Impaired Glucose Tolerance In Mice Research Articles

Dopamine signalling in pancreatic islet cells and role in adaptations to metabolic stress.

Dopamine and related receptors are evidenced in pancreatic endocrine tissue, but the impact on islet β-cell stimulus-secretion as well as (patho)physiological role are unclear. The present study has evaluated islet cell signalling pathways and biological effects of dopamine, as well as alterations of islet dopamine in rodent models of diabetes of different aetiology. The dopamine precursor l-DOPA partially impaired glucose tolerance in mice and attenuated glucose-, exendin-4, and alanine-induced insulin secretion. The latter effect was echoed by the attenuation of glucose-induced [Ca2+]i dynamics and elevation of ATP levels in individual mouse islet cells. l-DOPA significantly decreased β-cell proliferation rates, acting predominantly via the D2 receptor, which was most abundant at the mRNA level. The administration of streptozotocin (STZ) or high-fat diet (HFD) in mice significantly elevated numbers of dopamine-positive islet cells, with HFD also increasing colocalization of dopamine with insulin. At the same time, colocalization of dopamine with glucagon was increased in STZ-treated and pregnant mice, but unaffected by HFD. These findings highlight a role for dopamine receptor signalling in islet cell biology adaptations to various forms of metabolic stress.

Let7b-5p inhibits insulin secretion and decreases pancreatic β-cell mass in mice

MicroRNAs are crucial regulators for the development, mass and function of pancreatic β-cells. MiRNA dysregulation is associated with β-cell dysfunction and development of diabetes. The members of let7 family are important players in regulating cellular growth and metabolism. In this study we investigated the functional role of let7b-5p in the mouse pancreatic β-cells. We generated pancreatic β-cell-specific let7b-5p transgenic mouse model and analyzed the glucose metabolic phenotype, β-cells mass and insulin secretion in vivo. Luciferase reporter assay, immunofluorescence staining and western blot were carried out to study the target genes of let7b-5p in β-cells. Let7b-5p overexpression impaired the insulin production and secretion of β-cells and resulted impaired glucose tolerance in mice. The overexpressed let7b-5p inhibited pancreatic β-cell proliferation and decreased the expression of cyclin D1 and cyclin D2. Our findings demonstrated that let7b-5p was critical in regulating the proliferation and insulin secretion of pancreatic β-cells.

Suppressor of cytokine signalling-2 controls hepatic gluconeogenesis and hyperglycemia by modulating JAK2/STAT5 signalling pathway

Hepatic gluconeogenesis plays a crucial role in maintaining blood glucose homeostasis in mammals. Globe knockout of suppressor of cytokine signalling-2 (SOCS2), a feedback inhibitor of cytokine signalling, has been shown resistant to high-fat-diet (HFD)-induced hepatic steatosis with impaired glucose tolerance in mice. However, the underlying mechanism of SOCS2 regulates hepatic glucose homeostasis still undefined. In the present study, we demonstrated that the hepatic SOCS2 expression is markedly reduced in fasted C57BL/6 J mice or db/db mice. Moreover, hepatic SOCS2 expression levels are induced by metformin treatment. Ablation of SOCS2 attenuates suppressing effects of metformin on gluconeogenesis in hepatocytes. Gain- and loss-of-function studies indicated that SOCS2 regulates hepatic gluconeogenic genes expression and glucose output by mediating JAK2/STAT5 signalling pathway in db/db mice. Mechanistically, we observed that SOCS2 inactivates STAT5 by attenuating the interaction between JAK2 and STAT5, which in turn reduces hepatic gluconeogenesis. The present study reveals a critical role of SOCS2 in regulating hepatic gluconeogenesis. The inhibitory effect of metformin on gluconeogenesis is mediated, at least in part, by upregulating SOCS2 and therefore reducing hepatic gluconeogenic genes expression. SOCS2 may represent a new therapeutic target for the treatment of diabetes.

Amelioration of Olanzapine-Induced Impaired Glucose Tolerance in Mice with Methanol Extract of Steamed Brassica Oleracea (Cabbage) Leaves

Olanzapine is an antipsychotic drug and has been reported to induce impaired glucose tolerance leading to high blood glucose levels. In oral glucose tolerance tests (OGTT), methanolic extract of steamed cabbage (Brassica oleracea L. var. capitata) (MEBO) leaves have been shown to reduce elevated blood glucose levels in glucose-loaded mice. It was thus of interest to determine whether MEBO leaves can ameliorate olanzapineinduced impaired glucose tolerance in mice, which have been administered olanzapine for 28 days. Impaired glucose tolerance was measured through OGTT in mice. Olanzapine (28 days)-administered mice showed elevated blood glucose in OGTT. MEBO leaves showed significant reduction of blood glucose level in OGTT in mice (compared to vehicle or olanzapine treated mice for 28 days, Groups 2 and 3, respectively) both when administered for 28 days along with olanzapine, as well when administered 60 min prior to glucose loading in OGTT (positive control glibenclamide administered at 10 mg/kg). A single dose of MEBO (400 mg/kg) was used based on previous studies. Thus, MEBO leaves can be beneficial for improving glucose tolerance and reduce blood glucose levels in olanzapine-induced elevated blood glucose levels.

Anacardic acid mitigates liver fat accumulation and impaired glucose tolerance in mice fed a high-fat and high-sucrose diet.

In this study, we evaluated the effects of anacardic acid (AA), a phenolic lipid found in cashew nuts (Anacardium occidentale), on metabolic disorders related to obesity, fatty liver disease, and diabetes using both in vitro and in vivo models. The application of AA led to a reduction in lipid accumulation in 3T3‐L1 cells without observable cytotoxicity. Results from Western blot analysis revealed that AA treatment also led to decreased expression of fatty acid synthase and peroxisome proliferator‐activated receptor gamma. In vivo studies were performed to evaluate the effects of AA treatment on fatty liver disease and diabetes. Mice fed a high‐fat and high‐sucrose diet had significantly higher body and liver weights, and higher levels of liver fat, cholesterol, fasting glucose, and homeostasis model assessment of insulin resistance (HOMA‐IR). However, 12 weeks of oral treatment with 500 μg/kg BW AA slowed down lipid accumulation rates in the liver and mitigated insulin resistance in these mice. Thus, AA may reduce lipid levels and have an antidiabetic effect.

SAT-024 Adipose Specific Inactivation of Aromatase is Sufficient to Cause Adiposity and Impaired Glucose Tolerance in Mice

Aromatase synthesizes estrogens from androgens. While the gonads are major sites of aromatase expression, it is expressed in multiple other organs including adipose tissue, brain and bone. In global aromatase knockouts in mice, studied by us, or man aromatase deficiency there are phenotypes of adiposity and low bone mass as well as varying degrees of insulin resistance. Breast cancer patients treated with aromatase inhibitors (AIs) also develop adiposity and low bone mass. Thus, it remains unclear the role of site specific aromatase activity in the phenotypes observed in chemically or genetically induced aromatase deficiency. To begin to understand the relative contributions of tissue specific aromatase activity and develop a model that will be useful for understanding and developing therapeutic strategies for ailments in aromatase inhibitor therapy, we have developed mouse models with tissue specific aromatase inactivation. Embryonic stem cells were purchased from EUCOMM. Using standard methods and appropriate breeding strategies we generated C57Bl6 mice with Lox P sites flanking Exon 2 in the aromatase gene (Arom fl/fl). Mice with adipose specific inactivation of aromatase were generated by interbreeding Arom fl/fl mice with mice expressing Cre recombinase driven by the adiponectin promoter (AdipoCre). We measured body composition by dual energy x-ray absorptiometry (Lunar PIXIMus 2). As a first step in examining glucose homeostasis we performed oral glucose tolerance tests. All mice were fed normal chow and the study was approved by the local IACUC. Female mice aged 4-6 months were studied with at least 4 animals in each group. Arom fl/fl mice were compared as controls for the AdipoCre+; Arom fl/fl mice. Mean body weight was higher in fat specific aromatase knockout female mice (33.6 vs 21.6, p<0.05). On DEXA Mean %Body fat was higher in female AdipoCre+;Arom fl/fl mice (29.3% vs. 17.3%, p<0.05). Following a 16h fast female mice with adipose specific aromatase inactivation had higher fasting blood glucose levels (93.3+/-10.3 vs 74.5+/-10, p=0.03). For the glucose tolerance test, a bolus of glucose (2 g/kg) was delivered into the stomach by a gavage needleand blood was sampled at 0, 5, 10, 20, 30, 60, 90, 120, and 180 min for plasma glucose analyses. On OGTT peak glucose levels were higher and did not return to baseline. Insulin tolerance tests will be performed to further understand the mechanism of impaired glucose homeostasis. Male mice with fat specific aromatase inactivation showed similar patterns in higher body weight, percent body fat and glucose levels but the phenotype was more variable. In summary, fat specific aromatase inactivation is sufficient to cause adiposity and impaired glucose homeostasis. The observed phenotype in females is more robust than we have observed in the global knockout.

Glimepiride Administered in Chow Reversibly Impairs Glucose Tolerance in Mice.

Sulfonylureas are a class of antidiabetes medications prescribed to millions of individuals worldwide. Rodents have been used extensively to study sulfonylureas in the laboratory. Here, we report the results of studies treating mice with a sulfonylurea (glimepiride) in order to understand how the drug affects glucose homeostasis and tolerance. We tested the effect of glimepiride on fasting blood glucose, glucose tolerance, and insulin secretion, using glimepiride sourced from a local pharmacy. We also examined the effect on glucagon, gluconeogenesis, and insulin sensitivity. Unexpectedly, glimepiride exposure in mice was associated with fasting hyperglycemia, glucose intolerance, and decreased insulin. There was no change in circulating glucagon levels or gluconeogenesis. The effect was dose-dependent, took effect by two weeks, and was reversed within three weeks after removal. Glimepiride elicited the same effects in all strains evaluated: four wild-type strains, as well as the transgenic Grn−/− and diabetic db/db mice. Our findings suggest that the use of glimepiride as a hypoglycemic agent in mice should proceed with caution and may have broader implications about mouse models as a proxy to study the human pharmacopeia.

Multidrug resistance protein 4 (Mrp4), a drug transporter is a novel genetic determinant for adipogenesis

Multidrug resistance protein 4 (Mrp4; encoded by Abcc4) is a basolateral; efflux pump that transports several xenobiotic and endogenous molecules such as prostaglandins and cyclic nucleotides. Mrp4 is highly expressed in kidney and bladder, with minimal expression in liver. Recently, our laboratory determined that mice lacking Mrp4 develop hepatic steatosis during compensatory tissue regeneration following partial hepatectomy. These studies indicate that Mrp4 may play a significant role in lipid homeostasis. Recent studies also showed that Mrp4 is expressed in adipose tissue, yet the role of Mrp4 in adipose tissue physiology and lipid metabolism is unknown. To begin addressing this, WT and Mrp4 knockout (Mrp4−/−) mice were subjected to 24‐hr fasting. The mouse embryonic fibroblast cell line 3T3‐L1 was also used to investigate the role of Mrp4 in adipogenesis. The results showed that both male and female Mrp4−/− mice have increased adipose tissue to body weight ratio and adipocyte cell size in both fed and fasted conditions. Additionally, the lack of Mrp4 is associated with increased serum glucose levels and impaired glucose tolerance in mice challenged with the glucose tolerance test. In parallel to increased adipose tissue weights, Mrp4−/− mice also showed increase in leptin gene expression in adipose tissue. In vitro studies using 3T3‐L1 cells showed that pharmacological inhibition of Mrp4 function using either MK571 or C1 increases adipogenesis and expression of adipose tissue markers such as fatty acid binding protein 4 (Fabp4), lipoprotein lipase (Lpl), glucose transporter type 4 (Glut4) and peroxisome proliferator‐activated receptor gamma (Ppar‐γ). In conclusion, our studies show that the absence of Mrp4 or chemical inhibition promotes adipogenesis, in addition to its well‐established role as a drug transporter. The implications of these findings are that alterations in Mrp4 expression and function can now be considered as a potential risk factor in the pathogenesis of metabolic diseases such as fatty liver disease, obesity and diabetes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A2A adenosine receptors control pancreatic dysfunction in high-fat-diet-induced obesity.

Adenosine, a key extracellular signaling mediator, regulates several aspects of metabolism by activating 4 G-protein-coupled receptors, the A1, A2A, A2B, and A3 adenosine receptors (ARs). The role of A2AARs in regulating high-fat-diet (HFD)-induced metabolic derangements is unknown. To evaluate the role of A2AARs in regulating glucose and insulin homeostasis in obesity, we fed A2AAR-knockout (KO) and control mice an HFD for 16 wk to initiate HFD-induced metabolic disorder. We found that genetic deletion of A2AARs caused impaired glucose tolerance in mice fed an HFD. This impaired glucose tolerance was caused by a decrease in insulin secretion but not in insulin sensitivity. Islet size and insulin content in pancreata of A2AAR-deficient mice were decreased compared with control mice after consuming an HFD. A2AAR-KO mice had decreased expression of the β-cell-specific markers pdx1, glut2, mafA, and nkx6.1 and increased expression of the dedifferentiation markers sox2 and hes1. Ex vivo islet experiments confirmed the role of A2AARs in protecting against decreased insulin content and release caused by HFD. Other experiments with bone marrow chimeras revealed that inflammation was not the primary cause of decreased insulin secretion in A2AAR-KO mice. Altogether, our data showed that A2AARs control pancreatic dysfunction in HFD-induced obesity.-Csóka, B., Törő, G., Vindeirinho, J., Varga, Z. V., Koscsó, B., Németh, Z. H., Kókai, E., Antonioli, L., Suleiman, M., Marchetti, P., Cseri, K., Deák, Á., Virág, L., Pacher, P., Bai, P., Haskó, G. A2A adenosine receptors control pancreatic dysfunction in high-fat-diet-induced obesity.

In vivo Evaluation of Two Thiazolidin-4-one Derivatives in High Sucrose Diet Fed Pre-diabetic Mice and Their Modulatory Effect on AMPK, Akt and p38 MAP Kinase in L6 Cells.

We had previously demonstrated the anti-diabetic potential and pancreatic protection of two thiazolidin-4-one derivatives containing nicotinamide moiety (NAT-1 and NAT-2) in STZ-induced diabetic mice. However, due to the limitations of the STZ model, we decided to undertake a detailed evaluation of anti-diabetic potential of the molecules on a high sucrose diet (HSD) fed diabetic mouse model. Further, in vitro mechanistic studies on the phosphorylation of AMPK, Akt and p38 MAP kinase in L6 myotubes and anti-inflammatory studies in RAW264.7 mouse monocyte macrophage cells were performed. 15 months of HSD induced fasting hyperglycaemia and impaired glucose tolerance in mice. Treatment with NAT-1 and NAT-2 (100 mg/kg) for 45 days significantly improved the glucose tolerance and lowered fasting blood glucose levels compared to untreated control. An improvement in the elevated triglycerides and total cholesterol levels, and favorable rise in HDL cholesterol were also observed with test drug treatment. Also, no major changes were observed in the liver (albumin, AST and ALT) and kidney (creatinine and urea) parameters. This was further confirmed in their respective histology profiles which revealed no gross morphological changes. In L6 cells, significant phosphorylation of Akt and p38 MAP kinase proteins were observed with 100 μM of NAT-1 and NAT-2 with no significant changes in phosphorylation of AMPK. The molecules failed to exhibit anti-inflammatory activity as observed by their effect on the generation of ROS and nitrite, and nuclear levels of NF-κB in LPS-stimulated RAW264.7 cells. In summary, the molecules activated Akt and p38 MAP kinase which could have partly contributed to their anti-hyperglycaemic and hypolipidemic activities in vivo.

BAIBA attenuates insulin resistance and inflammation induced by palmitate or a high fat diet via an AMPK-PPARδ-dependent pathway in mice.

We explored the effects of β-aminoisobutyric acid (BAIBA) on hyperlipidaemic-condition-induced insulin resistance and inflammation as mediated through a signalling pathway involving AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor δ (PPARδ). Mouse skeletal muscle C2C12 cells and C57BL/6J mice were treated with palmitate or a high-fat diet (HFD) and BAIBA. Inflammation and the expression of genes associated with insulin signalling were determined by western blot and quantitative real-time PCR. Selected genes from candidate pathways were evaluated by small interfering (si)RNA knockdown and specific inhibitors. BAIBA treatment ameliorated impairment of insulin receptor substrate (IRS)-1/Akt-mediated insulin signalling in palmitate-treated C2C12 myocytes and in skeletal muscle of HFD-fed mice. In addition, BAIBA treatment reversed HFD-induced increases in body weight and improved impaired glucose tolerance in mice. In vitro and in vivo, inhibitory κBα (IκBα) phosphorylation, nuclear factor κB (NFκB) nuclear translocation and downstream inflammatory cytokines were significantly suppressed by BAIBA. Furthermore, BAIBA treatment significantly induced AMPK phosphorylation and expression of PPARδ in C2C12 myocytes and in skeletal muscle of mice. Both compound C, an AMPK inhibitor, and Pparδ (also known as Ppard) siRNA abrogated the inhibitory effects of BAIBA on palmitate-induced inflammation and insulin resistance. BAIBA significantly induced the expression of genes associated with fatty acid oxidation, such as carnitine palmitoyltransferase 1 (Cpt1), acyl-CoA oxidase (Aco; also known as Acox1) and fatty acid binding protein 3 (Fabp3); this effect of BAIBA was significantly reduced by compound C and Pparδ siRNA. These results are the first to demonstrate that BAIBA attenuates insulin resistance, suppresses inflammation and induces fatty acid oxidation via the AMPK-PPARδ pathway in skeletal muscle.

MR blockade protects against diet induced obesity, adipocyte dysfunction and cardiac inflammation in mice, through browning of the adipose organ and modulation of autophagy

Obesity is a key factor in the development of insulin resistance (IR), cardiovascular disease, hypertension, type 2 diabetes etc. Given the near epidemic incidence of obesity in western society there is a clear need for effective treatment options. Mineralocorticoid receptor (MR) blockade has shown significant promise in transgenic mouse models of obesity in limiting IR and adipocyte dysfunction, a disease that is independent of classical MR actions (renal). Female 10-weekold C57bl6 mice were fed with normal chow or a high fat (HF) diet for 12 weeks. Mice fed HF diet were concomitantly treated for 12 weeks with drospirenone (DRSP, 6 mg/kg/day), a potent MR antagonist with antiadipogenic activity, or spironolactone (SPIRO, 20 mg/kg/day). Mice fed HF diet showed a significant increase in total body weight, fat mass, mean adipocyte size, expression of white adipose tissue (WAT) marker genes and showed impaired glucose tolerance after intraperitoneal plasma glucose tolerance test. DRSP and SPIRO prevented weight gain and white fat mass expansion induced by HF diet in parametrial, perivescical, and inguinal depots without affecting interscapular fat pad weight. Magnetic Resonance Imaging (MRI) confirmed that MR antagonists blocked the HF dietdriven expansion of abdomino-pelvic (parametrial and perivescical) fat volume. High levels of MR mRNA were detected in all depots of adipose tissue. HF fed mice showed no increase in heart or kidney weight and tissue fibrosis. Cardiac macrophage recruitment and osteopontin staining was increased in hearts of HF fed mice and reversed by both MR antagonists. Moreover, both DRSP and SPIRO prevented the impaired glucose tolerance in mice fed HF diet, and countered HF diet-induced up-regulation of WAT markers transcripts and adipocyte hypertrophy. Importantly, MR antagonists increased uncoupling protein 1 (UCP-1) positive brown-like adipocyte content in WAT, and improved metabolic activity of adipose tissue, as indicated by PET/CT imaging. In keeping with this, MR antagonism significantly increased expression of brown-like adipocyte marker genes such PRDM16, CIDEA, beta-3 adrenergic receptor (ADRB3) and UCP-1 in all WAT depots analysed. In exploring the mechanism, we demonstrated that MR antagonism induced brown adipose tissue (BAT) markers, and reduced the autophagic rate, a key remodelling process in adipocyte differentiation, in WAT depots in vivo as well as in primary cultured adipocytes. We conclude that adipocyte MR regulates BAT-like remodeling of WAT through modulation of autophagy. MR blockade therefore has promise as a novel therapeutic option for the prevention of metabolic dysfunctions and the cardiac consequences of obesity. doi:10.1016/j.ijcme.2015.05.012 Transcriptional control of ICAM-1 in human coronary artery endothelial cells by Mineralocorticoid Receptor (MR): Implications for the protective effects of MR antagonists in cardiovascular diseases V. Marzolla, A. Armani, A. Fabbri, I.Z. Jaffe, M. Caprio Laboratory of Cardiovascular Endocrinology, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) San Raffaele Pisana, Rome, Italy Department of Medicina dei Sistemi, Endocrinology Unit, S. Eugenio & CTO A. Alesini Hospitals, University Tor Vergata, Rome, Italy Molecular Cardiology Research Institute, Tufts Medical Center, Boston,

Transcriptional control of ICAM-1 in human coronary artery endothelial cells by Mineralocorticoid Receptor (MR): Implications for the protective effects of MR antagonists in cardiovascular diseases

Obesity is a key factor in the development of insulin resistance (IR), cardiovascular disease, hypertension, type 2 diabetes etc. Given the near epidemic incidence of obesity in western society there is a clear need for effective treatment options. Mineralocorticoid receptor (MR) blockade has shown significant promise in transgenic mouse models of obesity in limiting IR and adipocyte dysfunction, a disease that is independent of classical MR actions (renal). Female 10-weekold C57bl6 mice were fed with normal chow or a high fat (HF) diet for 12 weeks. Mice fed HF diet were concomitantly treated for 12 weeks with drospirenone (DRSP, 6 mg/kg/day), a potent MR antagonist with antiadipogenic activity, or spironolactone (SPIRO, 20 mg/kg/day). Mice fed HF diet showed a significant increase in total body weight, fat mass, mean adipocyte size, expression of white adipose tissue (WAT) marker genes and showed impaired glucose tolerance after intraperitoneal plasma glucose tolerance test. DRSP and SPIRO prevented weight gain and white fat mass expansion induced by HF diet in parametrial, perivescical, and inguinal depots without affecting interscapular fat pad weight. Magnetic Resonance Imaging (MRI) confirmed that MR antagonists blocked the HF dietdriven expansion of abdomino-pelvic (parametrial and perivescical) fat volume. High levels of MR mRNA were detected in all depots of adipose tissue. HF fed mice showed no increase in heart or kidney weight and tissue fibrosis. Cardiac macrophage recruitment and osteopontin staining was increased in hearts of HF fed mice and reversed by both MR antagonists. Moreover, both DRSP and SPIRO prevented the impaired glucose tolerance in mice fed HF diet, and countered HF diet-induced up-regulation of WAT markers transcripts and adipocyte hypertrophy. Importantly, MR antagonists increased uncoupling protein 1 (UCP-1) positive brown-like adipocyte content in WAT, and improved metabolic activity of adipose tissue, as indicated by PET/CT imaging. In keeping with this, MR antagonism significantly increased expression of brown-like adipocyte marker genes such PRDM16, CIDEA, beta-3 adrenergic receptor (ADRB3) and UCP-1 in all WAT depots analysed. In exploring the mechanism, we demonstrated that MR antagonism induced brown adipose tissue (BAT) markers, and reduced the autophagic rate, a key remodelling process in adipocyte differentiation, in WAT depots in vivo as well as in primary cultured adipocytes. We conclude that adipocyte MR regulates BAT-like remodeling of WAT through modulation of autophagy. MR blockade therefore has promise as a novel therapeutic option for the prevention of metabolic dysfunctions and the cardiac consequences of obesity. doi:10.1016/j.ijcme.2015.05.012 Transcriptional control of ICAM-1 in human coronary artery endothelial cells by Mineralocorticoid Receptor (MR): Implications for the protective effects of MR antagonists in cardiovascular diseases V. Marzolla, A. Armani, A. Fabbri, I.Z. Jaffe, M. Caprio Laboratory of Cardiovascular Endocrinology, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) San Raffaele Pisana, Rome, Italy Department of Medicina dei Sistemi, Endocrinology Unit, S. Eugenio & CTO A. Alesini Hospitals, University Tor Vergata, Rome, Italy Molecular Cardiology Research Institute, Tufts Medical Center, Boston,

Evidence for inhibitory autocrine effects of proinsulin C‐peptide on pancreatic β‐cell function and insulin secretion

Autocrine and paracrine regulatory mechanisms ensure integrated secretion of islet hormones that respond efficiently to changes in metabolic need. As proinsulin C-peptide exerts various biological effects and binds to cell membranes including insulin-secreting β cells, its physiological role in insulin release was examined. Insulin releasing activity of human and rat C-peptides were studied in the clonal pancreatic cell line, BRIN-BD11, with findings substantiated using isolated islets and in vivo studies employing SWISS TO mice. Acute exposure of clonal β cells to human C-peptide resulted in concentration-dependent inhibitory effects on insulin secretion at 5.6 mM (p < 0.05-p < 0.001) and 16.7 mM (p < 0.01-p < 0.001) glucose. At physiologically relevant intra-islet concentrations (10(-9) -10(-6) M), C-peptide suppressed the insulin-secretory responses to a range of secretagogues acting at different points in the β cell stimulus-secretion coupling pathway including alanine (p < 0.05), Ca(2+) (p < 0.001), arginine (p < 0.05), tolbutamide (p < 0.001), glucagon-like peptide 1 (GLP-1) (p < 0.001), isobutylmethylxanthine (IBMX) (p < 0.01) and KCl (p < 0.05). Similar results were obtained using isolated mouse pancreatic islets. Human C-peptide (3 × 10(-7) M, p < 0.001), somatostatin-14 (3 × 10(-7) M, p < 0.01) and diazoxide (300 µM, p < 0.001) reduced both alanine and glucose-stimulated insulin release by 43, 25 and 48%, respectively. The effects of human C-peptide were reproduced using rat C-peptide I and II. C-peptide also inhibited in vivo glucose-stimulated insulin release and impaired glucose tolerance in mice. C-peptide is a biologically active endogenous peptide hormone that exerts inhibitory autocrine effects on pancreatic β-cell function. Mechanisms involving the activation of K(+) channels and a distal effect downstream of increased cytoplasmic Ca(2+) appear to be implicated in the inhibition of insulin secretion.

Sympathetic Innervation during Development Is Necessary for Pancreatic Islet Architecture and Functional Maturation

Sympathetic neurons depend on target-derived neurotrophic cues to control their survival and growth. However, whether sympathetic innervation contributes reciprocally to the development of target tissues is less clear. Here, we report that sympathetic innervation is necessary for the formation of the pancreatic islets of Langerhans and for their functional maturation. Genetic or pharmacological ablation of sympathetic innervation during development resulted in altered islet architecture, reduced insulin secretion, and impaired glucose tolerance in mice. Similar defects were observed with pharmacological blockade of β-adrenergic signaling. Conversely, the administration of a β-adrenergic agonist restored islet morphology and glucose tolerance in deinnervated animals. Furthermore, in neuron-islet cocultures, sympathetic neurons promoted islet cell migration in a β-adrenergic-dependent manner. This study reveals that islet architecture requires extrinsic inductive cues from neighboring tissues such as sympathetic nerves and suggests that early perturbations in sympathetic innervation might underlie metabolic disorders.

Mutations in Mll2, an H3K4 Methyltransferase, Result in Insulin Resistance and Impaired Glucose Tolerance in Mice

We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5–14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level.

Control of glucose homeostasis and insulin sensitivity by the Let-7 family of microRNAs

Diabetes mellitus is the most common metabolic disorder worldwide and a major risk factor for cardiovascular disease. MicroRNAs are negative regulators of gene expression that have been implicated in many biological processes, including metabolism. Here we show that the Let-7 family of microRNAs regulates glucose metabolism in multiple organs. Global and pancreas-specific overexpression of Let-7 in mice resulted in impaired glucose tolerance and reduced glucose-induced pancreatic insulin secretion. Mice overexpressing Let-7 also had decreased fat mass and body weight, as well as reduced body size. Global knockdown of the Let-7 family with an antimiR was sufficient to prevent and treat impaired glucose tolerance in mice with diet-induced obesity, at least in part by improving insulin sensitivity in liver and muscle. AntimiR treatment of mice on a high-fat diet also resulted in increased lean and muscle mass, but not increased fat mass, and prevented ectopic fat deposition in the liver. These findings demonstrate that Let-7 regulates multiple aspects of glucose metabolism and suggest antimiR-induced Let-7 knockdown as a potential treatment for type 2 diabetes mellitus. Furthermore, our Cre-inducible Let-7-transgenic mice provide a unique model for studying tissue-specific aspects of body growth and type 2 diabetes.

Inhibition of Adenylyl Cyclase Type 5 Protects Against Obesity and Diabetes

We reported adenylyl cyclase type 5 (AC5) disruption (knock out, KO) increases food intake, but reduces weight, implying a metabolic profile consistent with protection against obesity and diabetes. We compared AC5 KO and wild type littermates (WTL) on a normal diet and examined the effects of a pharmacological inhibitor, specific for AC5, on WT mice on a high fat diet (HFD). The AC5 KO mice, despite eating more, weighed 12% less compared against WTL (P<0.05). At sacrifice, fat pads were isolated and adiposity index calculated. In the KO mice adiposity index was 34% less than WTL. The pharmacolgical inhibitor of AC5 (Vidarabine), was administered to WT mice via a subcutaneous osmotic pump (30 mg/kg/day). The specificity of this inhibitor was demonstrated by the degree of inhibition of forskolin-stimulated AC activity in cardiac myocytes as follows: AC5 overexpression (OE) > WT, AC6 OE > AC5 KO. Mice on HFD treated with the AC5 inhibitor ate similarly but gained 41% less weight (p<0.05), compared to vehicle treated mice and the adiposity index was reduced by 30% (p<0.05), with a 32% reduction in visceral fat cell size (p<0.05). The AC5 inhibitor also normalized the impaired glucose tolerance in mice on the HFD. Thus, inhibiting AC5 reduces obesity and improves glucose tolerance, suggesting a potentially novel therapeutic approach to obesity and diabetes.

Anti-obesity and anti-diabetic effects of ethanol extract of Artemisia princeps in C57BL/6 mice fed a high-fat diet

Artemisia princeps is commonly used as a food ingredient and in traditional Asian medicine. In this study, we examined the effects of long-term administration of an ethanol extract of A. princeps (APE) on body weight, white adipose tissue, blood glucose, insulin, plasma and hepatic lipids, and adipocytokines in C57BL/6 mice fed a high-fat diet. Daily feeding of a 1% APE diet for 14 weeks normalized elevated body weight, white adipose tissue, and plasma glucose and insulin levels, and delayed impaired glucose tolerance in mice a fed high-fat diet. These events were not observed in mice fed a control diet containing 1% APE. Liver triglyceride and cholesterol levels were similar in mice fed a 1% APE-diet and those fed a control diet. In the high-fat diet groups, APE inhibited hepatic fatty acid synthase (FAS) and suppressed the elevation of plasma leptin, but had no effect on adiponectin levels. These findings suggest that the regulation of leptin secretion by APE may inhibit FAS activity with subsequent suppression of triglyceride accumulation in the liver and adipose tissues. Inhibition of lipid accumulation can, in turn, lead to improvements in impaired glucose tolerance.

Examination of the effects of arsenic on glucose homeostasis in cell culture and animal studies: Development of a mouse model for arsenic-induced diabetes

Previous epidemiologic studies found increased prevalences of type 2 diabetes mellitus in populations exposed to high levels of inorganic arsenic (iAs) in drinking water. Although results of epidemiologic studies in low-exposure areas or occupational settings have been inconclusive, laboratory research has shown that exposures to iAs can produce effects that are consistent with type 2 diabetes. The current paper reviews the results of laboratory studies that examined the effects of iAs on glucose metabolism and describes new experiments in which the diabetogenic effects of iAs exposure were reproduced in a mouse model. Here, weanling male C57BL/6 mice drank deionized water with or without the addition of arsenite (25 or 50 ppm As) for 8 weeks. Intraperitoneal glucose tolerance tests revealed impaired glucose tolerance in mice exposed to 50 ppm As, but not to 25 ppm As. Exposure to 25 and 50 ppm As in drinking-water resulted in proportional increases in the concentration of iAs and its metabolites in the liver and in organs targeted by type 2 diabetes, including pancreas, skeletal muscle and adipose tissue. Dimethylarsenic was the predominant form of As in the tissues of mice in both 25 and 50 ppm groups. Notably, the average concentration of total speciated arsenic in livers from mice in the 50 ppm group was comparable to the highest concentration of total arsenic reported in the livers of Bangladeshi residents who had consumed water with an order of magnitude lower level of iAs. These data suggest that mice are less susceptible than humans to the diabetogenic effects of chronic exposure to iAs due to a more efficient clearance of iAs or its metabolites from target tissues.