the protective effect of Idd9 against 8.3-CD8+ T-cell-enhanced T1D was mediated by Idd9.1. Although Idd9.2 and Idd9.3 afforded some protection, the effects were small, and did not enhance the greater protective effect of Idd9.1. B10 Idd9.1 afforded T1D resistance without impairing the developmental biology or intrinsic diabetogenic potential of autoreactive CD8+ T-cells. Studies in T- and B-cell-deficient 8.3-NOD.B10 Idd9.1 mice revealed that this anti-diabetogenic effect was mediated by endogenous, non-transgenic T-lymphocytes in a B-cell-independent manner. Consistent with this, B10 Idd9.1 increased the suppressive function and anti-diabetogenic activity of the FoxP3+CD4+CD25+ T-cell subset, both in TCR-transgenic and non-transgenic mice.

A gene(s) within Idd9.1 regulates the development and function of FoxP3+CD4+CD25+ Treg cells and, in turn, the activation of CD8+ effector T-cells in the pancreas draining lymph nodes, without affecting their development or intrinsic diabetogenic potential.

Carbohydrate-Responsive Element-Binding Protein (ChREBP) is a transcription factor which has been shown to regulate carbohydrate metabolism in the liver and pancreatic β cells in response to elevated glucose concentrations. Since very few genes have been identified so far as bona fida ChREBP-target genes, we have performed a genome-wide analysis of the ChREBP transcriptome in pancreatic β cells.

Chromatin immunoprecipitation and high density oligonucleotide tiling arrays (ChIP-chip, Agilent technologies) using MIN6 pancreatic β cell extracts was performed together with transcriptional and other analysis using standard techniques.

One of the genes identified by ChIP-chip and linked to glucose-sensing and insulin secretion was ARNT/HIF1β, a transcription factor implicated in altered gene expression and pancreatic-islet dysfunction in type 2 diabetes. We first confirmed that elevated glucose concentrations decreased ARNT/HIF-1β levels in INS-1 (832/13) cells and primary mouse islets. Demonstrating a role for ChREBP in ARNT gene regulation, ChREBP silencing increased ARNT mRNA levels in INS-1 (832/13) cells and ChREBP over-expression decreased ARNT mRNA in INS-1 (832/13) cells and primary mouse islets. We demonstrated that ChREBP and MLX bind on ARNT/HIF-1β promoter on the proximal region that also confers the negative glucose responsiveness.

These results demonstrate that ChREBP acts as a novel repressor of the ARNT/HIF-1β gene and might contribute to β cell dysfunction induced by glucotoxicity.

In vitro models suggest that free fatty acid-induced apoptotic β-cell death is mediated through PKC. To examine the role of PKC signaling in vivo, transgenic mice over-expressing a kinase negative PKC (PKCKN) selectively in β-cells were generated and analyzed for glucose homeostasis and β-cell survival.

Mice were fed a standard or high fat diet (HFD). Blood glucose and insulin levels were determined after glucose loads. Islet size, cleaved caspase-3 and PKC expression were estimated by immunohistochemistry. In isolated islet cells apoptosis was assessed with TUNEL/TO-PRO3 DNA staining and the mitochondrial potential by rhodamine-123 staining. Changes in phosphorylation and subcellular distribution of FoxO1 were analyzed by Western blotting and immunohistochemistry.

PKCKN mice were protected from HFD–induced glucose intolerance. This was accompanied by increased insulin levels in vivo, by an increased islet size and by a reduced staining of β-cells for cleaved caspase-3 compared to wild-type (WT) littermates. In accordance, long-term treatment with palmitate increased apoptotic cell death of isolated islet cells from WT but not from PKCKN mice. PKCKN over-expression protected islet cells from palmitate-induced mitochondrial dysfunction and inhibited nuclear accumulation of FoxO1 in mouse islet and INS-1E cells. The inhibition of nuclear accumulation of FoxO1 by PKCKN was accompanied by an increased phosphorylation of FoxO1 at Ser256 and a significant reduction of FoxO1 protein.

Over-expression of PKCKN in β-cells protects from HFD-induced β-cell failure in vivo by a mechanism that involves inhibition of fatty acid-mediated apoptosis, inhibition of mitochondrial dysfunction and inhibition of FoxO1 activation.

At least twenty type 2 diabetes loci have now been identified and several of these are associated with altered beta-cell function. In this study we have investigated the combined effects of eight known beta cell loci on insulin secretion stimulated by three different secretagogues during hyperglycemic clamps.

447 subjects originating from four independent studies in the Netherlands and Germany (256 NGT/191 IGT) underwent a hyperglycemic clamp. A subset had an extended clamp with additional GLP-1 and arginine (n=224). We next genotyped SNPs in TCF7L2, KCNJ11, CDKAL1, IGF2BP2, HHEX/IDE, CDKN2A/B, SLC30A8 and MTNR1B and calculated a risk allele score by risk allele counting.

The risk allele score was associated with lower 1^st phase glucose stimulated insulin secretion (GSIS) (p=7.1*10^-6). The effect size was equal in NGT and IGT subjects. We also noted an inverse correlation with the disposition index (p=1.6*l10^-3). When we stratified the study population according to the number of risk alleles into three groups those with a medium or high risk allele score had 9% and 23% lower 1^st phase GSIS. 2^nd phase GSIS, ISI and GLP-1 or arginine stimulated insulin release were not significantly different.

A combined risk allele score for eight known beta cell genes is associated with the rapid 1^st phase GSIS and the disposition index. The slower 2^nd phase GSIS, GLP-1 and arginine stimulated insulin secretion are not associated suggesting that especially processes involved in rapid granule recruitment and exocytosis are affected in the majority of risk loci.

The intraislet insulin hypothesis proposes that glucagon secretion during hypoglycemia is triggered by a decrease in intraislet insulin secretion. A more recent hypothesis based on in vivo data from hypoglycemic rats is that it is the decrease in zinc co-secreted with insulin from β-cells, rather than the decrease in insulin itself, that signals glucagon secretion from -cells during hypoglycemia. These studies were designed to determine whether closure of the -cell K_ATP channel is the mechanism through which the zinc switch-off signal triggers glucagon secretion during glucose deprivation.

All studies were performed using perifused isolated islets.

In control experiments, the expected glucagon response to an endogenous insulin switch-off signal during glucose deprivation was observed in wild type mouse islets. In experiments with streptozotocin-treated wild type islets, a glucagon response to an exogenous zinc switch-off signal was observed during glucose deprivation. However, this glucagon response to the zinc switch-off signal during glucose deprivation was not seen in the presence of nifedipine, diazoxide, or tolbutamide, or if K_ATP channel knockout mouse islets were used. All islets had intact glucagon responses to epinephrine.

These data demonstrate that closure of K_ATP channels and consequent opening of calcium channels is the mechanism through which the zinc switch-off signal triggers glucagon secretion during glucose deprivation.

Activation of ERK1/2 by cytokines in adipocytes is involved in the alterations of adipose tissue functions participating in insulin resistance. This study aims at identifying proteins regulating ERK1/2 activity specifically in response to inflammatory cytokines to provide new insights into mechanisms leading to abnormal adipose tissue function.

Kinase activities were inhibited with pharmacological inhibitors or siRNA. Lipolysis was monitored through glycerol production. Gene expression in adipocytes and adipose tissue of obese mice and subjects was measured by real-time PCR.

IKKβ inhibition prevented MEK/ERK1/2 activation in response to IL-1β and TNF- but not insulin in 3T3-L1 and human adipocytes suggesting that IKKβ regulated a MAP kinase kinase kinase (MAP3K) involved in ERK1/2 activation induced by inflammatory cytokines. We show that the MAP3K8 called Tpl2 was expressed in adipocytes and that IL-1β and TNF- activated Tpl2 and regulated its expression through an IKKβ pathway. Pharmacological inhibition or silencing of Tpl2 prevented MEK/ERK1/2 activation by these cytokines but not by insulin demonstrating its involvement in ERK1/2 activation specifically in response to inflammatory stimuli. Importantly, Tpl2 was implicated in cytokines-induced lipolysis and in IRS1 serine phosphorylation. Tpl2 mRNA expression was up-regulated in adipose tissue of obese mice and patients and correlated with TNF- expression.

Tpl2 is selectively involved in inflammatory cytokines-induced ERK1/2 activation in adipocytes and is implicated in their deleterious effects on adipocytes functions. The deregulated expression of Tpl2 in adipose tissue suggests that Tpl2 may be a new actor in adipose tissue dysfunction in obesity.

Extra cellular nucleotides and nucleosides are involved in regulation of skeletal muscle blood flow. Diabetes induces cardiovascular dysregulation but the extent to which the vasodilatatory capacity of nucleotides and nucleosides are affected in type 2 diabetes is unknown. The present study investigated: 1) the vasodilatatory effect of ATP, UTP, and adenosine (ADO) and 2) the expression and distribution of P2Y₂ and P2X₁ receptors in skeletal muscles of diabetic subjects.

In 10 diabetic patients and 10 age-matched controls, leg blood flow (LBF) was measured during intrafemoral artery infusion of ATP, UTP, and ADO eliciting a blood flow equal to knee-extensor exercise at 12 watts (~2.6 L/min).

The vasodilatatory effect of the purinergic system was 50 % lower in the diabetic group as exemplified by a LBF increase by 274±37 vs. 143±26 ml/µ;mol ATP x kg; by 494±80 vs. 234±39 ml/µmol UTP x kg; and by 14.9±2.7 vs. 7.5±0.6 ml/µmol ADO x kg in control and diabetic subjects, respectively, thus making the vasodilator potency: UTP-controls (100) > ATP-controls (55) > UTP-DM (47) > ATP-DM (29) > ADO-controls (3) > ADO-DM (1.5). The distribution and mRNA-expression of receptors were similar in the two groups.

The vasodilatatory effect of the purinergic system is severely reduced in type 2 diabetic patients. The potency of nucleotides varies with the following rank order: UTP>ATP>>>ADO. This is not due to alterations in receptor distribution and mRNA expression, but may be due to differences in receptor sensitivity.

To investigate the role of the endoplasmic reticulum (ER) chaperone GRP78/BiP in the pathogenesis of obesity, insulin resistance, and type 2 diabetes.

Male Grp78+/– mice and their wild-type littermates were subjected to high-fat diet (HFD) regimen. Pathogenesis of obesity and type 2 diabetes was examined by multiple approaches of metabolic phenotyping. Tissue-specific insulin sensitivity was analyzed by hyperinsulinemic-euglycemic clamps. Molecular mechanism was explored via immunoblotting and tissue culture manipulation.

Grp78 heterozygosity increases energy expenditure and attenuates HFD-induced obesity. Grp78+/– mice are resistant to diet-induced hyperinsulinemia, liver steatosis, white adipose tissue (WAT) inflammation and hyperglycemia. Hyperinsulinemic-euglycemic clamp studies revealed that Grp78 heterozygosity improves glucose metabolism independent of adiposity, and following HFD increases insulin sensitivity predominantly in WAT. As mechanistic explanations, Grp78 heterozygosity in WAT under HFD stress promotes adaptive unfolded protein response (UPR), attenuates translational block and upregulates EDEM and ER chaperones, thus improving ER quality control and folding capacity. Further, overexpression of the active form of ATF6 induces protective UPR and improves insulin signaling upon ER stress.

HFD-induced obesity and type 2 diabetes are improved in Grp78+/– mice. Adaptive UPR in WAT could contribute to this improvement, linking ER homeostasis to energy balance and glucose metabolism.

The effect of diabetes on neovascularization varies between different organ systems. While excessive angiogenesis complicates diabetic retinopathy, impaired neovascularization contributes to coronary and peripheral complications of diabetes. However, how diabetes influences cerebral neovascularization is not clear. Our aim was to determine diabetes-mediated changes in the cerebrovasculature and its impact on the short-term outcome of cerebral ischemia.

Angiogenesis (capillary density) and arteriogenesis (number of collaterals and intratree anostomoses) were determined as indices of neovascularization in the brain of control and type 2 diabetic Goto-Kakizaki (GK) rats. The infarct volume, edema, hemorrhagic transformation (HT), and short-term neurological outcome were assessed after permanent middle cerebral artery occlusion (MCAO).

The number of collaterals between middle and anterior cerebral arteries, the anastomoses within MCA trees, the vessel density, and the level of brain derived neurotrophic factor were increased in diabetes. Cerebrovascular permeability, matrix metalloproteinase (MMP)-9 protein level, and total MMP activity was augmented while occludin was decreased in isolated cerebrovessels of the GK group. Following permanent MCAO, infarct size was smaller, edema was greater, and there was no macroscopic HT in GK rats.

The augmented neovascularization in GK model includes both angiogenesis and arteriogenesis. While adaptive arteriogenesis of the pial vessels and angiogenesis at the capillary level may contribute to smaller infarction, changes in the tight junction proteins may lead to the greater edema following cerebral ischemia in diabetes.

An emerging model of metabolic syndrome and type-2 diabetes is of adipose dysfunction with leukocyte recruitment into adipose leading to chronic inflammation and insulin resistance (IR). This study sought to explore potential mechanisms of inflammatory-induced IR in humans with a focus on adipose tissue.

We performed a sixty-hour endotoxemia protocol (3 ng/kg intravenous bolus) in healthy adults (N=20, 50% male, 80% Caucasian, age 27.3±4.8). Before and after endotoxin, whole blood sampling, subcutaneous adipose biopsies, and frequently-sampled intravenous glucose tolerance (FSIGT) testing were performed. The primary outcome was the FSIGT insulin sensitivity index (SI). Secondary measures included inflammatory and metabolic markers, and whole blood and adipose mRNA and protein expression.

Endotoxemia induced systemic IR as demonstrated by a 35% decrease in SI (3.17 ± 1.66 to 2.06 ± 0.73 x 10^–4 (µU/ml)^–1·min^–1, p<0.005) while there was no effect on pancreatic beta-cell function. In adipose, endotoxemia suppressed insulin receptor substrate-1 and markedly induced suppressor of cytokine signaling (SOCS) proteins (1 and 3) coincident with local activation of innate (IL-6, TNF) and adaptive (monocyte chemoattractant protein-1 and CXCL10 chemokines) inflammation. These changes are known to attenuate insulin receptor signaling in model systems.

We demonstrate, for the first time in humans, that acute inflammation induces systemic IR following modulation of specific adipose inflammatory and insulin signaling pathways. It also provides a rationale for focused mechanistic studies and a model for human proof-of-concept trials of novel therapeutics targeting adipose inflammation in IR and related consequences in humans.

Aggregation of human amylin/islet amyloid polypeptide (hA/hIAPP) into small soluble β-sheet-containing oligomers is linked to islet β-cell degeneration and the pathogenesis of type 2 diabetes. Here, we employed tetracycline, which modifies hA/hIAPP oligomerization, to probe mechanisms whereby hA/hIAPP causes diabetes in hemizygous hA/hIAPP-transgenic mice.

We chronically treated hemizygous hA/hIAPP transgenic mice with oral tetracycline to determine its effects on rates of diabetes initiation, progression and survival.

Homozygous mice developed severe spontaneous diabetes due to islet β-cell loss. Hemizygous transgenic animals also developed spontaneous diabetes, although severity was less and progression rates slower. Pathogenesis was characterised by initial islet β-cell dysfunction followed by progressive β-cell loss. Islet amyloid was absent from hemizygous animals with early-onset diabetes and correlated positively with longevity. Some long-lived non-diabetic hemizygous animals also had large islet-amyloid areas showing that amyloid itself was not intrinsically cytotoxic. Administration of tetracycline dosage-dependently ameliorated hyperglycemia and polydipsia, delayed rates of diabetes initiation and progression, and increased longevity compared with water-treated controls.

This is the first report to show that treating hA/hIAPP transgenic mice with a modifier of hA/hIAPP-misfolding can ameliorate their diabetic phenotype. Fibrillar amyloid was neither necessary nor sufficient to cause diabetes and indeed was positively correlated with longevity therein, whereas early- to mid-stage diabetes was associated with islet β-cell dysfunction followed by β-cell loss. Interventions capable of suppressing misfolding in soluble hA/hIAPP oligomers rather than mature fibrils may have potential for treating or preventing type-2 diabetes.

An emerging model of metabolic syndrome and type-2 diabetes is of adipose dysfunction with leukocyte recruitment into adipose leading to chronic inflammation and insulin resistance (IR). This study sought to explore potential mechanisms of inflammatory-induced IR in humans with a focus on adipose tissue.

We performed a sixty-hour endotoxemia protocol (3 ng/kg intravenous bolus) in healthy adults (N=20, 50% male, 80% Caucasian, age 27.3±4.8). Before and after endotoxin, whole blood sampling, subcutaneous adipose biopsies, and frequently-sampled intravenous glucose tolerance (FSIGT) testing were performed. The primary outcome was the FSIGT insulin sensitivity index (SI). Secondary measures included inflammatory and metabolic markers, and whole blood and adipose mRNA and protein expression.

Endotoxemia induced systemic IR as demonstrated by a 35% decrease in SI (3.17 ± 1.66 to 2.06 ± 0.73 x 10^–4 (µU/ml)^–1·min^–1, p<0.005) while there was no effect on pancreatic beta-cell function. In adipose, endotoxemia suppressed insulin receptor substrate-1 and markedly induced suppressor of cytokine signaling (SOCS) proteins (1 and 3) coincident with local activation of innate (IL-6, TNF) and adaptive (monocyte chemoattractant protein-1 and CXCL10 chemokines) inflammation. These changes are known to attenuate insulin receptor signaling in model systems.

We demonstrate, for the first time in humans, that acute inflammation induces systemic IR following modulation of specific adipose inflammatory and insulin signaling pathways. It also provides a rationale for focused mechanistic studies and a model for human proof-of-concept trials of novel therapeutics targeting adipose inflammation in IR and related consequences in humans.

Hyperglycemia impairs angiogenesis in response to ischemia, leading to ventricular remodeling. Although the effects of overexpressing angiogenic growth factors have been studied in inducing angiogenesis, the formation of functional vessels remains a challenge. The present study evaluates the reversal of diabetes mediated impairment of angiogenesis in the infarcted diabetic rat myocardium by pro-angiogenic gene therapy.

Ad.VEGF and Ad.Ang1 were intramyocardially administered in combination immediately after myocardial infarction to non-diabetic and diabetic rats. Ad.LacZ was similarly administered to the respective control groups. The hearts were excised for molecular and immunohistochemical analysis at predetermined time points. The myocardial function was measured by echocardiography 30 days after the intervention.

We observed reduced fibrosis and increased capillary/arteriolar density along with reduced ventricular remodeling, as assessed by echocardiography in the treated diabetic animals when compared to the non-treated diabetic controls. We have also observed increased p-MK2, 2 days after the treatment and increased expression of VEGF, Flk-1, Ang-1, Tie-2, and survivin, 4 days after treatment in the diabetic animals. Gel shift analysis revealed that the combination gene therapy stimulated the DNA binding activity of NFB in the diabetic animals.

Our preclinical data demonstrates the efficacy of co-administration of adenoviral VEGF and Ang-1 in increasing angiogenesis and reducing ventricular remodeling in the infarcted diabetic myocardium. These unique results calls for the initiation of a clinical trial to assess the efficacy of this therapeutic strategy in the treatment of diabetes related human heart failure.

The contribution of antecedent viral infection to the development of type 1 diabetes in humans is controversial. Using a newer rat model of the disease, we sought to 1) identify viruses capable of modulating diabetes penetrance, 2) identify conditions that increase or decrease the diabetogenicity of infection, and 3) determine if maternal immunization would prevent diabetes.

About 2% of LEW.1WR1 rats develop spontaneous autoimmune diabetes, but disease penetrance is much higher if weanling rats are exposed to environmental perturbants including Kilham rat virus (KRV). We compared KRV with other viruses for diabetogenic activity.

Both KRV and rat cytomegalovirus (RCMV) induced diabetes in up to 60% of LEW.1WR1 rats, whereas H-1, vaccinia, and Coxsackie B4 viruses did not. Simultaneous inoculation of KRV and RCMV induced diabetes in 100% of animals. Pretreatment of rats with an activator of innate immunity increased the diabetogenicity of KRV but not RCMV, and was associated with a moderate rate of diabetes after Coxsackie B4 and vaccinia virus infection. Inoculation of LEW.1WR1 dams with both KRV and RCMV prior to pregnancy protected weanling progeny from virus-induced diabetes in a virus-specific manner.

Exposure to viruses can affect the penetrance of autoimmune diabetes in genetically susceptible animals. The diabetogenicity of infection is virus-specific and is modified by immunomodulation prior to inoculation. Maternal immunization protects weanlings from virus-induced diabetes, suggesting that modification of immune responses to infection could provide a means of preventing islet autoimmunity.

We have hypothesised that variation in imprinted growth promoting fetal genes may affect maternal glucose concentrations in pregnancy. In order to test this hypothesis we evaluated the effects of fetal disruption of murine H19¹³ on maternal glucose concentrations in pregnancy.

Experimental mice were pregnant females, who had inherited the disrupted H19¹³ from their fathers so were phenotypically wild type due to imprinting, where approximately half of their litter were null for H19¹³ through maternal inheritance of the disrupted gene. In control mice approximately half the litter paternally-inherited the disrupted H19¹³, so the pups were either genetically wild type or phenotypically wild type due to imprinting. Blood glucose concentrations were assessed by intra-peritoneal glucose tolerance tests on days 1, 16 and 18 of pregnancy.

There were no differences in the glucose concentrations of control and experimental pregnant mice at day 1 (e1). However at e16 mothers carrying H19¹³-null pups had a significantly higher area under the glucose tolerance test curves than controls (1845 ± 378 v. 1386 ± 107 mmol.min/L (p=0.01)) in association with increasing pregnancy-related insulin resistance. Although this difference lessened towards term, overall mothers of maternally-inherited H19¹³ mutants had significantly higher glucose concentrations during the last trimester (1602 ± 321 (n=17) v. 1359 ± 147 (n=18) mmol.min/L (p=0.009)).

This study provides evidence that maternal glucose concentrations in pregnant mice can be affected by targeted disruption of fetal H19¹³. This implies that variable fetal IGF2 expression could affect risk for gestational diabetes.

Mutations in the HNF1A gene are the most common cause of MODY. There is a substantial variation in the age at diabetes diagnosis, even within families where diabetes is caused by the same mutation. We investigated the hypothesis that common polygenic variants that predispose to type 2 diabetes might account for the difference in age at diagnosis.

Fifteen robustly associated T2D variants were successfully genotyped in 410 individuals from 203 HNF1A-MODY families, from two study centers in the UK and Norway. We assessed their effect on the age at diagnosis both individually and in a combined genetic score by summing the number of T2D risk alleles carried by each patient.

We confirmed the effects of environmental and genetic factors known to modify the age at HNF1A-MODY diagnosis, namely intrauterine hyperglycemia (–5.1 years if present, P=1.6x10^–10), and HNF1A mutation position (–5.2 years if at least two isoforms affected, P=1.8x10^–2). Additionally, our data showed strong effects of gender (females diagnosed 3.0 years earlier, P=6.0x10^–4), and age at study (0.3 years later diagnosis per year increase in age, P=4.7x10^–38). There were no strong individual SNP effects; however, in the combined genetic score model, each additional risk allele was associated with 0.35 years earlier diabetes diagnosis (P=5.1x10^–3).

We show that T2D risk variants of modest effect sizes reduce the age at diagnosis in HNF1A-MODY. This is one of the first studies to demonstrate that clinical characteristics of a monogenic disease can be modified by common polygenic variants.

Alteration in endoplasmic reticulum (ER) stress in diabetic hearts and its effect on cytoprotective signaling are unclear. Here we examine the hypothesis that ER stress in diabetic hearts impairs phospho-GSK-3β-mediated suppression of mitochondrial permeability transition pore (mPTP) opening, compromising myocardial response to cytoprotective signaling.

A rat model of type 2 diabetes (OLETF) and its control (LETO) were treated with TUDCA (100 mg/kg/day for 7 days), an ER stress modulator. Infarction was induced by 20-min coronary occlusion and 2-h reperfusion.

Levels of ER chaperones (GRP78 and GRP94) in the myocardium and level of non-phoshopho-GSK-3β in the mitochondria were significantly higher in OLETF than in LETO. TUDCA normalized levels of GRP78 and GRP94 and mitochondrial GSK-3β in OLETF. Administration of EPO induced phosphorylation of Akt and GSK-3β and reduced infarct size (% risk area) from 47.4±5.2% to 23.9±3.5% in LETO hearts. However, neither phosphorylation of Akt and GSK-3β nor infarct size limitation was induced by EPO in OLETF. The threshold for mPTP opening was significantly lower in mitochondria from EPO-treated OLETF than in those from EPO-treated LETO. TUDCA restored responses of GSK-3β, mPTP opening threshold and infarct size to EPO receptor activation in OLETF. There was a significant correlation between mPTP opening threshold and phospho-GSK-3β-to-total-GSK-3β ratio in the mitochondrial fraction.

Disruption of protective signals leading to GSK-3β phosphorylation and increase in mitochondrial GSK-3β are dual mechanisms by which increased ER stress inhibits EPO-induced suppression of mPTP opening and cardioprotection in diabetic hearts.

Insulin represses the expression of gluconeogenic genes at the mRNA level, but the hormone appears to have only weak inhibitory effects in vivo. The aims of this study were to determine 1) the maximal physiologic effect of insulin, 2) the relative importance of its effects on gluconeogenic regulatory sites and to 3) correlate those changes with alterations at the cellular level.

Conscious 60-h-fasted dogs were studied at three insulin levels (near basal, 4x, or 16x) during a 5-h euglycemic clamp. Pancreatic hormones were controlled using somatostatin with portal insulin and glucagon infusions. Glucose metabolism was assessed using the arterio-venous difference technique and molecular signals were assessed.

Insulin reduced gluconeogenic flux to G6P but only at the near maximal physiological level (16x basal). The effect was modest compared to its inhibitory effect on net hepatic glycogenolysis, it occurred within 30 min, and was associated with a marked decrease in hepatic fat oxidation, increased liver fructose-2,6-bisphosphate level, and reductions in lactate, glycerol and amino acid extraction. No further diminution in gluconeogenic flux to G6P occurred over the remaining 4.5-h of the study, despite a marked decrease in PEPCK content, suggesting poor control strength for this enzyme in gluconeogenic regulation in the dog.

Gluconeogenic flux can be rapidly inhibited by high insulin levels in the dog. Initially decreased hepatic lactate extraction is important and later reduced gluconeogenic precursor availability plays a role. Changes in PEPCK appear to have little or no acute effect on gluconeogenic flux.

As data on the predictive characteristics of diabetes-associated autoantibodies for type 1 diabetes in the general population are scarce, we assessed the predictive performance of islet cell autoantibodies (ICA) in combination with autoantibodies against insulin (IAA), glutamic acid decarboxylase (GADA), and/or islet antigen 2 (IA-2A) for type 1 diabetes in children with HLA-defined disease predisposition recruited from the general population.

We observed 7410 children from birth (median 9.2 years) for β-cell autoimmunity and diabetes. If a child developed ICA positivity or diabetes, also the three other antibodies were measured in all samples available from that individual. Persistent autoantibody positivity was defined as continued positivity in at least two sequential samples including the last available sample.

Prediabetic ICA positivity was observed in 1173 subjects (15.8%), 155 of whom developed type 1 diabetes. With ICA screening 86% of the 180 progressors (median age at diagnosis 5.0 years) were identified. Positivity for four antibodies was associated with the highest disease sensitivity (54.4%) and NPV (98.3%) and the lowest negative likelihood ratio (0.5). The combination of persistent ICA and IAA positivity resulted in the highest PPV (91.7%), positive LR (441.8), cumulative disease risk (100%), and specificity (100%). Young age at seroconversion, high ICA level, multipositivity, and persistent positivity for IAA were significant risk markers for type 1 diabetes.

Within the general population the combination of HLA and autoantibody screening resulted in disease risks that are likely to be as high as those reported among autoantibody-positive siblings of children with type 1 diabetes.

The medial hypothalamus mediates leptin-induced glucose uptake in peripheral tissues, and brain melanocortin receptors (MCRs) mediate certain central effects of leptin. However, the contributions of the leptin receptor and MCR in individual medial hypothalamic nuclei to regulation of peripheral glucose uptake have remained unclear. We examined the effects of injection of leptin and the MCR agonist MT-II into medial hypothalamic nuclei on glucose uptake in peripheral tissues.

Leptin or MT-II was injected into the ventromedial (VMH), dorsomedial (DMH), arcuate (ARC), or paraventricular (PVH) hypothalamus or the lateral ventricle (i.c.v.) in freely moving mice. The MCR antagonist SHU9119 was injected i.c.v. Glucose uptake was measured by the 2-[³H]deoxy-D-glucose method.

Leptin injection into VMH increased glucose uptake in skeletal muscle, brown adipose tissue (BAT), and heart, whereas that into ARC increased glucose uptake in BAT and that into DMH or PVH had no effect. SHU9119 abolished these effects of leptin injected into VMH. Injection of MT-II either into VMH or i.c.v. increased glucose uptake in skeletal muscle, BAT, and heart, whereas that into PVH increased glucose uptake in BAT and that into DMH or ARC had no effect.

VMH mediates leptin- and MT-II-induced glucose uptake in skeletal muscle, BAT, and heart. These effects of leptin are dependent on MCR activation. The leptin receptor in ARC and MCR in PVH regulate glucose uptake in BAT. Medial hypothalamic nuclei thus play distinct roles in leptin- and MT-II-induced glucose uptake in peripheral tissues.

An increase in the rate of hepatic glucose production is the major determinant of fasting hyperglycemia in type 2 diabetes. A better understanding of the signaling pathways and molecules that regulate hepatic glucose metabolism is therefore of great clinical importance. Recent studies suggest that an increase in vagal outflow to the liver leads to decreased hepatic glucose production and reduced blood glucose levels. Since acetylcholine (ACh) is the major neurotransmitter of the vagus nerve and exerts its parasympathetic actions via activation of muscarinic ACh receptors (mAChRs), we examined the potential metabolic relevance of hepatocyte mAChRs.

We initially demonstrated that the M₃ mAChR is the only mAChR subtype expressed by mouse liver/hepatocytes. To assess the physiological role of this receptor subtype in regulating hepatic glucose fluxes and glucose homeostasis in vivo, we used gene targeting and transgenic techniques to generate mutant mice lacking or overexpressing M₃ receptors in hepatocytes only.

Strikingly, detailed in vivo phenotyping studies failed to reveal any significant metabolic differences between the M₃ receptor mutant mice and their control littermates, independent of whether the mice were fed regular chow or a high-fat diet. Moreover, the expression levels of genes for various key transcription factors, signaling molecules, and enzymes regulating hepatic glucose fluxes were not significantly altered in the M₃ receptor mutant mice.

This rather surprising finding suggests that the pronounced metabolic effects mediated by activation of hepatic vagal nerves are mediated by non-cholinergic signaling pathways.

Nur77 is an orphan nuclear receptor with pleotropic functions. Previous studies have identified Nur77 as a transcriptional regulator of glucose utilization genes in skeletal muscle and gluconeogenesis in liver. However, the net functional impact of these pathways is unknown. To examine the consequence of Nur77 signaling for glucose metabolism in vivo, we challenged Nur77 null mice with high-fat feeding.

Wildtype and Nur77 null mice were fed a high-fat diet (60% calories from fat) for 3 months. We determined glucose tolerance, tissue-specific insulin sensitivity, oxygen consumption, muscle and liver lipid content, muscle insulin-signaling, and expression of glucose and lipid metabolism genes.

Mice with genetic deletion of Nur77 exhibited increased susceptibility to diet-induced obesity and insulin resistance. Hyperinsulinemic-euglycemic clamp studies revealed greater HFD-induced insulin resistance in both skeletal muscle and liver of Nur77 null mice compared to controls. Loss of Nur77 expression in skeletal muscle impaired insulin signaling and markedly reduced Glut4 protein expression. Muscles lacking Nur77 also exhibited increased triglyceride content and accumulation of multiple even-chained acylcarnitine species. In the liver, Nur77 deletion led to hepatic steatosis and enhanced expression of lipogenic genes, likely reflecting the lipogenic effect of hyperinsulinemia.

Collectively, these data demonstrate that loss of Nur77 impacts systemic glucose metabolism and highlight the physiological contribution of muscle Nur77 to this regulatory pathway.

GCK and G6PC2 regulate the glucose cycling step in pancreatic β-cells and may regulate insulin secretion. GCK rs1799884 and G6PC2 rs560887 have been independently associated with fasting glucose, but their interaction on glucose-insulin relationships is not well characterized.

We tested whether these variants are associated with diabetes-related quantitative traits in Mexican Americans from the BetaGene study and attempted to replicate our findings in Finnish men from the METSIM study.

rs1799884 was not associated with any quantitative trait (corrected p>0.1), whereas rs560887 was significantly associated with the OGTT 30-minute incremental insulin response (30' Insulin, corrected p=0.021). We found no association between quantitative traits and the multiplicative interaction between rs1799884 and rs560887 (p>0.26). However, the additive effect of these SNPs was associated with fasting glucose (corrected p=0.03) and 30' Insulin (corrected p=0.027). This additive association was replicated in METSIM (fasting glucose, p=3.5x10^–10 30' Insulin, p=0.028). When we examined the relationship between fasting glucose and 30' Insulin stratified by GCK and G6PC2, we noted divergent changes in these quantitative traits for GCK, but parallel changes for G6PC2. A similar pattern was observed in METSIM.

We conclude that variation in GCK and G6PC2 have additive effects on both fasting glucose and insulin secretion.

SIRT1 has pleiotropic metabolic functions. We investigated whether SIRT1 genetic variation is associated with obesity.

In 6251 elderly subjects from the prospective, population-based Rotterdam Study (RS), three Single Nucleotide Polymorphisms (SNPs) in the SIRT1 gene were studied in relation with body mass index (BMI) and risk of obesity (BMI ≥ 30kg/m²) and prospectively with BMI-change after 6.4 years follow-up. We used cross-sectional data from 2347 participants from the Erasmus Rucphen Family (ERF) study for replication.

Minor alleles of rs7895833 (G=20.2%) and rs1467568 (A=36.8%) were associated with lower BMI in the RS (P=0.02 and 0.04) and in the replication cohort ERF (P=0.03 and 0.008) and in studies combined (P=0.002 for both SNPs) with 0.2-0.4 kg/m² decrease in BMI per allele copy. Carriers of these alleles had 13-18% decreased risk of obesity; odds ratio (95%CI) for rs7895833 the RS: 0.79 (0.67-0.94), P=0.007; in ERF: 0.93 (0.73-1.19), P=0.37 and in the studies combined 0.87 (0.77-0.97), P=0.02; for rs1467568 in the RS: 0.80 (0.68-0.94), P=0.007; in ERF: 0.85 (0.72-0.99), P=0.04 and in the studies combined: 0.82 (0.73-0.92), P=0.0009. In the RS, the two variants were also associated with lower BMI increase during 6.4 years of follow-up (P=0.01 and 0.08).

Two common variants in SIRT1 are associated with lower BMI in two independent Dutch populations. Carriers of these variants have 13-18% decreased risk of obesity and gain less weight over time. The availability of SIRT1 stimulators makes these findings relevant in light of the growing obesity epidemic.

To investigate whether the effects of common genetic variants associated with fasting glucose in adults are detectable in healthy children.

Single nucleotide polymorphisms in MTNR1B (rs10830963), G6PC2 (rs560887) and GCK (rs4607517) were genotyped in 2,025 healthy European children aged 9-11 and 14-16 years. Associations with fasting glucose (FG), insulin, HOMA-IR and HOMA-B were investigated along with those observed for type 2 diabetes (T2D) variants available in this study (CDKN2A/B, IGF2BP2, CDKAL1, SLC30A8, HHEX-IDE, Chr 11p12).

Strongest associations were observed for G6PC2 and MTNR1B, with FG levels (95% CI) being 0.084 (0.06; 0.11) mmol/L, p=7.9x10^–11 and 0.069 (0.04; 0.09) mmol/L, p=1.9x10^–7 higher per risk allele copy. A similar, but weaker trend was observed for GCK (0.028 (–0.006; 0.06) mmol/L, p=0.11). All three variants were associated with lower β-cell function (HOMA-B p=9.38x10^–5, 0.004 and 0.04, respectively). SLC30A8 (rs13266634) was the only T2D variant associated with higher FG (0.033 mmol/L (0.01; 0.06); p=0.01). Calculating a genetic predisposition score adding the number of risk alleles of G6PC2, MTNR1B, GCK and SLC30A8 showed that glucose levels were successively higher in children carrying a greater number of risk alleles (p=7.1x10^–17), with mean levels of 5.34 versus 4.91 mmol/L comparing children with 7 alleles (0.6% of all children) to those with none (0.5%). No associations were found for fasting insulin or HOMA-IR with any of the variants.

The effects of common polymorphisms influencing FG are apparent in healthy children, where the presence of multiple risk alleles amounts to a difference of more than a standard deviation of FG.

Glucagon-like peptide-1 (GLP-1) is a regulatory peptide synthesized in the gut and the brain that plays an important role in the regulation of food intake. Both GLP-1 and exendin-4 (Ex4), a long-acting GLP-1 receptor (GLP-1r) agonist, reduce food intake when administered intracerebroventricularly (i3vt), whereas Ex4 is much more potent at suppressing food intake when given peripherally. It has generally been hypothesized that this difference is due to the relative pharmacokinetic profiles of GLP-1 and Ex4, but it is possible that the two peptides control feeding via distinct mechanisms.

In this study, the anorectic effects of i3vt GLP-1 and Ex4, and the sensitivity of these effects to GLP-1r antagonism, were compared in rats. In addition, the GLP-1r-dependence of the anorectic effect of i3vt Ex4 was assessed in GLP-1r-/- mice.

I3vt Ex4 was 100-fold more potent than GLP-1 at reducing food intake, and this effect was insensitive to GLP-1r antagonism. However, GLP-1r antagonists completely blocked the anorectic effect of intraperitoneal Ex4. Despite the insensitivity of i3vt Ex4 to GLP-1r antagonism, i3vt Ex4 failed to reduce food intake in GLP-1r-/- mice.

These data suggest that, although GLP-1r are required for the actions of Ex4, there appear to be key differences in how GLP-1 and Ex4 interact with CNS GLP-1r, and in how Ex4 interacts with GLP-1r in the brain versus the periphery. A better understanding of these unique differences may lead to expansion and/or improvement of GLP-1-based therapies for type 2 diabetes and obesity.

We studied how tolerance to GAD65 affected the development of autoimmunity to other ß-cell autoantigens (ß-CAAs) in GAD65-transgenic (GAD-tg) NOD mice.

We used ELISPOT to characterize the frequency and functional phenotype of T cell responses to GAD65 and other ß-CAAs at different ages in GAD-tg mice and their NOD mouse littermates.

In young GAD-tg mice, Th1 responses to GAD65's dominant determinants were 13% - 18% of those in young NOD mice. This coincided with a great reduction in Th1 responses to other ß-CAAs. Evidently, GAD65-reactive T cells are important for activating and/or expanding early autoreactivities in NOD mice. As GAD-tg mice aged, their T cell responses to GAD65 remained low, but they developed supernormal splenic and pancreatic lymph node T cell autoimmunity to other ß-CAAs. Apparently, the elimination/impairment of many GAD65-reactive T cells allowed other ß-CAA-reactive T cells to eventually expand to a greater extent, perhaps by reducing competition for APCs, or homeostatic proliferation in the target tissue, which may explain the GAD-tg mice's usual disease incidence.

Transgenically-induced reduction of GAD65 autoreactivity curtailed the development of early T cell responses to other ß-CAAs. However, later in life, ß-CAA-reactive T cells expanded to supernormal levels. These data suggest that early ß-cell autoreactivities are mutually dependent for support to activate and expand, while later in the disease process, autoantigen-specific T cell pools can expand autonomously. These findings have implications for understanding T1D immunopathogenesis and for designing antigen-based immunotherapeutics.

Chronic exposure of pancreatic β-cells to saturated free fatty acids (FFAs) causes endoplasmic reticulum (ER) stress and apoptosis, and may contribute to β-cell loss in type 2 diabetes. Here, we evaluated the molecular mechanisms involved in the protection of β-cells from lipotoxic ER stress by GLP-1 agonists, utilized in the treatment of type 2 diabetes.

INS-1E or FACS-purified primary rat β-cells were exposed to oleate or palmitate with or without the GLP-1 agonist exendin-4 or forskolin. CPA was used as a synthetic ER stressor, while the ATF4-CHOP branch was selectively activated with salubrinal. The ER stress signaling pathways modulated by GLP-1 agonists were studied by real time PCR and Western blot. Knockdown by RNA interference was used to identify mediators of the anti-apoptotic GLP-1 effects in the ER stress response and downstream mitochondrial cell death mechanisms.

Exendin-4 and forskolin protected β-cells against FFAs via the induction of the ER chaperone BiP and the anti-apoptotic protein JunB that mediate β-cell survival under lipotoxic conditions. On the other hand, exendin-4 and forskolin protected against synthetic ER stressors by inactivating caspase 12 and upregulating Bcl-2 and XIAP that inhibit mitochondrial apoptosis.

These observations suggest that GLP-1 agonists increase in a context-dependent way β-cell defense mechanisms against different pathways involved in ER stress-induced apoptosis. The identification of the pathways modulated by GLP-1 agonists allows for targeted approaches to alleviate β-cell ER stress in diabetes.

The aim of this study was to investigate the impact of nine days of bed rest on insulin secretion, insulin action and whole-body glucose and fat metabolism in first-degree relative (FDR) and matched control (CON) subjects.

13 FDR and 20 CON subjects participated in the study. All were studied before and after nine days of bed rest using the clamp technique combined with indirect calorimetry preceded by an intravenous glucose tolerance test. Glucose and glycerol turnover rates were studied using stable isotope kinetics.

Bed rest caused a significant decrease in whole body insulin sensitivity in both groups. Hepatic insulin resistance was elevated in FDR subjects prior to bed rest, and was significantly augmented by bed rest in FDR (P < 0.01) but not in CON subjects (P = NS). The rate of whole body lipolysis decreased during bed rest in both FDR and CON subjects, with no significant differences between the groups. Insulin resistance induced by bed rest was fully accounted for by the impairment of non-oxidative glucose metabolism in both groups (overall P < 0.001).

Whole body insulin action in both insulin resistant FDR and healthy CON subjects deteriorates with nine days of bed rest, converging towards similar degrees of whole body insulin resistance. FDR subjects exhibit hepatic insulin resistance, which, in contrast to CON subjects, deteriorates in response to physical inactivity. FDR subjects exhibit reduced insulin secretion when seen in relation to their degree of hepatic, but not peripheral, insulin resistance.

Digenic causes of human disease are rarely reported. Insulin via its receptor, encoded by INSR, plays a key role in both metabolic and growth signaling pathways. Heterozygous INSR mutations are the most common cause of monogenic insulin resistance. However, growth retardation is only reported with homozygous or compound heterozygous mutations. We describe a novel translocation [t(7;19)(p15.2;p13.2)] co-segregating with insulin resistance and pre- and post-natal growth deficiency. Chromosome translocations present a unique opportunity to identify modifying loci therefore our objective was to determine the mutational mechanism resulting in this complex phenotype.

Breakpoint mapping was performed by Fluorescence in-situ hybridisation (FISH) on patient chromosomes. Sequencing and gene expression studies of disrupted and adjacent genes were performed on patient derived tissues.

Affected individuals had increased insulin, c-peptide, insulin/c-peptide ratio and adiponectin levels consistent with an insulin receptoropathy. FISH mapping established that the translocation breakpoints disrupt INSR on chromosome 19p15.2 and CHN2 on chromosome 7p13.2. Sequencing demonstrated INSR haploinsufficiency accounting for elevated insulin levels and dysglycaemia. CHN2 encoding beta-2 chimerin was shown to be expressed in insulin sensitive tissues and its disruption to result in decreased gene expression in patient derived adipose tissue.

We present a likely digenic cause of insulin resistance and growth deficiency resulting from the combined heterozygous disruption of INSR and CHN2; implicating CHN2 for the first time as a key element of proximal insulin signaling in-vivo.

Low-fat hypocaloric diets reduce insulin resistance and prevent type 2 diabetes in those at risk. Low-carbohydrate high-fat diets are advocated as an alternative but reciprocal increases in dietary fat may have detrimental effects on insulin resistance and offset the benefits of weight reduction.

We investigated a low-fat (20% fat, 60% carbohydrate) versus a low-carbohydrate (60% fat, 20% carbohydrate) weight reduction diet in 24 overweight/obese subjects, BMI 33.6±3.7 kg/m², age 39±10 years (mean ± SD), in an 8 week randomized controlled trial. All food was weighed and distributed and intake calculated to produce a 500kcal/day energy deficit. Insulin action was assessed by the euglycaemic clamp and insulin secretion by meal tolerance test. Body composition, adipokine levels and vascular compliance by pulse-wave analysis were also measured.

Significant weight loss occurred in both groups (P<0.01), with no difference between groups (P=0.40). Peripheral glucose uptake increased but there was no difference between groups (P=0.28) and suppression of endogenous glucose production was also similar between groups. Meal tolerance-related insulin secretion decreased with weight loss with no difference between groups (P=0.71). The change in overall systemic arterial stiffness, was, however, significantly different between diets (P=0.04); this reflected a significant decrease in augmentation index following the low-fat diet, compared to a non-significant increase within the low-carbohydrate group.

This study demonstrates comparable effects on insulin resistance of low-fat and low-carbohydrate diets independent of macronutrient content. The difference in augmentation index may imply a negative effect of low-carbohydrate diets on vascular risk.

To identify genes that confer susceptibility to autoimmune diabetes following viral infection in the LEW.1WR1 rat.

About 2% of LEW.1WR1 rats develop spontaneous autoimmune diabetes. Immunological perturbants including viral infection increase both the frequency and tempo of diabetes onset. To identify diabetes susceptibility genes, (LEW.1WR1 x WF) F2 rats were infected with Kilham rat virus following brief pre-treatment with polyinosinic:polycytidylic acid. This treatment induces diabetes in 100% of parental LEW.1WR1 rats and 0% of parental WF rats. Linkage to diabetes was analyzed by genome-wide scanning.

Among 182 F2 rats, 57 (31%) developed autoimmune diabetes after a mean latency of 16 days. All diabetic animals and ~20% of non-diabetic animals exhibited pancreatic insulitis. Genome-wide scanning revealed a requirement for the Iddm14 locus, long known to be required for diabetes in the BB rat. In addition, a new locus near the RT1 major histocompatibility complex was found to be a major determinant of disease susceptibility. Interestingly, one gene linked to autoimmune diabetes in mouse and human, UBD, lies within this region.

The Iddm14 diabetes locus in the rat is a powerful determinant of disease penetrance in the LEW.1WR1 rat following viral infection. In addition, a locus near the MHC (Iddm37) conditions diabetes susceptibility in these animals. Other, as yet unidentified, genes are required to convert latent susceptibility to overt diabetes. These data provide insight into the polygenic nature of autoimmune diabetes in the rat and the interplay of genetic and environmental factors underlying disease expression.

AMP-activated protein kinase (AMPK) and the ATP-sensitive K⁺ (K_ATP) channel are metabolic sensors that become activated during metabolic stress. AMPK is an important regulator of metabolism, whereas the K_ATP channel is a regulator of cellular excitability. Crosstalk between these systems is poorly understood.

Rat pancreatic β-cells or INS-1 cells were pretreated for 2 h at various concentrations of glucose. Maximum K_ATP conductance (G_max) was monitored by whole-cell measurements following intracellular ATP washout using ATP-free internal solutions. K_ATP channel activity (NPo) was monitored by inside-out patch recordings in the presence of diazoxide. Distributions of K_ATP channel proteins (Kir6.2 and SUR1) were examined using immunofluorescence imaging and surface biotinylation studies. Insulin secretion from rat pancreatic islets was measured using an enzyme immunoassay.

G_max and NPo in cells pretreated with glucose-free or 3 mM glucose solutions were significantly higher than those in cells pretreated in 11.1 mM glucose solutions. Immunofluorescence imaging and biotinylation studies revealed that glucose deprivation induced an increase in the surface level of Kir6.2 without affecting the total cellular amount. Increases in G_max and the surface level of Kir6.2 were inhibited by compound C, an AMPK inhibitor, and siAMPK transfection. The effects of glucose deprivation on K_ATP channels were mimicked by an AMPK activator. Glucose deprivation reduced insulin secretion, but this response was attenuated by compound C.

K_ATP channel trafficking is regulated by energy status via AMPK, and this mechanism may play a key role in inhibiting insulin secretion under low energy status.

In skeletal muscles, dantrolene (Dan) inhibits the exercise-induced membrane translocation of glucose transporter 4 (GLUT4). It has been postulated that the inhibitory action of Dan on Ca²⁺ release from the sarcoplasmic reticulum (SR) causes the inhibition of exercise-induced glucose uptake (EIGU); however, the precise mechanism has not been adequately studied.

We discovered that Dan can bind to skeletal-type neuroendocrine-specific protein like 1 (sk-NSPl1) with photo-reactive Dan derivatives. In sk-NSPl1-deficient muscles, we examined the change in glucose uptake and the membrane translocation of GLUT4. In addition, we examined the change in blood glucose and also measured the glycogen level in isolated and in situ skeletal muscles after electrical stimulation using our mutant mouse.

In sk-NSPl1-deficient muscles, EIGU was totally abolished with no change in insulin-induced glucose uptake. The Ca²⁺ release mechanism and its inhibition by Dan were completely preserved in these muscles. The expression of GLUT4 in the mutant muscles also appeared unchanged. Confocal imaging and results using the membrane isolation method showed that exercise/contraction did not enhance GLUT4 translocation in these sk-NSPl1-deficient muscles under conditions of adequate muscle glycogen consumption. The blood glucose level in normal mice was reduced by electrical stimulation of the hindlimbs, but that in mutant mice was unchanged.

Sk-NSPl1 is a novel Dan receptor that plays an important role in membrane translocation of GLUT4 induced by contraction/exercise. The 23 kDa sk-NSPl1 may also be involved in the regulation of glucose levels in the whole body.

Haptoglobin (Hp) binds free hemoglobin, inhibiting hemoglobin-induced oxidative damage. As oxidative stress has been associated with microvascular complications, we evaluated the relationship between Hp genotype and microalbuminuria (MA), macroalbuminuria (ON), end-stage renal disease (ESRD), and early renal function decline in type 1 diabetes.

Participants from the Epidemiology of Diabetes Complications Study with DNA available were studied for the incidence of MA (albumin excretion rate (AER) 20-200 µg/min), ON (AER>200 µg/min), ESRD (renal dialysis or transplantation) and renal function decline (a decline ≥30 mL/min/1.73 m² from baseline estimated (by the Cockcroft-Gault equation) glomerular filtration rate (eGFR) in those with baseline eGFR >60 mL/min/1.73 m²).

The proportions with the Hp 2/2, 2/1 and 1/1 genotype were 43.4%, 44.4% and 12.1%, respectively. During 18 years of follow-up, the incidence of eGFR decline, MA, ON and ESRD were 42.0%, 40.5%, 16.7%, and 12.2%, respectively. No significant univariate differences were observed by Hp genotype. However, in multivariable Cox models, an approximately twofold increased risk was observed for the Hp 2/2 compared to the Hp 1/1 genotype for eGFR decline (HR=1.79, 95% CI=1.06-3.00) and end-stage renal disease (HR=2.74, 95% CI=1.17-6.45); no significant associations were observed for MA or ON.

These data suggest that whereas Hp genotype is not associated with albuminuria per se, it may be an independent determinant of early renal function decline and progression to ESRD. Understanding these apparent contradictory findings may provide further insight into the pathogenesis of renal disease in type 1 diabetes.

Cross-sectional studies found less microalbuminuria in type 2 diabetic patients (T2D) with the Ala12 allele of the peroxisome proliferator-activated receptor-2 (PPAR-2) Pro12Ala polymorphism. We prospectively evaluated the association between Pro12Ala polymorphism (rs1801282) and new-onset microalbuminuria

Pro12Ala polymorphism was genotyped by TaqMan-based assay in genomic DNA of 1119 consenting patients from BENEDICT, a prospective, randomized trial evaluating ACE inhibition effect on new-onset microalbuminuria (albuminuria 20-200 µg/min in at least 2 of 3 consecutive overnight urine collections in 2 consecutive visits) in hypertensive T2D with albuminuria <20 µg/min at inclusion.

Baseline characteristics of Ala (Ala/Ala or Ala/Pro) carriers and Pro/Pro homozygotes were similar, with a non-significant trend to lower albuminuria (p=0.1107) in the 177 Ala carriers. Over a median (interquartile range) of 44.0 (17.1-51.9) months, 7 (4%) Ala carriers and 86 (9.1%) Pro/Pro homozygotes developed microalbuminuria [HR (95%CI)=0.45 (0.21-0.97), p=0.042]. Final albuminuria was significantly lower in Ala carriers (7.3±9.1 vs 10.5±24.9 µg/min, respectively), even after adjustment for baseline albuminuria (p=0.048). Baseline and follow-up blood pressure and metabolic control were similar in both groups. Incidence of microalbuminuria was significantly decreased by ACE vs non-ACE inhibitor therapy in Pro/Pro homozygotes [6.3% vs 11.9%, respectively, HR (95%CI)=0.46 (0.29-0.72), p<0.001].

In type 2 diabetes the Ala allele protects from worsening albuminuria and new-onset microalbuminuria, and ACE inhibition blunts the excess risk of microalbuminuria associated with the Pro/Pro genotype. Evaluating Pro12Ala polymorphism may help identifying patients at risk who may benefit the most of early renoprotective therapy.

Chemerin is an adipokine that affects adipogenesis and glucose homeostasis in adipocytes and increases with BMI in humans. This study aimed at investigating the regulation of chemerin release and its effects on glucose metabolism in skeletal muscle cells.

Human skeletal muscle cells were treated with chemerin to study insulin signaling, glucose uptake and activation of stress kinases. The release of chemerin was analyzed from in vitro differentiated human adipocytes and adipose tissue explants from 27 lean and 26 obese patients.

Human adipocytes express chemerin and CMKLR1 differentiation-dependently and secrete chemerin (15 ng/ml from 10⁶ cells). This process is slightly but significantly increased by TNF and markedly inhibited by over 80 % by PPAR activation. Adipose tissue explants from obese patients are characterized by significantly higher chemerin secretion as compared to lean controls (21 ng and 8 ng from 10⁷ cells, respectively). Chemerin release is correlated with BMI, waist-hip-ratio and adipocyte volume. Furthermore, higher chemerin release is associated with insulin resistance at the level of lipogenesis and insulin-induced antilipolysis in adipocytes. Chemerin induces insulin resistance in human skeletal muscle cells at the level of IRS1, Akt and GSK3 phosphorylation and glucose uptake. Furthermore, chemerin activates p38 MAPK, NF-B and ERK1/2. Inhibition of ERK prevents chemerin-induced insulin resistance pointing to participation of this pathway in chemerin action.

Adipocyte-derived secretion of chemerin may be involved in the negative crosstalk between adipose tissue and skeletal muscle contributing to the negative relationship between obesity and insulin sensitivity.

To characterize glycation adducts formed both in vivo in extracellular matrix (ECM) proteins of endoneurium from streptozotocin (STZ)-induced diabetic rats, and in vitro by glycation of laminin and fibronectin with methylglyoxal (MG) and glucose. To investigate the impact of advanced glycation endproduct (AGE) residue content of ECM on neurite outgrowth from sensory neurons.

Glycation, oxidation and nitration adducts of ECM proteins extracted from the endoneurium of control and STZ-diabetic rat sciatic nerve (3-24 weeks post-STZ), and of laminin and fibronectin that had been glycated using glucose or MG, were examined by liquid chromatography with tandem mass spectrometry. MG-glycated or unmodified ECM proteins were used as substrata for dissociated rat sensory neurons as in vitro models of regeneration.

STZ-induced diabetes produced a significant increase in early glycation N-fructosyl-lysine (FL) and AGE residue contents of endoneurial ECM. Glycation of laminin and fibronectin by MG and glucose increased glycation adduct residue contents with MG-derived hydroimidazolone (MG-H1) and FL, respectively, of greatest quantitative importance. Glycation of laminin caused a significant decrease in both neurotrophin-stimulated and preconditioned sensory neurite-outgrowth. This decrease was prevented by aminoguanidine. Glycation of fibronectin also decreased preconditioned neurite-outgrowth, which was prevented by aminoguanidine and NGF.

Early glycation and AGE residue content of endoneurial ECM proteins increase markedly in STZ-induced diabetes. Glycation of laminin and fibronectin causes a reduction in neurotrophin-stimulated neurite outgrowth and preconditioned neurite-outgrowth. This may provide a mechanism for the failure of collateral sprouting and axonal regeneration in diabetic neuropathy.

Recent studies in humans and animal models of obesity have shown increased adipose tissue activity of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which amplifies local tissue glucocorticoid concentrations. The reasons for this 11β-HSD1 dysregulation are unknown. Here, we tested whether 11β-HSD1 expression, like the metabolic syndrome, is ‘programmed’ by prenatal environmental events in a non-human primate model, the common marmoset monkey.

We used a ‘fetal programming’ paradigm where brief antenatal exposure to glucocorticoids leads to the metabolic syndrome in the offspring. Pregnant marmosets were given the synthetic glucocorticoid dexamethasone (DEX) orally for one week in either early or late gestation or vehicle. Tissue 11β-HSD1 and glucocorticoid receptor (GR) mRNA expression were examined in the offspring at 4 and 24 months of age.

Prenatal DEX treatment, selectively during late gestation, resulted in early and persistent elevations in 11β-HSD1 mRNA expression and activity in the liver, pancreas and subcutaneous, but not visceral fat. The increase in 11β-HSD1 occurred before animals developed obesity or overt features of the metabolic syndrome. In contrast to rodents, in utero DEX exposure did not alter GR expression in metabolic tissues in marmosets.

These data suggest that longterm up-regulation of 11β-HSD1 in metabolically active tissues may follow prenatal ‘stress’ hormone exposure and indicates a novel mechanism for fetal origins of adult obesity and the metabolic syndrome.

Fibroblast growth factor–21 (FGF-21) is a potent metabolic regulator, which in animal models has been shown to improve glucose metabolism and insulin sensitivity. Recently, FGF-21 was shown to be expressed and secreted from murine muscle cells in response to insulin stimulation.

We studied muscular FGF-21 expression and plasma FGF-21 after acute insulin stimulation in young healthy men during a hyperinsulinemic euglycemic clamp. Furthermore, we investigated systemic levels and muscle FGF-21 expression in humans with or without insulin resistance and chronic elevated insulin.

FGF-21 was barely detectable in young healthy males before insulin infusion. After 3 or-4 hours of insulin infusion during a hyperinsulinemic euglycemic clamp muscular FGF-21 expression increased significantly. Plasma FGF-21 followed the same pattern. In individuals with chronic elevated insulin, muscular FGF-21 expression was associated with hyperinsulinemia in men, but not in women. In plasma, hyperinsulinemia as well as fasting glucose was positively associated with plasma FGF-21, while plasma FGF-21 correlated negatively with HDL-cholesterol. No associations between muscle and plasma FGF-21 were found in the individuals with chronic hyperinsulinemia.

In conclusion, FGF-21 is expressed in human skeletal muscle in response to insulin stimulation, suggesting that FGF-21 is an insulin-regulated myokine. In support, we found an association between chronic hyperinsulinemia and levels of FGF-21.

In obesity, an increased macrophage infiltration in adipose tissue occurs, contributing to the low grade inflammation and insulin resistance. Epidermal growth factor receptor (EGFR) mediates both chemotaxis and proliferation in monocytes and macrophages. However, the role of EGFR inhibitors on this subclinical inflammation was not yet investigated. We investigated, herein, in vivo efficacy and associated molecular mechanisms by which PD153035, an EGFR tyrosine kinase inhibitor, improved diabetes control and insulin action.

The effect of PD153035 was investigated on insulin sensitivity, insulin signaling and JNK and NF-B activity in tissues of high-fat diet-fed mice, and also on infiltration and the activation state of adipose tissue macrophage (ATM) in these mice.

PD153035 treatment for 1 day decreased the protein expression of iNOS, TNF- and IL-6 in the stroma vascular fraction, suggesting that this drug reduces the M1 proinflammatory state in ATM, as an initial effect, in turn reducing the circulating levels of TNF- and IL-6 initiating an improvement in insulin signaling and sensitivity. After 14 days of drug administration, there was a marked improvement in glucose tolerance, a reduction in insulin resistance, a reduction in macrophage infiltration in adipose tissue and in TNF-, IL-6 and FFAs, accompanied by an improvement in insulin signaling in liver, muscle and adipose tissue, and also a decrease in IRS-1 Ser³⁰⁷ phosphorylation, in JNK and IKKβ activation in these tissues

Treatment with PD153035 improves glucose tolerance, insulin sensitivity and signaling and reduces subclinical inflammation in HFD mice.

Natriuretic peptides (NP) have been characterized as vascular hormones which regulate vascular tone via guanylyl cyclase (GC), cyclic GMP and cyclic GMP-dependent protein kinase (cGK). Recent clinical studies have shown that plasma NP levels were lower in persons with the metabolic syndrome. The present study was aimed to elucidate the roles for NP/cGK cascades in energy metabolism.

We used three types of genetically engineered mice: BNP transgenic (BNP-Tg), cGK-Tg, and GCA-heterozygous knockout (GCA+/–) mice and analyzed the metabolic consequences of chronic activation of NP/cGK cascades in vivo. We also examined the effect of NP in cultured myocytes.

BNP-Tg mice fed on high-fat diet were protected against diet-induced obesity and insulin resistance, and cGK-Tg mice had reduced body weight even on standard chow, and surprisingly, giant mitochondria were densely packed in the skeletal muscle. Both mice showed an increase in muscle mitochondrial content and fat oxidation through up-regulation of PGC-1 and PPAR. The functional NP-receptors, GCA and GCB, were down-regulated by feeding high-fat diet; while GCA+/– mice showed increases in body weight and glucose intolerance when fed on high-fat diet. NP directly increased the expression of PGC-1 and PPAR, and mitochondrial content in cultured myocytes.

The findings together suggest that NP/cGK cascades can promote muscle mitochondrial biogenesis and fat oxidation, as to prevent obesity and glucose intolerance. The vascular hormone, NP, would contribute to coordinated regulation of oxygen supply and consumption.

GPR40 is a G protein-coupled receptor regulating free fatty acid-induced insulin secretion. We have generated transgenic mice overexpressing the human GPR40 gene (hGPR40-Tg) under control of the mouse insulin II promoter and have used them to examine the role of GPR40 in the regulation of insulin secretion and glucose homeostasis.

Normal (C57BL/6J) and diabetic (KK) mice overexpressing the human GPR40 gene under control of the insulin II promoter were generated, and their glucose metabolism and islet function were analyzed.

In comparison with nontransgenic littermates, hGPR40-Tg mice exhibited improved oral glucose tolerance with an increase in insulin secretion. Although islet morphological analysis showed no obvious differences between hGPR40-Tg and nontransgenic (NonTg) mice, isolated islets from hGPR40-Tg mice enhanced insulin secretion in response to high glucose (16 mM) than those from NonTg mice with unchanged low glucose (3 mM)-stimulated insulin secretion. In addition, hGPR40-Tg islets significantly increased insulin secretion against a naturally occurring agonist palmitate in the presence of 11 mM glucose. hGPR40-Tg mice were also found to be resistant to high fat diet-induced glucose intolerance, and hGPR40-Tg harboring KK mice showed augmented insulin secretion and improved oral glucose tolerance compared to nontransgenic littermates.

Our results suggest that GPR40 may have a role in regulating glucose-stimulated insulin secretion and plasma glucose levels in vivo, and that pharmacological activation of GPR40 may provide a novel insulin secretagogue beneficial for the treatment of type 2 diabetes.