To evaluate the direct impact of -3 polyunsaturated fatty acids (-3 PUFAs) on the functions and viability of pancreatic β-cells.

We developed an mfat-1 transgenic mouse model in which endogenous production of -3 PUFAs was achieved through overexpressing a C. elegans -3 fatty acid desaturase gene, mfat-1. The islets and INS-1 cells expressing mfat-1 were analyzed for insulin secretion and viability in response to cytokine treatment.

The transgenic islets contain much higher levels of -3 PUFAs, lower levels of -6 PUFAs, than those of the wild type. Insulin secretion stimulated by glucose-, amino acids, and GLP-1 was significantly elevated in the transgenic islets. When challenged with TNF-, IL-1β, and IFN-, the transgenic islets completely resisted cytokine-induced cell death. Adenoviral transduction of mfat-1 gene in wild-type islets and in INS-1 cells led to acute changes in the cellular levels of -3- and -6 PUFAs, and recapitulated the results in the transgenic islets. The expression of mfat-1 led to decreased production of PGE₂, which in turn contributed to the elevation of insulin secretion. We further found that cytokine-induced activation of NF-B and ERK_1/2 was significantly attenuated, and that the expression of PDX-1, glucokinase, and insulin-1 was increased as a result of -3 PUFA production.

Stable cellular production of -3 PUFAs via mfat-1 can enhance insulin secretion and confers strong resistance to cytokine-induced β-cell destruction. The utility of mfat-1 gene in deterring type-1 diabetes should be further explored in vivo.

A number of studies have found that body mass index (BMI) in early life influences the risk of developing type 2 diabetes (T2D) later in life. Our goal was to investigate if any T2D variants uncovered through genome wide association studies (GWAS) impact BMI in childhood.

Utilizing data from an ongoing GWAS of pediatric BMI in our cohort, we investigated the association of pediatric BMI with 20 SNPs at 18 T2D loci uncovered through GWAS, consisting of ADAMTS9, CDC123-CAMK1D, CDKAL1, CDKN2A/B, EXT2, FTO, HHEX-IDE, IGF2BP2, the intragenic region on 11p12, JAZF1, KCNQ1, LOC387761, MTNR1B, NOTCH2, SLC30A8, TCF7L2, THADA and TSPAN8-LGR5. We randomly partitioned our cohort exactly in half in order to have a ‘discovery’ cohort (n=3592) and a ‘replication’ cohort (n=3592).

Our data show that the major, T2D-risk conferring G allele of rs7923837 at the HHEX-IDE locus was associated with higher pediatric BMI in both the discovery (P=0.0013; and survived correction for 20 tests) and replication (P=0.023) sets (combined P=1.01x10^–4). Association was not detected with any other known type 2 diabetes loci uncovered to date through GWAS except for the well established FTO.

Our data show that the same genetic HHEX-IDE variant which is associated with type 2 diabetes from previous studies also influences pediatric BMI.

Prokineticin 2 (PK2) is a hypothalamic neuropeptide expressed in CNS areas known to be involved in food intake. We therefore hypothesized that PK2 plays a role in energy homeostasis.

We investigated the effect of nutritional status on hypothalamic PK2 expression and effects of PK2 on the regulation of food intake by intracerebroventricular (ICV) injection of PK2 and anti-PK2 antibody. Subsequently we investigated the potential mechanism of action by determining sites of neuronal activation following ICV injection of PK2, the hypothalamic site of action of PK2 and interaction between PK2 and other hypothalamic neuropeptides regulating energy homeostasis. To investigate PK2's potential as a therapeutic target we investigated the effect of chronic administration in lean and obese mice.

Hypothalamic PK2 expression was reduced by fasting. ICV administration of PK2 to rats potently inhibited food intake whilst anti-PK2 antibody increased food intake suggesting that PK2 is an anorectic neuropeptide. ICV administration of PK2 increased c-fos expression in proopiomelanocortin neurons of the arcuate nucleus (ARC) of the hypothalamus. In keeping with this, PK2 administration into the ARC reduced food intake and PK2 increased the release of alpha-MSH from ex-vivo hypothalamic explants. In addition ICV co-administration of the alpha-MSH antagonist agouti related peptide blocked the anorexigenic effects of PK2. Chronic peripheral administration of PK2 reduced food and bodyweight in lean and obese mice.

This is the first report showing that PK2 has a role in appetite regulation and its anorectic effect is partly mediated via the melanocortin system.

The sites of insulin action in the central nervous system that regulate glucose metabolism and energy expenditure are incompletely characterized. We have shown that mice with hypothalamic deficiency (L1) of insulin receptors (InsR) fail to regulate hepatic glucose production (HGP) in response to insulin.

To distinguish neurons that mediate insulin's effects on HGP from those that regulate energy homeostasis, we used targeted knock-ins to express InsR in AgRP or POMC neurons of L1 mice.

Restoration of insulin action in AgRP neurons normalized insulin suppression of HGP. Surprisingly, POMC-specific InsR knock-in increased energy expenditure and locomotor activity, exacerbated insulin resistance and increased HGP, associated with decreased expression of the K_ATP channel SUR1 subunit and decreased inhibitory synaptic contacts on POMC neurons.

The contrasting phenotypes of InsR knock-ins in POMC and AgRP neurons suggest a branched-pathway model of hypothalamic insulin signaling, in which InsR signaling in AgRP neurons decreases HGP, while InsR activation in POMC neurons promotes HGP and activates the melanocortinergic energy expenditure program.

The endogenous cannabinoid (or endocannabinoid) system (ECS) is part of a central neuromodulatory system thought to play a key role in the regulation of feeding behaviour and energy balance. However, increasing evidence suggests that modulation of the ECS may also act to regulate peripheral mechanisms involved in these processes, including lipogenesis in adipose tissue and liver, insulin release from pancreatic β-cells and glucose uptake into skeletal muscle. It was recently shown that cannabinoid receptor type 1 (CB1) and type 2 (CB2), both key components of the ECS, are expressed in human and rodent skeletal muscle. However, their role in modulating insulin sensitivity in this metabolically active tissue has yet to be determined. Our aim was to establish the role, if any, of these receptors in modulating insulin sensitivity in skeletal muscle cells.

Cultured skeletal muscle cells were exposed to CB1 and/or CB2 pharmacological agonists/antagonists/inverse agonists and the resulting effects on insulin-regulated PI3K-PKB and ERK1/2-directed signalling were determined.

Here, we report that modulating the activity of the ECS in skeletal muscle regulates both insulin-dependent MAP Kinase (ERK1/2) and the canonical PI3K-PKB signalling pathways. We show that pharmacological activation or inhibition of CB1 receptor activity exerts a differential effect with regards to MAP Kinase and PKB-directed signalling.

Our study provides evidence that signalling via cannabinoid receptors can significantly modulate mitogenic and metabolic signalling in skeletal muscle with important implications for muscle growth and differentiation as well as the regulation of glucose and lipid metabolism.

Common variants in the gene TCF7L2 confer the largest effect on the risk of T2D. The present study was undertaken to increase our understanding of the mechanisms by which this gene affects T2D risk.

Eight subjects with risk-conferring TCF7L2 genotypes (TT or TC at rs7903146) and ten-matched subjects with wild-type genotype (CC) underwent 5-h oral glucose tolerance test (OGTT), isoglycemic intravenous glucose infusion, and graded glucose infusion (GGI). Mathematical modeling was used to quantify insulin-secretory profiles during OGTT and glucose infusion protocols. The incretin effect was assessed from ratios of the insulin secretory rates (ISR) during oral and isoglycemic glucose infusions. Dose-response curves relating insulin secretion to glucose concentrations were derived from the GGI.

β-cell responsivity to oral glucose was 50% lower (47±4 vs. 95±15x10⁹ min^–1; P=0.01) in the group of subjects with risk-conferring TCF7L2 genotypes compared with controls. The incretin effect was also reduced by 30% (32±4 vs. 46±4%; P=0.02) in the at-risk group. The lower incretin effect occurred despite similar GIP and GLP-1 responses to oral glucose. The ISR response over a physiologic glucose concentration range (5–9 mmol/L) was similar between groups.

The TCF7L2 variant rs7903146 appears to affect risk of T2D, at least in part, by modifying the effect of incretins on insulin secretion. This is not due to reduced secretion of GLP-1 and GIP but rather due to the effect of TCF7L2 on the sensitivity of the β-cell to incretins. Treatments that increase incretin sensitivity may decrease the risk of T2D.

Increased activity of the innate immune system has been implicated in the pathogenesis of the dyslipidemia and insulin resistance associated with obesity and Type 2 diabetes mellitus (T2DM). In this study we addressed the potential role of Kupffer cells (liver-specific macrophages, KC) in these metabolic abnormalities.

Rats were depleted of KC by administration of gadolinium chloride after which all animals were exposed to a 2-week high fat or high sucrose diet. Subsequently, the effects of these interventions on the development of hepatic insulin resistance and steatosis were assessed. In further studies, the effects of M1 polarized KC on hepatocyte lipid metabolism and insulin sensitivity were addressed.

As expected, a high fat or high sucrose diet induced steatosis and hepatic insulin resistance. However, these metabolic abnormalities were prevented when liver was depleted of KC. In vitro, KC recapitulated the in vivo effects of diet by increasing hepatocyte triglyceride accumulation and fatty acid esterification, and decreasing fatty acid oxidation and insulin responsiveness. To address the mechanisms(s) of KC action we inhibited a panel of cytokines using neutralizing antibodies. Only neutralizing antibodies against TNF attenuated KC-induced alterations in hepatocyte fatty acid oxidation, triglyceride accumulation, and insulin responsiveness. Importantly, KC TNF levels were increased by diet in vivo and in isolated M1-polarized KC in vitro.

These data demonstrate a role for liver macrophages in diet-induced alterations in hepatic lipid metabolism and insulin sensitivity, and suggest a role for these cells in the etiology of the metabolic abnormalities of obesity/T2DM.

In type 1 diabetes (T1D), allogeneic pancreatic islet transplantation restores insulin production, but life-threatening immunosuppression is required to avoid graft rejection. Induction of antigen (Ag)-specific tolerance by cell therapy with regulatory T cells (Tregs) represents an attractive alternative approach but its therapeutic efficacy in islet transplantation remains to be determined. Among the different subsets of CD4⁺ Tregs, the T inducible regulatory type 1 (Tr1) cells can be generated from naïve T cells in the presence of IL-10 and represent one promising therapeutic choice. This study was designed to define the efficacy of Tr1-cell therapy in preclinical models of islet transplantation.

Non Ag-specific polyclonal Tr1 cells and donor Ag-specific Tr1 cells were transferred, in the absence of any pharmacological treatment, in two distinct mouse models of islet transplantation. The two models differed in their therapeutic stringency, based on the mean rejection time of transplanted untreated mice.

Transfer of polyclonal Tr1 cells engendered graft tolerance only in the non-stringent mouse model. Conversely, cell therapy with Ag-specific Tr1 cells induced an IL-10--dependent tolerance in the stringent mouse model of islet transplantation. The therapeutic advantage of Ag-specific Tr1 cells over polyclonal Tr1 cells was due to their donor Ag-specificity.

These results demonstrate that Tr1-cell therapy leads to tolerance in settings of islet transplantation and that its therapeutic efficacy is highly dependent on the antigen specificity of these cells.

Obesity is associated with monocyte-macrophage accumulation in adipose tissue. Previously, we showed that glucose-stimulated production by adipocytes of serum amyloid A (SAA), monocyte chemoattractant protein-1 (MCP-1) and hyaluronan (HA) facilitated monocyte accumulation. The current objective was to determine how the other major nutrient, free fatty acids (FFA), affects these molecules and monocyte recruitment by adipocytes.

Differentiated 3T3-L1, SGBS adipocytes and MEFs were exposed to various FFAs (250M) in either 5mM or 25mM (high) glucose for evaluation of SAA, MCP-1 and HA regulation in vitro.

Saturated fatty acids (SFA) such as laurate, myristate and palmitate increased cellular triglyceride accumulation, SAA and MCP-1 expression, generated reactive oxygen species (ROS) and increased NFB translocation in both 5 and 25mM glucose. Conversely, polyunsaturated fatty acids (PUFA) such as arachidonate, eicosapentaenate (EPA) and docosahexaenate (DHA) decreased these events. Gene expression could be dissociated from triglyceride accumulation. Although excess glucose increased HA content, SFAs, oleate and linoleate did not. Antioxidant treatment repressed glucose and palmitate-stimulated ROS generation and NFB translocation and decreased SAA and MCP-1 expression and monocyte chemotaxis. Silencing toll-like receptor-4 (TLR4) markedly reduced SAA and MCP-1 expression in response to palmitate but not glucose. DHA suppressed NFB translocation stimulated by both excess glucose and palmitate via a PPAR-dependent pathway.

Excess glucose and SFAs regulate chemotactic factor expression by a mechanism that involves ROS generation, NFB and PPAR, and which is repressed by PUFAs. Certain SFAs, but not excess glucose, trigger chemotactic factor expression via a TLR4-dependent pathway.

Cytokines contribute to pancreatic β-cell death in T1D. This effect is mediated by complex gene networks that remain to be characterized. We presently utilized array analysis to define the global expression pattern of genes, including spliced variants, modified by the cytokines IL-1β+IFN- and TNF-+IFN- in primary rat β-cells.

FACS-purified rat β-cells were exposed to IL-1β+IFN- or TNF-+IFN- for 6 or 24 h and global gene expression was analyzed by microarray. Key results were confirmed by RT-PCR, and siRNAs were used to investigate the mechanistic role of novel and relevant transcription factors identified by pathway analysis.

Nearly 16.000 transcripts were detected as present in β-cells, with temporal differences in the number of genes modulated by IL-1β+IFN or TNF-+IFN-. These cytokine combinations induced differential expression of inflammatory response genes, which is related to differential induction of IRF-7. Both treatments decreased the expression of genes involved in the maintenance of β-cell phenotype and growth/regeneration. Cytokines induced HIF-1, which in this context has a pro-apoptotic role. Cytokines also modified the expression of >20 genes involved in RNA splicing, and exon array analysis showed cytokine-induced changes in alternative splicing of more than 50% of the cytokine-modified genes.

The present study doubles the number of known genes expressed in primary β-cells, modified or not by cytokines, and indicates the biological role for several novel cytokine-modified pathways in β-cells. It also shows that cytokines modify alternative splicing in β-cells, opening a new avenue of research for the field.

Diabetic nephropathy is one of the most common causes of end-stage renal failure. Inhibition of ACE2 function accelerates diabetic kidney injury while renal ACE2 is downregulated in diabetic nephropathy. We examined the ability of human recombinant ACE2 (hrACE2) to slow the progression of diabetic kidney injury.

Male 12-week old diabetic Akita mice (Ins2^WT/C96Y) and control C57BL/6J mice (Ins2^WT/WT) were injected daily with placebo or with rhACE2 (2 mg/kg, i.p.) for four weeks. Albumin excretion, gene expression, histomorphometry, NADPH oxidase activity and peptide levels were examined. The effect of hrACE2 on high glucose and Ang II-induced changes was also examined in cultured mesangial cells.

Treatment with hrACE2 increased plasma ACE2 activity, normalized blood pressure, and reduced the urinary albumin excretion in Akita Ins2^WT/C96Y mice in association with a decreased glomerular mesangial matrix expansion and normalization of increased alpha-SMA and collagen III expression. Human recombinant ACE2 increased Ang 1-7 levels, lowered Ang II levels and reduced NADPH oxidase activity. mRNA levels for p47^phox and NOX2 and protein levels for PKC and PKCβ1 were also normalized by treatment with hrACE2. In vitro, hrACE2 attenuated both high glucose and ANG II-induced oxidative stress and NADPH oxidase activity.

Treatment with hrACE2 attenuates diabetic kidney injury in the Akita mouse in association with a reduction in blood pressure and a decrease in NADPH oxidase activity. In vitro studies show that the protective effect of hrACE2 is due to reduction in Ang II and an increase in Ang 1-7 levels.

Resveratrol, a natural polyphenolic compound that is found in grapes and red wine, increases metabolic rate, insulin sensitivity, mitochondrial biogenesis and physical endurance and reduces fat accumulation in mice. Although it is thought that resveratrol targets Sirt1, this is controversial because resveratrol also activates 5'-AMP activated kinase (AMPK), which also regulates insulin sensitivity and mitochondrial biogenesis. Here, we use mice deficient in AMPK1 or 2 to determine if the metabolic effects of resveratrol are mediated by AMPK.

Mice deficient in the catalytic subunit of AMPK (1 or 2) and wild type mice were fed high-fat diet or high-fat supplemented with resveratrol for 13 weeks. Body weight was recorded by weekly and metabolic parameters were measured. We also used mouse embryonic fibroblasts (mefs) deficient in AMPK to study the role of AMPK in resveratrol-mediated effects in vitro.

Resveratrol increased the metabolic rate and reduced fat mass in wild-type mice but not in AMPK1–/– mice. In the absence of either AMPK1 or 2, resveratrol failed to increase insulin sensitivity, glucose tolerance, mitochondrial biogenesis and physical endurance. Consistent with this, the expression of genes important for mitochondrial biogenesis was not induced by resveratrol in AMPK-deficient mice. In addition, resveratrol increased the NAD/NADH ratio in an AMPK-dependent manner, which may explain how resveratrol may activate Sirt1 indirectly.

We conclude that AMPK, which was thought to be an off-target hit of resveratrol, is the central target for the metabolic effects of resveratrol.

The response of ventromedial hypothalamic (VMH) glucose-inhibited (GI) neurons to decreased glucose is impaired under conditions where the counter-regulatory response (CRR) to hypoglycemia is impaired (e.g., recurrent hypoglycemia). This suggests a role for GI neurons in the CRR. We recently showed that decreased glucose increases nitric oxide (NO) production in cultured VMH GI neurons. These in vitro data lead us to hypothesize that NO release from VMH GI neurons is critical for the CRR.

The CRR was evaluated in rats and mice in response to acute insulin-induced hypoglycemia and hypoglycemic clamps after modulation of brain NO signaling. The glucose sensitivity of VMN GI neurons was also assessed.

Hypoglycemia increased hypothalamic constitutive NO synthase (NOS) activity and nNOS but not eNOS phosphorylation in rats. Intracerebroventricular (ICV) and VMH injection of the non-selective NOS inhibitor N^G-Nitro-L-arginine (LNMMA) slowed the recovery to euglycemia following hypoglycemia. VMH LNMMA injection also increased the glucose infusion rate (GIR) and decreased epinephrine secretion during hyperinsulinemic/hypoglycemic clamp in rats. The GIR required to maintain the hypoglycemic plateau was higher in nNOS knockout (KO) than wildtype (WT) or eNOS KO mice. Finally, VMH GI neurons were virtually absent in nNOS KO mice.

We conclude that VMH NO production is necessary for glucose sensing in GI neurons and full generation of the CRR to hypoglycemia. These data suggest that potentiating NO signaling may improve the defective CRR resulting from recurrent hypoglycemia in patients using intensive insulin therapy.

Type 2 diabetes (T2D) is caused by both environmental and genetic factors. To better understand the genetic factors we used forward genetics to discover genes that have not previously been implicated in the development of hyperglycemia or diabetes.

Offspring of ENU-mutagenized C57BL/6 mice were bred to homozygosity, maintained on high fat diet (HFD), and screened for hyperglycemia. The phenotype in one diabetic family of mice was mapped among hybrid F2s with single nucleotide polymorphic markers, followed by candidate gene sequencing to identify the gene harboring the causative mutation. Subsequent analysis was done on wild-type, heterozygous and homozygous mutant mice on a pure C57BL/6 background.

Diabetes mapped to a point mutation in the Sec61a1 gene that encodes a histidine to tyrosine substitution at amino acid 344 (Y344H). Metabolic profiling, histological examination and electron microscopy revealed that hyperglycemia was a result of insulin insufficiency due to β-cell apoptosis brought on by ER stress. Transgenic β cell specific expression of Sec61a1 in mutant mice rescued diabetes, β cell apoptosis and ER stress. In vitro experiments showed that Sec611 plays a critical role in the β cell response to glucose.

Here we phonotypically characterize diabetes in mice with a novel point mutation in a basic component of the cell's ER protein translocation machinery, Sec611. Translocation by the mutant protein does not appear to be affected. Rather, ER homeostasis is perturbed leading to β-cell death and diabetes.

To evaluate retrograde axonal transport of VEGF-A protein to sensory neurons following intramuscular administration of an engineered zinc finger protein activator of endogenous VEGF-A (VZ+434) in an experimental model of diabetes, and to characterize the VEGF-A target neurons.

We compared the expression of VEGF-A in lumbar (L)4/5 dorsal root ganglia (DRG) of control rats and VZ+434 treated and untreated streptozotocin (STZ)-induced diabetic rats. In addition, axonal transport of VEGF-A, activation of signal transduction pathways in the DRG, and mechanical sensitivity was assessed.

VEGF-A-immunoreactivity (-IR) was detected in small-medium diameter neurons in DRG of control rats. Fewer VEGF-A-IR neurons were observed in DRG from STZ-diabetic rats, this decrease was confirmed and quantified by Western blotting. VZ+434 administration resulted in a significant increase in VEGF-A protein expression in ipsilateral DRG, 24 hours following injection. VEGF-A was axonally transported to the DRG via the sciatic nerve. VZ+434 administration resulted in significant activation of AKT in the ipsilateral DRG by 48 hours which was sustained for 1 week post-injection. VZ+434 protected against mechanical allodynia 8 weeks post-STZ.

Intramuscular administration of VZ+434 increases VEGF-A protein levels in L4/5 DRG correcting the deficit observed following induction of diabetes and protects against mechanical allodynia. Elevated VEGF-A levels result from retrograde axonal transport and are associated with altered signal transduction, via the phosphatidylinositol 3'-kinase pathway. These data support a neuroprotective role for VEGF-A in the therapeutic actions of VZ+434 and suggest a mechanism by which VEGF-A exerts this activity.

The inability of pancreatic β-cells to appropriately respond to glucose and secrete insulin are primary defects associated with β-cell failure in type 2 diabetes. Mitochondrial dysfunction has been implicated as a key factor in the development of type 2 diabetes; however a link between mitochondrial dysfunction and defective insulin secretion is unclear.

We investigated the changes in islet mitochondrial function and morphology during progression from insulin resistance (3 week-old), immediately prior to hyperglycemia (5 week-old) and after diabetes onset (10 week-old) in transgenic MKR mice compared to control. The molecular and protein changes at 10 weeks were determined using microarray and iTRAQ proteomic screens.

At 3 weeks MKR mice were hyperinsulinemic but normoglycemic and β-cells showed negligible mitochondrial or morphological changes. At 5 weeks MKR islets displayed abrogated hyperpolarization of mitochondrial membrane potential (_m), reduced mitochondrial Ca²⁺ uptake, slightly enlarged mitochondria and reduced glucose-stimulated insulin secretion. By 10 weeks, MKR mice were hyperglycemic, hyperinsulinemic and β-cells contained swollen mitochondria with disordered cristae. β-cells displayed impaired stimulus-secretion coupling including reduced hyperpolarization of _m, impaired Ca²⁺-signaling, and reduced glucose-stimulated ATP/ADP and insulin release. Furthermore, decreased cytochrome c oxidase-dependent oxygen consumption and enhanced oxidative stress were observed in diabetic islets. Protein profiling of diabetic islets revealed that 36 mitochondrial proteins were differentially expressed, including inner membrane proteins of the electron transport chain.

We provide novel evidence for a critical role of defective mitochondrial oxidative phosphorylation and morphology in the pathology of insulin resistance-induced β-cell failure.

In part, activation of invariant natural killer T (iNKT)-cells with the superagonist -galactosylceramide (-GalCer) inhibits the development of T-cell-mediated autoimmune type 1 diabetes (T1D) in NOD mice by inducing the downstream differentiation of antigen-presenting dendritic cells (DC) to an immunotolerogenic state. However, in other systems iNKT-cell activation has an adjuvant-like effect that enhances rather than suppresses various immunological responses. Thus, we tested if in some circumstances genetic variation would enable activated iNKT-cells to support rather than inhibit T1D development.

We tested whether iNKT-conditioned DC in NOD mice and a major histocompatibility complex matched C57BL/6 (B6) background congenic stock differed in capacity to inhibit T1D induced by the adoptive transfer of pathogenic AI4 CD8 T-cells.

Unlike those of NOD origin, iNKT-conditioned DC in the B6 background stock matured to a state that actually supported rather than inhibited AI4 T-cell induced T1D. The induction of a differing activity pattern of T-cell co-stimulatory molecules varying in capacity to override programmed death-ligand-1 (PD-L1) inhibitory effects contributes to the respective ability of iNKT-conditioned DC in NOD and B6 background mice to inhibit or support T1D development. Genetic differences inherent to both iNKT-cells and DC contribute to their varying interactions in NOD and B6.H2^g7 mice.

This great variability in the interactions between iNKT-cells and DC in two inbred mouse strains should raise a cautionary note about considering manipulation of this axis as a potential T1D prevention therapy in genetically heterogeneous humans.

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.

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.