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Aims/hypothesis: Obesity and type 2 diabetes are concomitant with low-grade inflammation affecting insulin sensitivity and insulin secretion. Recently, the thioredoxin interacting protein (TXNIP) has been implicated in the activation process of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. In this study, we aim to determine whether the expression of TXNIP is altered in the circulating immune cells of individuals with type 2 vs type 1 diabetes and whether this can be related to specific causes and consequences of inflammation., Methods: The expression of TXNIP, inflammatory markers, markers of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress and enzymes involved in sphingolipid metabolism was quantified by quantitative reverse transcription real-time PCR (qRT-PCR) in peripheral blood mononuclear cells (PBMCs) of 13 non-diabetic individuals, 23 individuals with type 1 diabetes and 81 with type 2 diabetes. A lipidomic analysis on the plasma of 13 non-diabetic individuals, 35 individuals with type 1 diabetes and 94 with type 2 diabetes was performed. The effects of ER stress or of specific lipids on TXNIP and inflammatory marker expression were analysed in human monocyte-derived macrophages (HMDMs) and THP-1 cells., Results: The expression of TXNIP and inflammatory and UPR markers was increased in the PBMCs of individuals with type 2 diabetes when compared with non-diabetic individuals or individuals with type 1 diabetes. TXNIP expression was significantly correlated with plasma fasting glucose, plasma triacylglycerol concentrations and specific UPR markers. Induction of ER stress in THP-1 cells or cultured HMDMs led to increased expression of UPR markers, TXNIP, NLRP3 and IL-1β. Conversely, a chemical chaperone reduced the expression of UPR markers and TXNIP in PBMCs of individuals with type 2 diabetes. The lipidomic plasma analysis revealed an increased concentration of saturated dihydroceramide and sphingomyelin in individuals with type 2 diabetes when compared with non-diabetic individuals and individuals with type 1 diabetes. In addition, the expression of specific enzymes of sphingolipid metabolism, dihydroceramide desaturase 1 and sphingomyelin synthase 1, was increased in the PBMCs of individuals with type 2 diabetes. Palmitate or C2 ceramide induced ER stress in macrophages as well as increased expression of TXNIP, NLRP3 and IL-1β., Conclusions/interpretation: In individuals with type 2 diabetes, circulating immune cells display an inflammatory phenotype that can be linked to ER stress and TXNIP expression. Immune cell ER stress can in turn be linked to the specific exogenous and endogenous lipid environment found in type 2 diabetes.

A series of benzothiazol-2-one containing α-ethoxyphenylpropionic acid derivatives incorporating resveratrol or butein scaffolds were designed as fused full PPARγ agonist ligands and SIRT1-activating compounds for the treatment of type 2 diabetes (T2D) and its complications. Compound 14d displayed the best in vitro pharmacological profile with full PPARγ agonist activity (Emax = 98%, EC 50  = 200 nM), SIRT1 enzymatic activation (+128%) and SGK1 expression inhibition (- 57%) which is known to limit side effects as fluid retention and body-weight gain. Compound 14d showed high efficacy in an ob/ob mice model with significant decreases in serum triglyceride, glucose and insulin levels but mostly with limited body-weight gain by mimicking calorie restriction (CR) and inhibiting SGK1 expression., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Plasma metabolite concentrations reflect the activity of tissue metabolic pathways and their quantitative determination may be informative about pathogenic conditions. We searched for plasma lipid species whose concentrations correlate with various parameters of glucose homeostasis and susceptibility to type 2 diabetes (T2D). Shotgun lipidomic analysis of the plasma of mice from different genetic backgrounds, which develop a pre-diabetic state at different rates when metabolically stressed, led to the identification of a group of sphingolipids correlated with glucose tolerance and insulin secretion. Quantitative analysis of these and closely related lipids in the plasma of individuals from two population-based prospective cohorts revealed that specific long-chain fatty-acid-containing dihydroceramides were significantly elevated in the plasma of individuals who will progress to diabetes up to 9 years before disease onset. These lipids may serve as early biomarkers of, and help identify, metabolic deregulation in the pathogenesis of T2D., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: In type 2 diabetes (T2D), pancreatic β cells become progressively dysfunctional, leading to a decline in insulin secretion over time. In this study, we aimed to identify key genes involved in pancreatic beta cell dysfunction by analyzing multiple mouse strains in parallel under metabolic stress., Methods: Male mice from six commonly used non-diabetic mouse strains were fed a high fat or regular chow diet for three months. Pancreatic islets were extracted and phenotypic measurements were recorded at 2 days, 10 days, 30 days, and 90 days to assess diabetes progression. RNA-Seq was performed on islet tissue at each time-point and integrated with the phenotypic data in a network-based analysis., Results: A module of co-expressed genes was selected for further investigation as it showed the strongest correlation to insulin secretion and oral glucose tolerance phenotypes. One of the predicted network hub genes was Elovl2 , encoding Elongase of very long chain fatty acids 2. Elovl2 silencing decreased glucose-stimulated insulin secretion in mouse and human β cell lines., Conclusion: Our results suggest a role for Elovl2 in ensuring normal insulin secretory responses to glucose. Moreover, the large comprehensive dataset and integrative network-based approach provides a new resource to dissect the molecular etiology of β cell failure under metabolic stress.

7-Azaindole has been identified as a novel bidentate anchor point for allosteric glucokinase activators. A systematic investigation around three principal parts of the new small molecule glucokinase activators led to a robust SAR in agreement with structural data that also helped to assess the conformational flexibility of the allosteric activation site. The increase in glucose uptake resulting from glucokinase activation in hepatocytes in vitro translated into the efficient lowering of glucose levels in vivo with the best compounds.

The respective roles of predisposing genetic factors and environmental factors in the development of type 2 diabetes (T2D) in obese subjects is poorly documented. Rodent models have been set up in an attempt to better understand of the differential effect of a prolonged metabolic stress induced by a high fat diet on glycaemic control according to the genetic background. In utero growth retardation resulting from a hypocaloric diet in pregnant rats induces a dramatic alteration of the development of islet cells leading to diabetes and insulin secretory defects in adult age. Experimentally induced diabetes in rodents results in hyperglycaemia and hyperinsulinemia in the fetus related to accelerated endocrine pancreas maturation responsible for the onset of diabetes in the adult. Deranged metabolic environment during fetal life may therefore further contribute to the onset of diabetes in the adult. Normal mouse strains with different genetic backgrounds show a wide range of responses to a high fat diet, with strains resistant to the diet and other more or less sensitive to the diet, the most sensitive exhibiting obesity diabetes and, insulin deficiency. The inability of the β cell to respond to the increased insulin demand related to insulin resistance seems to be pivotal in the pathophysiologic process and a new notion is emerging: "nutritional genetics" which studies the influence of nutrients on gene expression., (© 2013 médecine/sciences – Inserm.)

Background and Purpose: Small molecule glucokinase activators (GKAs) have been associated with potent antidiabetic efficacy and hepatic steatosis in rodents. This study reports the discovery of S 50131 and S 51434, two novel GKAs with an original scaffold and an atypical pharmacological profile., Experimental Approach: Activity of the compounds was assessed in vitro by measuring activation of recombinant glucokinase, stimulation of glycogen synthesis in rat hepatocytes and increased insulin secretion from rat pancreatic islets of Langerhans. Efficacy and safety in vivo were evaluated after oral administration in db/db mice by measuring glycaemia, HbA1c and dyslipidaemia-associated events., Key Results: S 50131 and S 51434 activated GK and stimulated glycogen synthesis in hepatocytes and insulin secretion from pancreatic islets. Unexpectedly, while both compounds effectively lowered glycaemia after acute oral administration, they did not decrease HbA1c after a 4-week treatment in db/db mice. This lack of antidiabetic efficacy was associated with increased plasma free fatty acids (FFAs), contrasting with the effect of GKA50 and N00236460, two GKAs with sustained HbA1c lowering activity but neutral regarding plasma FFAs. S 50131, but not S 51434, also induced hepatic steatosis, as did GKA50 and N00236460. However, a shorter, 4-day treatment resulted in increased hepatic triglycerides without changing the plasma FFA levels, demonstrating dynamic alterations in the lipid profile over time., Conclusions and Implications: In addition to confirming the occurrence of dyslipidaemia with GKAs, these findings provide new insights into understanding how such compounds may sustain or lose efficacy over time., (© 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.)

Background and Purpose: Small-molecule glucokinase activators (GKAs) are currently being investigated as therapeutic options for the treatment of type 2 diabetes (T2D). Because liver overexpression of glucokinase is thought to be associated with altered lipid profiles, this study aimed at assessing the potential lipogenic risks linked to oral GKA administration., Experimental Approach: Nine GKA candidates were qualified for their ability to activate recombinant glucokinase and to stimulate glycogen synthesis in rat hepatocytes and insulin secretion in rat INS-1E cells. In vivo activity was monitored by plasma glucose and HbA1c measurements after oral administration in rodents. Risk-associated effects were assessed by measuring hepatic and plasma triglycerides and free fatty acids, as well as plasma aminotransferases, and alkaline phosphatase., Key Results: GKAs, while efficiently decreasing glycaemia in acute conditions and HbA1c levels after chronic administration in hyperglycemic db/db mice, were potent inducers of hepatic steatosis. This adverse outcome appeared as soon as 4 days after daily oral administration at pharmacological doses and was not transient. GKA treatment similarly increased hepatic triglycerides in diabetic and normoglycaemic rats, together with a pattern of metabolic phenotypes including different combinations of increased plasma triglycerides, free fatty acids, alanine and aspartyl aminotransferases, and alkaline phosphatase. GKAs belonging to three distinct structural families induced hepatic steatosis in db/db mice, arguing in favour of a target-mediated, rather than a chemical class-mediated, effect., Conclusion and Implications: Given the risks associated with fatty liver disease in the general population and furthermore in patients with T2D, these findings represent a serious warning for the use of GKAs in humans., Linked Article: This article is commented on by Rees and Gloyn, pp. 335-338 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02201.x., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Background: The sterol regulatory element-binding protein (SREBP) 1c contributes to the transcriptional coordination of cholesterol, fatty acid, and carbohydrate metabolisms. Alterations in these processes accelerate the progression of hepatic steatosis and insulin resistance during aging and obesity., Methods: Using an ex vivo chromatin immunoprecipitation coupled to microarray (ChIP-on-chip) technique combined with genome-wide gene expression analysis, we analyzed the transcriptomic adaptations mediated by Srebp-1c binding to gene promoters in the liver of mice fed with a low-fat diet or a high-fat diet (HFD) for either 1 or 12 months., Results: Aging had a higher transcriptional impact than HFD and modified the expression of genes involved in fatty acid oxidation and oxidative stress. HFD was associated with a marked induction of genes involved in lipid and cholesterol metabolism. The prolonged high-fat feeding together with the aging effects stimulates inflammatory pathways. ChIP-on-chip applied to aging and HFD analyses revealed that the binding of SREBP-1c to a series of promoters accompanied a paralleled modification of gene expression. Therefore, SREBP-1c could play a role in aging and high-fat feeding through the regulation of genes involved in lipid metabolism and inflammatory response., Conclusions: This study represents an original ex vivo experiment to elucidate the molecular events involved in metabolic disorders., (Copyright © 2013 S. Karger AG, Basel.)

The compound S38151 is a nanomolar antagonist that acts at the melanin-concentrating hormone receptor 1 (MCH(1)). S38151 is more stable than its purely peptide counterpart, essentially because of the blockade of its N-terminus. Therefore, its action on various models of obesity was studied. Acute intra-cerebroventricular (i.c.v.) administration of S38151 in wild-type rats counteracted the effect of the stable precursor of melanin-concentrating hormone (MCH), NEI-MCH, in a dose-dependent manner (from 0.5 to 50 nmol/kg). In genetically obese Zucker fa/fa rats, daily i.c.v. administration of S38151 induced dose-dependent (5, 10, and 20 nmol/kg) inhibition of food intake, water intake, and body weight gain, as well as increased motility (maximal effect observed at 20 nmol/kg). In Zucker fa/fa rats, intraperitoneal injection of S38151 (30 mg/kg) induced complete inhibition of food consumption within 1 h. Daily intraperitoneal injection of S38151 (10 and 30 mg/kg) into genetically obese ob/ob mice or diet-induced obese mice is able to limit body weight gain. Furthermore, S38151 administration (10 and 30 mg/kg) does not affect food intake, water intake, or body weight gain in MCHR1-deleted mice, demonstrating that its effects are linked to its interaction with MCH(1). These results validate MCH(1) as a target of interest in obesity. S38151 cannot progress to the clinical phase because it is still too poorly stable in vivo.

Compounds that simultaneously activate peroxisome proliferator-activated receptor (PPAR) subtypes α and γ have the potential to effectively treat dyslipidemia and type 2 diabetes (T2D) in a single pharmaceutically active molecule. The frequently observed side effects of selective PPARγ agonists, such as edema and weight gain, were expected to be overcome by using additive PPARα activity, leading to dual PPARα/γ agonists with balanced activity for both subtypes. Herein we report the discovery, synthesis, and optimization of a new series of α-ethoxyphenylpropionic acid bearing 5- or 6-substituted indoles. The incorporation of oxime ethers on the carbonyl portion of the benzoyl group can bring the PPARα/γ potency ratio equal to or slightly greater than one, as is the case for compounds 20 c and 21 a. Compound 20 c shows high efficacy in an ob/ob mouse model of T2D and dyslipidemia, similar to that of rosiglitazone and tesaglitazar, but with a significant increase in body weight gain. In contrast, compound 21 a, less potent as a dual PPARα/γ activator than 20 c, showed an interesting pharmacological profile, as it elicits a decrease in body weight relative to reference compounds., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In order to identify new leads for the treatment of type 2 diabetes, polyenic molecules A and B derived from nipecotic acid and dienol derivatives C have been prepared and their effect on PPARs transcriptional activity evaluated and compared to that of rosiglitazone, WY14,643 and GW501516. Among the synthesized compounds, dienol 39 is the most active, increasing WY14,643 PPARα response and demonstrating partial agonist properties on rosiglitazone PPARγ.

Although interleukin (IL)-7 is mostly known as a key regulator of lymphocyte homeostasis, we recently demonstrated that it also contributes to body weight regulation through a hypothalamic control. Previous studies have shown that IL-7 is produced by the human obese white adipose tissue (WAT) yet its potential role on WAT development and function in obesity remains unknown. Here, we first show that transgenic mice overexpressing IL-7 have reduced adipose tissue mass associated with glucose and insulin resistance. Moreover, in the high-fat diet (HFD)-induced obesity model, a single administration of IL-7 to C57BL/6 mice is sufficient to prevent HFD-induced WAT mass increase and glucose intolerance. This metabolic protective effect is accompanied by a significant decreased inflammation in WAT. In lymphocyte-deficient HFD-fed SCID mice, IL-7 injection still protects from WAT mass gain. However, IL-7-triggered resistance against WAT inflammation and glucose intolerance is lost in SCID mice. These results suggest that IL-7 regulates adipose tissue mass through a lymphocyte-independent mechanism while its protective role on glucose homeostasis would be relayed by immune cells that participate to WAT inflammation. Our observations establish a key role for IL-7 in the complex mechanisms by which immune mediators modulate metabolic functions.

In healthy conditions, insulin-like growth factor-I (IGF-I) acts in a coordinated fashion with insulin to lower glycemia, mainly by increasing insulin sensitivity in peripheral tissues. The aim of this study was to explore the relationship between glucose homeostasis and the endocrine IGF-I axis in Zucker diabetic fatty (ZDF) rats. The plasma levels of glucose, insulin, growth hormone, free IGF-I, total IGF-I (associated to insulin-like growth factor binding proteins plus free), and corticosterone were measured in 13-week-old ZDF rats and in age-matched controls under fasting and postprandial conditions. The plasma IGF-I binding capacity was measured by radioligand binding. In ZDF rats, fasting total and free IGF-I levels were reduced by 22% and 92%, respectively, compared with controls. Postprandial free IGF-I was reduced by 35%, whereas total IGF-I was unaffected. The plasma IGF-I binding capacity in ZDF rats was reduced by 24% after fasting and by 13% under postprandial conditions. A clear correlation between free IGF-I and insulin was observed in postprandial controls but not in ZDF rats. A principal component analysis clearly separated ZDF and control rats into 2 main components under both fasting and postprandial conditions. The first component was determined equally by total IGF-I, bound IGF-I, the free to total IGF-I ratio, and the IGF-I binding capacity. The second component was determined mostly by glucose and insulin. Our results show a marked alteration of the plasma IGF-I levels and of the capacity of plasma to bind IGF-I, and a disturbed relationship between IGF-I and postprandial insulinemia in a rat model of type 2 diabetes mellitus., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Structural modifications around 8-HETE (8-hydroxyeicosatetraenoic acid), a natural agonist of the PPAR (peroxisome proliferator-activated receptor) nuclear receptors have led previously to the identification of a promising analog, the quinoline S 70655. Series of novel quinoline or benzoquinoline derivatives were designed through the modification of this lead. Variations of the nature of the aromatic core and of the side chains were carried out. The SAR studies indicated the high sensitivity of the upper acid chain to modifications as well as the strong effect of the length and size of the lipophilic side chain. They afforded several new promising PPARalpha/gamma dual agonists with a high PPARalpha activity in vitro.

Insulin resistance and altered endocrine pancreas function are central pathophysiological features of type 2 diabetes mellitus (T2DM). The Goto-Kakizaki (GK) rat is a model of spontaneous T2DM characterised by reduced beta cell mass and genetically determined glucose intolerance and altered insulin secretion. To identify genetic determinants of endocrine pancreas histopathology, we carried out quantitative trait locus (QTL) mapping of histological phenotypes (beta cell mass -BCM and insulin-positive cell area -IPCA) and plasma concentration of hormones and growth factors in a F2 cohort derived from GK and normoglycemic Brown Norway rats. Although IPCA and BCM in the duodenal region of the pancreas were highly positively correlated (P < 10(-6)), and similarly in the splenic region, both measures were poorly correlated when comparing duodenal and splenic phenotypes. Strongest evidence of linkage to pancreas morphological traits was obtained between BCM and chromosome 10 (LOD 3.2). Evidence of significant linkage (LOD 4.2) to plasma corticosterone was detected in a region of chromosome 1 distal to other QTLs previously identified in the GK. Male-specific genetic effects were detected, including linkages (LOD > 4) to growth hormome (GH) on chromosome 6 and prolactin on chromosome 17. These data suggest independent genetic control of the structure and function of ontologically different regions of the endocrine pancreas. Novel QTLs for corticosterone, prolactin and GH may contribute to diabetes in the GK. The QTLs that we have identified in this, and previous genetic studies collectively underline the complex and multiple mechanisms involved in diabetes in the GK strain.

Obese patients have chronic, low-grade inflammation that predisposes to type 2 diabetes and results, in part, from dysregulated visceral white adipose tissue (WAT) functions. The specific signaling pathways underlying WAT dysregulation, however, remain unclear. Here we report that the PPARgamma signaling pathway operates differently in the visceral WAT of lean and obese mice. PPARgamma in visceral, but not subcutaneous, WAT from obese mice displayed increased sensitivity to activation by its agonist rosiglitazone. This increased sensitivity correlated with increased expression of the gene encoding the ubiquitin hydrolase/ligase ubiquitin carboxyterminal esterase L1 (UCH-L1) and with increased degradation of the PPARgamma heterodimerization partner retinoid X receptor alpha (RXRalpha), but not RXRbeta, in visceral WAT from obese humans and mice. Interestingly, increased UCH-L1 expression and RXRalpha proteasomal degradation was induced in vitro by conditions mimicking hypoxia, a condition that occurs in obese visceral WAT. Finally, PPARgamma-RXRbeta heterodimers, but not PPARgamma-RXRalpha complexes, were able to efficiently dismiss the transcriptional corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) upon agonist binding. Increasing the RXRalpha/RXRbeta ratio resulted in increased PPARgamma responsiveness following agonist stimulation. Thus, the selective proteasomal degradation of RXRalpha initiated by UCH-L1 upregulation modulates the relative affinity of PPARgamma heterodimers for SMRT and their responsiveness to PPARgamma agonists, ultimately activating the PPARgamma-controlled gene network in visceral WAT of obese animals and humans.

Rosiglitazone (RSG), developed for the treatment of type 2 diabetes mellitus, is known to have potent effects on carbohydrate and lipid metabolism leading to the improvement of insulin sensitivity in target tissues. To further assess the capacity of RSG to normalize gene expression in insulin-sensitive tissues, we compared groups of 18-day-treated db/db mice with increasing oral doses of RSG (10, 30, and 100 mg/kg/d) with untreated non-diabetic littermates (db/+). For this aim, transcriptional changes were measured in liver, inguinal adipose tissue (IAT) and soleus muscle using microarrays and real-time PCR. In parallel, targeted metabolomic assessment of lipids (triglycerides (TGs) and free fatty acids (FFAs)) in plasma and tissues was performed by UPLC-MS methods. Multivariate analyses revealed a relationship between the differential gene expressions in liver and liver trioleate content and between blood glucose levels and a combination of differentially expressed genes measured in liver, IAT, and muscle. In summary, we have integrated gene expression and targeted metabolomic data to present a comprehensive overview of RSG-induced changes in a diabetes mouse model and improved the molecular understanding of how RSG ameliorates diabetes through its effect on the major insulin-sensitive tissues.

Type-2 diabetes (T2D) is a complex metabolic disease characterized by insulin resistance in the liver and peripheral tissues accompanied by a deficiency in pancreatic beta-cells. Since their discovery, three subtypes of peroxisome proliferator activated receptors have been identified, namely PPARalpha, PPARgamma and PPARbeta/(delta). In this study, we were interested in designing novel PPARgamma selective agonists and/or dual PPARalpha/gamma agonists. Based on the typical topology of synthetic PPAR agonists, we focused our design approach on using 4,4-dimethyl-1,2,3,4-tetrahydroquinoline as a novel cyclic scaffold with oxime and acidic head group structural variations.

We examined whether S26948, a new specific peroxisome proliferator activated receptor gamma modulator prevented insulin-resistance induced by a 48 h intralipid-infusion in normal rat (IL rats). The effect of S26948 (30 mg/kg) was compared to rosiglitazone (10 mg/kg). Rats were catheterized in the right jugular vein 4 days before the beginning of the 48 h lipid or saline infusions. Animals were intraperitoneally injected once daily with vehicle, S26948 or rosiglitazone. At the end of the infusion the rats underwent either a glucose tolerance test or a euglycemic-hyperinsulinemic clamp. Finally isolation and incubation of hepatocytes in another series of rats were performed. Intralipid infusion leads to a 4-fold increase in plasma free fatty acid concentration compared to controls (C). Both S26948 and rosiglitazone decreased plasma free fatty acid concentration in IL rats compared to vehicle treated IL rats. Glucose-induced insulin secretion was significantly increased in IL compared to C and was associated with insulin resistance. Both S26948 and rosiglitazone treatments normalized glucose-induced insulin secretion and improved insulin action in IL rats. However, S26948 specifically improved hepatic insulin sensitivity whereas rosiglitazone improved both hepatic insulin sensitivity and insulin-stimulated glucose utilization. Finally, studies on isolated hepatocytes showed differential effect of both compounds on gene expression of key enzymes of glucose metabolism. Our data show that non thiazolidinedione S26948 may represent an alternative way for the management of dysregulated hepatic insulin sensitivity.

Objective: Insulin secretion involves complex events in which the mitochondria play a pivotal role in the generation of signals that couple glucose detection to insulin secretion. Studies on the mitochondrial generation of reactive oxygen species (ROS) generally focus on chronic nutrient exposure. Here, we investigate whether transient mitochondrial ROS production linked to glucose-induced increased respiration might act as a signal for monitoring insulin secretion., Research Design and Methods: ROS production in response to glucose was investigated in freshly isolated rat islets. ROS effects were studied using a pharmacological approach and calcium imaging., Results: Transient glucose increase from 5.5 to 16.7 mmol/l stimulated ROS generation, which was reversed by antioxidants. Insulin secretion was dose dependently blunted by antioxidants and highly correlated with ROS levels. The incapacity of beta-cells to secrete insulin in response to glucose with antioxidants was associated with a decrease in ROS production and in contrast to the maintenance of high levels of ATP and NADH. Then, we investigated the mitochondrial origin of ROS (mROS) as the triggering signal. Insulin release was mimicked by the mitochondrial-complex blockers, antimycin and rotenone, that generate mROS. The adding of antioxidants to mitochondrial blockers or to glucose was used to lower mROS reversed insulin secretion. Finally, calcium imaging on perifused islets using glucose stimulation or mitochondrial blockers revealed that calcium mobilization was completely reversed using the antioxidant trolox and that it was of extracellular origin. No toxic effects were present using these pharmacological approaches., Conclusions: Altogether, these complementary results demonstrate that mROS production is a necessary stimulus for glucose-induced insulin secretion.

Background: The mass of pancreatic beta-cells varies according to increases in insulin demand. It is hypothesized that functionally heterogeneous beta-cell subpopulations take part in this process. Here we characterized two functionally distinct groups of beta-cells and investigated their physiological relevance in increased insulin demand conditions in rats., Methods: Two rat beta-cell populations were sorted by FACS according to their PSA-NCAM surface expression, i.e. beta(high) and beta(low)-cells. Insulin release, Ca(2+) movements, ATP and cAMP contents in response to various secretagogues were analyzed. Gene expression profiles and exocytosis machinery were also investigated. In a second part, beta(high) and beta(low)-cell distribution and functionality were investigated in animal models with decreased or increased beta-cell function: the Zucker Diabetic Fatty rat and the 48 h glucose-infused rat., Results: We show that beta-cells are heterogeneous for PSA-NCAM in rat pancreas. Unlike beta(low)-cells, beta(high)-cells express functional beta-cell markers and are highly responsive to various insulin secretagogues. Whereas beta(low)-cells represent the main population in diabetic pancreas, an increase in beta(high)-cells is associated with gain of function that follows sustained glucose overload., Conclusion: Our data show that a functional heterogeneity of beta-cells, assessed by PSA-NCAM surface expression, exists in vivo. These findings pinpoint new target populations involved in endocrine pancreas plasticity and in beta-cell defects in type 2 diabetes.

Background: Complex etiology and pathogenesis of pathophysiological components of the cardio-metabolic syndrome have been demonstrated in humans and animal models., Methodology/principal Findings: We have generated extensive physiological, genetic and genome-wide gene expression profiles in a congenic strain of the spontaneously diabetic Goto-Kakizaki (GK) rat containing a large region (110 cM, 170 Mb) of rat chromosome 1 (RNO1), which covers diabetes and obesity quantitative trait loci (QTL), introgressed onto the genetic background of the normoglycaemic Brown Norway (BN) strain. This novel disease model, which by the length of the congenic region closely mirrors the situation of a chromosome substitution strain, exhibits a wide range of abnormalities directly relevant to components of the cardio-metabolic syndrome and diabetes complications, including hyperglycaemia, hyperinsulinaemia, enhanced insulin secretion both in vivo and in vitro, insulin resistance, hypertriglyceridemia and altered pancreatic and renal histological structures. Gene transcription data in kidney, liver, skeletal muscle and white adipose tissue indicate that a disproportionately high number (43-83%) of genes differentially expressed between congenic and BN rats map to the GK genomic interval targeted in the congenic strain, which represents less than 5% of the total length of the rat genome. Genotype analysis of single nucleotide polymorphisms (SNPs) in strains genetically related to the GK highlights clusters of conserved and strain-specific variants in RNO1 that can assist the identification of naturally occurring variants isolated in diabetic and hypertensive strains when different phenotype selection procedures were applied., Conclusions: Our results emphasize the importance of rat congenic models for defining the impact of genetic variants in well-characterised QTL regions on in vivo pathophysiological features and cis-/trans- regulation of gene expression. The congenic strain reported here provides a novel and sustainable model for investigating the pathogenesis and genetic basis of risks factors for the cardio-metabolic syndrome.

Type-2 diabetes (T2D) is a complex metabolic disease characterized by insulin resistance in the liver and peripheral tissues accompanied by a defect in pancreatic beta-cell. Since their discovery three subtypes of Peroxisomes Proliferators Activated Receptors were identified namely PPARalpha, PPARgamma and PPARbeta/(delta). We were interested in designing novel PPARgamma selective agonists and/or dual PPARalpha/gamma agonists. Based on the typical topology of synthetic PPAR agonists, we focused our design approach on 4,4-dimethyl-1,2,3,4-tetrahydroquinoline as novel cyclic tail.

The most common form of diabetes, type 2 diabetes (T2D) is a major Public Health issue which is receiving a great deal of attention both in industrial and public research, in order to develop new and more effective drugs. The hyperglycaemia of T2D is the result of two interdependent defects : decreased biological efficacy of insulin in target tissues (insulin resistance), and a decreased capacity for beta cells to secrete insulin in response to glucose. Furthermore, hyperglycaemia evolves with time and even with rigorous treatment there is a progressive deterioration of glucose homeostasis. Seventy five percent of DT2 patients are obese and show a perturbed lipid profile. beta-cell plasticity is a unique property of these cells to adapt their number and volume (beta-cell mass) and their function to the increased secretory demand linked to insulin resistance. This is well documented in physiological (pregnancy) as well in pathophysiological conditions (obesity, acromegaly). Although the lack of reliable techniques makes it very difficult to document it in humans, this property is likely altered in DT2, mainly as a consequence of the prolonged exposure of islet cells to high plasma levels of glucose and free fatty acids (gluco-lipotoxicity). The mechanisms by which hyperglycaemia and hyperlipidemia exert their deleterious effects on the beta-cell include the generation of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) and Advanced Glycosylation End Products (AGE). Altogether the prevailing clinical and experimental data urge us to consider that the pathophysiology of DT2 lies, at least in part, the inability of beta-cells to adapt their functional mass to the prevailing insulin demand. This re-evaluation of the pathophysiology of DT2 stimulates the research of new therapeutic approaches aimed at maintaining and/or restoring the functional beta-cell mass by targeting the mechanisms responsible for its decrease.

We have generated transgenic mouse lines expressing exclusively a human INS transgene on an Ins1/Ins2 double knockout (mIKO) background. The transgene expression was driven by either a 4000 bp or a 353 bp promoter. These transgenic lines, designated mIKO:INS4000 and mIKO:INS353, were viable and fertile. Determination of the amounts of insulin transcripts and total pancreatic insulin content revealed relative insulin underproduction in both lines, from birth to adulthood. Total pancreatic insulin stores in mIKO:INS4000 and mIKO:INS353 mice represented only about 50% and 27%, respectively, as compared to wild-type mice. Morphometric analysis of pancreas did not show any compensatory beta-cell hyperplasia. The majority of animals in both lines remained normoglycemic throughout their lives. Nevertheless, glucose tolerance tests revealed glucose intolerance in nearly half of mIKO:INS4000 male mice, likely due to impaired insulin secretion detected in those animals. In addition, a small fraction (2-4%) of male mice in both lines spontaneously developed diabetes with very distinct pathophysiological features. Diabetes was never seen in female animals. The diabetes developed by mIKO:INS353 mice was rapidly lethal, accompanied by a dramatic depletion of pancreatic insulin stores whereas the mIKO:INS4000 diabetic animals could live for several months. This suggests a possible link between the structure of the human INS gene promoter and the type of diabetes developed in these lines.

Imidazoline derivatives have been reported to show antihyperglycemic activity in vivo. In the present study, we first showed that there was no correlation between the in vivo antidiabetic activity and the in vitro affinities for the I1/I2 binding sites for several substituted aryl imidazolines. Among these compounds, 2-(alpha-cyclohexyl-benzyl)-4,5-dihydro-1H-imidazole 2 exhibited potent antihyperglycemic properties. It was then chosen as lead compound. Thirty-six new derivatives were synthesized by replacing the cyclohexyl/benzyl group by various cyclic systems or the imidazoline ring by isosteric heterocycles. These compounds were evaluated in vivo for their antihyperglycemic activity using an oral glucose tolerance test (OGTT) in a rat model of type-2 diabetes obtained by giving a single intravenous (iv) injection of a low dose of streptozotocin to rats (STZ rats) and in normal rats. Nine compounds with an imidazoline moiety, possibly substituted by a methyl group, had a potent effect on the glucose tolerance in normal or STZ-diabetic rats, after an oral (po) administration of the test compound at a dose of 30 or 10 mg kg(-1), without any hypoglycemia. Replacement of the imidazoline ring by isosteric heterocycles resulted in a total loss of activity.

The physiological signaling mechanisms that link glucose sensing to the electrical activity in metabolism-regulating hypothalamus are still controversial. Although ATP production was considered the main metabolic signal, recent studies show that the glucose-stimulated signaling in neurons is not totally dependent on this production. Here, we examined whether mitochondrial reactive oxygen species (mROS), which are physiologically generated depending on glucose metabolism, may act as physiological sensors to monitor the glucose-sensing response. Transient increase from 5 to 20 mmol/l glucose stimulates reactive oxygen species (ROS) generation on hypothalamic slices ex vivo, which is reversed by adding antioxidants, suggesting that hypothalamic cells generate ROS to rapidly increase glucose level. Furthermore, in vivo, data demonstrate that both the glucose-induced increased neuronal activity in arcuate nucleus and the subsequent nervous-mediated insulin release might be mimicked by the mitochondrial complex blockers antimycin and rotenone, which generate mROS. Adding antioxidants such as trolox and catalase or the uncoupler carbonyl cyanide m-chlorophenylhydrazone in order to lower mROS during glucose stimulation completely reverses both parameters. In conclusion, the results presented here clearly show that the brain glucose-sensing mechanism involved mROS signaling. We propose that this mROS production plays a key role in brain metabolic signaling.

Type 2 diabetes is characterized by insulin resistance and progressive beta-cell failure. Deficient insulin secretion, with increased proportions of insulin precursor molecules, is a common feature of type 2 diabetes; this could result from inappropriate beta-cell function and/or reduced beta-cell mass. Most studies using tissues from diabetic patients are retrospective, providing only limited information on the relative contribution of beta-cell dysfunction versus decreased beta-cell mass to the "beta-cell failure" of type 2 diabetes. The gerbil Psammomys obesus is a good model to address questions related to the role of insulin resistance and beta-cell failure in nutritionally induced diabetes. Upon a change from its natural low-calorie diet to the calorie-rich laboratory food, P. obesus develops moderate obesity associated with postprandial hyperglycemia. Continued dietary load, superimposed on its innate insulin resistance, results in depletion of pancreatic insulin stores, with increased proportions of insulin precursor molecules in the pancreas and the blood. Inadequate response of the preproinsulin gene to the increased insulin needs is an important cause of diabetes progression. Changes in beta-cell mass do not correlate with pancreatic insulin stores and are unlikely to play a role in disease initiation and progression. The major culprit is the inappropriate insulin production with depletion of insulin stores as a consequence. Similar mechanisms could operate during the evolution of type 2 diabetes in humans.

Recent studies ascribe a major role to pancreatic beta-cell loss in type 2 diabetes. We investigated the dynamics of beta-cell mass during diabetes evolution in Psammomys obesus, a model for nutrition-dependent type 2 diabetes, focusing on the very early and the advanced stages of the disease. P. obesus fed a high-calorie diet for 26 days developed severe hyperglycemia, beta-cell degranulation, and markedly reduced pancreatic insulin content. Reducing calories for 7 days induced normoglycemia in 90% of the animals, restoring beta-cell granulation and insulin content. To dissociate effects of diet from blood glucose reduction, diabetic animals received phlorizin for 2 days, which normalized glycemia and increased the pancreatic insulin reserve to 50% of control, despite a calorie-rich diet. During diabetes progression, beta-cell mass decreased initially but recovered spontaneously to control levels, despite persistent hyperglycemia. Strikingly, however, beta-cell mass did not correlate with degree of hyperglycemia or pancreatic insulin content. We conclude that reduced insulin reserve is the main cause of diabetes progression, whereas irreversible beta-cell mass reduction is a late event in P. obesus. The rapid recovery of the pancreas by phlorizin-induced normoglycemia implies a causal relationship between hyperglycemia and islet dysfunction. Similar mechanisms could be operative during the evolution of type 2 diabetes in humans.

We investigated the possible interplay between insulin and glucose signaling pathways in rat pancreatic beta-cell with a special focus on the role of glucose in IRS signaling in vivo. Three groups of rats were constituted by combining simultaneous infusion during 48 h either of glucose and/or insulin, or glucose+diazoxide: Hyperglycemic-Hyperinsulinemic (HGHI), euglycemic-Hyperinsulinemic (eGHI), Hyperglycemic-euinsulinemic (HGeI). Control rats were infused with 0,9%NaCl. In HGHI and HGeI rats plasma glucose levels were maintained at 20-22 mmol/l. In eGHI rats, plasma glucose was not different from that of controls, whereas plasma insulin was much higher than in controls. In HGHI rats, IRS-2 mRNA expression, total protein and phosphorylated protein amounts were increased compared to controls. In HGeI rats, only IRS-2 mRNA expression was increased. No change was observed in eGHI rats whatever the parameter considered. In all groups, mRNA concentration of IRS-1 was similar to that of controls. The quantity of total and phosphorylated IRS-1 protein was dramatically increased in HGHI rats and to a lesser extent in eGHI rats. Neither mRNA nor IRS-1 protein expression were modified in HGeI rats. The data suggest that glucose and insulin play at once a specific and a complementary role in islet IRSs signaling. Especially, glucose stimulates IRS-2 mRNA expression whatever the insulin status and independently of the secretory process. The differential regulation of IRS-1 and IRS-2 expressions is in agreement with their supposed different involvement in the control of beta-cell growth and function.

Genetic studies in human populations and rodent models have identified regions of human chromosome 1q21-25 and rat chromosome 2 showing evidence of significant and replicated linkage to diabetes-related phenotypes. To investigate the relationship between the human and rat diabetes loci, we fine mapped the rat locus Nidd/gk2 linked to hyperinsulinemia in an F2 cross derived from the diabetic (type 2) Goto-Kakizaki (GK) rat and the Brown Norway (BN) control rat, and carried out its genetic and pathophysiological characterization in BN.GK congenic strains. Evidence of glucose intolerance and enhanced insulin secretion in a congenic strain allowed us to localize the underlying diabetes gene(s) in a rat chromosomal interval of approximately 3-6 cM conserved with an 11-Mb region of human 1q21-23. Positional diabetes candidate genes were tested for transcriptional changes between congenics and controls and sequence variations in a panel of inbred rat strains. Congenic strains of the GK rats represent powerful novel models for accurately defining the pathophysiological impact of diabetes gene(s) at the locus Nidd/gk2 and improving functional annotations of diabetes candidates in human 1q21-23.

ID-1101 (4-hydroxyisoleucine), an amino acid extracted from fenugreek seeds, exhibits an interesting glucose-dependent insulin-stimulating activity. The present study was undertaken to investigate a possible extrapancreatic effect of ID-1101 on insulin signaling and action besides its previously described insulinotropic action. Insulin-sensitizing effects of ID-1101 were investigated in rat in vivo by three different approaches: 1) using euglycemic hyperinsulinemic clamps in two different rat models of insulin resistance, i.e., Zucker fa/fa rats and rats fed a sucrose-lipid diet; 2) measuring liver and muscle phosphatidylinositol (PI) 3-kinase activity after an acute injection of ID-1101 in normal and insulin-resistant diabetic rats; and 3) after chronic treatment in two rat models of insulin resistance. Euglycemic hyperinsulinemic clamp experiments revealed that ID-1101 can improve insulin resistance through an increase of peripheral glucose utilization rate in sucrose-lipid-fed rats and by decreasing hepatic glucose production in Zucker fa/fa rats. Moreover, we demonstrated that a single injection of ID-1101 activates the PI 3-kinase activity in liver and muscle from normal rats but also in muscle from diabetic rats. Finally, chronic ID-1101 treatment significantly reduced insulinemia in type 2 diabetic rats and reduced the progression of hyperinsulinemia in insulin-resistant obese Zucker fa/fa rats. These findings clearly demonstrate that ID-1101 can reduce insulin resistance through activation of the early steps of insulin signaling in peripheral tissues and in liver. In summary, ID-1101, besides its insulinotropic effect, directly improves insulin sensitivity, making it a potentially very valuable therapeutic agent for diabetes treatment.

Diabetic pregnancy results in several metabolic and hormonal disorders, both in the embryo and the fetus of different species, including humans. Insulin is a potent modulator of brain development and is suggested to promote the differentiation and maturation of hypothalamic or related extrahypothalamic structures, which are directly involved in neural inputs to the pancreas. Because these structures are known to be specifically responsive both to insulin and glucose, we examined the effects of 48-h hyperglycemic clamps in unrestrained pregnant rats on insulin binding and glucose transporter expression in hypothalamic and extrahypothalamic-related areas of their fetal offspring. The main result was an increase in insulin binding in the ventromedial hypothalamic nucleus (VMH), the arcuate nucleus (AN), and the lateral hypothalamus (LH), and in the nucleus of the tractus solitarius (NTS) for extrahypothalamic areas (+30% in the VMH, +37% in the AN, +25.8% in the LH, and +37.3% in the NTS). The deleterious effect of brain hyperinsulinism during the late gestational stage does not seem to act through glucose transporter (GLUT) expression, inasmuch as no relationship between GLUT level and hyperinsulinism in brain areas could be observed. The specific increase in insulin binding in areas involved in the nervous control of metabolism could be a factor in the increased glucose intolerance and impairment of insulin secretion that was previously observed in the adult rats from hyperglycemic mothers.

A series of GLP-1-[7-36]-NH(2) (tGLP-1) and GLP-1-[7-37] analogs modified in position 7, 8, 9 and 36, have been designed and evaluated on murine GLP-1 receptors expressed in RIN T3 cells for both their affinity and activity. Ten of the synthesized peptides were found full agonists with activities superior or at least equal to that of the native hormone. Five of them were investigated for their plasmatic stability and the most stable, [a(8)-desR(36)]GLP-1-[7-37]- NH(2) (Compound 8), evaluated in vivo in a glucose tolerance test which confirmed a clearly longer activity than that of the native hormone. We also performed circular dichroism study and propose a hypothetical structural model explaining the most part of observed activities of GLP-1 analogs on RIN T3 cells.

Beta-cell neogenesis triggers the generation of new beta-cells from precursor cells. Neogenesis from duct epithelium is the most currently described and the best documented process of differentiation of precursor cells into beta-cells. It is contributes not only to beta-cell mass expansion during fetal and nonatal life but it is also involved in the maintenance of the beta-cell mass in adults. It is also required for the increase in beta-cell mass in situations of increase insulin demand (obesity, pregnancy). A large number of factors controlling the differentiation of beta-cells has been identified. They are classified into the following main categories: growth factors, cytokine and inflammatory factors, and hormones such as PTHrP and GLP-1. The fact that intestinal incretin hormone GLP-1 exerts a major trophic role on pancreatic beta-cells provides insights into the possibility to pharmacologically stimulate beta-cell neogenesis. This could have important implications for the of treatment of type 1 and type 2 diabetes. Transdifferentiation, that is, the differentiation of already differentiated cells into beta-cells, remains controversial. However, more and more studies support this concept. The cells, which can potentially "transdifferentiate" into beta-cells, can belong to the pancreas (acinar cells) and even islets, or originate from extra-pancreatic tissues such as the liver. Neogenesis from intra-islet precursors also have been proposed and subpopulations of cell precursors inside islets have been described by some authors. Nestin positive cells, which have been considered as the main candidates, appear rather as progenitors of endothelial cells rather than beta-cells and contribute to angiogenesis rather than neogenesis. To take advantage of the different differentiation processes may be a direction for future cellular therapies. Ultimately, a better understanding of the molecular mechanisms involved in beta-cell neogenesis will allow us to use any type of differentiated and/or undifferentiated cells as a source of potential cell precursors.

We investigated the specific and associated effects of insulin and glucose on beta-cell growth and function in adult rats. By combining simultaneous infusion either of glucose and/or insulin or glucose and diazoxide, three groups of rats were constituted: hyperglycemic-hyperinsulinemic rats (high glucose-high insulin), hyperglycemic-euinsulinemic rats (high glucose), and euglycemic-hyperinsulinemic rats (high insulin). All the infusions lasted 48 h. Control rats were infused with 0.9% NaCl (saline controls). In all groups, beta-cell mass was significantly increased, compared with controls (by 70% in high glucose-high insulin rats, 65% in high glucose rats, and 50% in high insulin rats). The stimulation of neogenesis was suggested by the high number of islets budding from pancreatic ducts in high glucose-high insulin and high glucose rats and by the presence of numerous clusters of few beta-cells within the exocrine pancreas in high insulin rats. beta-Cell hypertrophy was observed only in high glucose-high insulin rats. The rate of beta-cell proliferation was similar to that of controls in high glucose-high insulin rats after a 48-h glucose infusion, dropped dramatically in high insulin rats, and dropped to a lesser extent in high glucose rats. In high glucose-high insulin and high glucose rats, beta-cell mass increase was related to a higher beta-cell responsiveness to glucose in vitro as measured by islet perifusion studies, whereas in high insulin rats, no significant enhancement of glucose induced insulin secretion could be noticed. The data show that glucose and insulin may have specific stimulating effects on beta-cell growth and function in vivo in adult rats independently of the influence they exert each other on their respective plasma concentration.

We investigated here whether non-esterified fatty acids (NEFA) influence insulin secretion and action through a direct effect on central nervous system sites involved in the control of glucose homeostasis. Normal Wistar rats received a 48-h intracerebroventricular infusion of either a 10% triglyceride (Intralipid, IL)/heparin emulsion (IL/h) or saline/heparin solution (control). At 48 h, insulin secretion as measured by an intravenous glucose tolerance test, was more elevated in IL/h than in control rats. Pancreatic noradrenaline turnover was decreased by 57% in IL/h rats, suggesting low pancreatic sympathetic output that could account partly for the elevated insulin secretion. The time course of glycaemia was similar in both groups, suggesting insulin resistance. Euglycaemic-hyperinsulinaemic clamps were imposed to assess peripheral and hepatic insulin sensitivity. At each insulin concentration glucose utilization was increased to a similar extent in both groups, whereas hepatic glucose production decreased much less in IL/h than in control rats. Hepatic insulin insensitivity could be related partly to activation of the hypothalamic-pituitary-adrenocortical axis, since plasma corticosterone concentration was significantly increased in IL/h rats compared with controls. Our data indicate that lipids may alter both insulin secretion and hepatic sensitivity to insulin through their effect on central nervous system.