bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological and Molecular Characteristics of Rat Hypothalamic Ventromedial Nucleus Glucosensing Neurons
Authors: Kang Ling , Routh Vanessa H. , Kuzhikandathil Eldo V. , Gaspers Larry D. , Levin Barry E.
Published: March 2007

To evaluate potential mechanisms for neuronal glucosensing, fura-2 Ca2+ imaging and single-cell RT-PCR were carried out in dissociated ventromedial hypothalamic nucleus (VMN) neurons. Glucose-excited (GE) neurons increased and glucose-inhibited (GI) neurons decreased intracellular Ca2+ ([Ca2+]i) oscillations as glucose increased from 0.5 to 2.5 mmol/l. The Kir6.2 subunit mRNA of the ATP-sensitive K+ channel was expressed in 42% of GE and GI neurons, but only 15% of nonglucosensing (NG) neurons. Glucokinase (GK), the putative glucosensing gatekeeper, was expressed in 64% of GE, 43% of GI, but only 8% of NG neurons and the GK inhibitor alloxan altered [Ca2+]i oscillations in ∼75% of GK-expressing GE and GI neurons. Insulin receptor and GLUT4 mRNAs were coexpressed in 75% of GE, 60% of GI, and 40% of NG neurons, although there were no statistically significant intergroup differences. Hexokinase-I, GLUT3, and lactate dehydrogenase-A and -B were ubiquitous, whereas GLUT2, monocarboxylate transporters-1 and -2, and leptin receptor and GAD mRNAs were expressed less frequently and without apparent relationship to glucosensing capacity. Thus, although GK may mediate glucosensing in up to 60% of VMN neurons, other regulatory mechanisms are likely to control glucosensing in the remaining ones.
Virally Induced Inflammation Triggers Fratricide of Fas-Ligand–Expressing β-Cells
Authors: Christen Urs , Darwiche Rima , Thomas Helen E. , Wolfe Tom , Rodrigo Evelyn , Chervonsky Alexander , Flavell Richard A. , von Herrath Matthias G.
Published: March 2007

Tissue-specific expression of Fas-ligand (Fas-L) can provide immune privilege by inducing apoptosis of “invading” lymphocytes expressing Fas. However, accelerated diabetes has been reported in transgenic mice expressing Fas-L in islets (RIP-Fas-L) as a result of Fas-dependent fratricide of β-cells after transfer of diabetogenic clones. Here we studied whether Fas-L could protect islets from autoaggressive CD8 lymphocytes in a transgenic model of virally induced diabetes (RIP-LCMV-NP transgenic mice), in which the autoaggressive response is directed to a viral nucleoprotein (NP) expressed as a transgene in β-cells. Indeed, disease incidence after viral (lymphocytic choriomeningitis virus [LCMV]) infection was reduced by ∼30%, which was associated with a decrease of autoaggressive CD8 NP-specific lymphocytes in islets and pancreatic draining lymph nodes. However, surprisingly, a high degree (50%) of diabetes was seen in mice that expressed only Fas-L but not the viral transgene (NP) in β-cells after infection with LCMV. This was due to induction of Fas on β-cells after LCMV infection of the pancreas, resulting in Fas/Fas-L–mediated fratricide. Thus, although Fas-L can lend some immune privilege to islet cells, local virus-induced inflammation will induce Fas on β-cells, leading to their mutual destruction if Fas-L is present. Expression of Fas-L therefore might not be protective in situations in which viral inflammation can be expected, resulting in Fas induction on the targeted cell itself.
Secretion, Degradation, and Elimination of Glucagon-Like Peptide 1 and Gastric Inhibitory Polypeptide in Patients with Chronic Renal Insufficiency and Healthy Control Subjects
Authors: Meier Juris J. , Nauck Michael A. , Kranz Daniel , Holst Jens J. , Deacon Carolyn F. , Gaeckler Dirk , Schmidt Wolfgang E. , Gallwitz Baptist
Published: March 2007

Glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP) are important factors in the pathogenesis of type 2 diabetes and have a promising therapeutic potential. Alterations of their secretion, in vivo degradation, and elimination in patients with chronic renal insufficiency (CRI) have not yet been characterized. Ten patients with CRI (aged 47 ± 15 years, BMI 24.5 ± 2.2 kg/m2, and serum creatinine 2.18 ± 0.86 mg/dl) and 10 matched healthy control subjects (aged 44 ± 12 years, BMI 24.9 ± 3.4 kg/m2, and serum creatinine 0.89 ± 0.10 mg/dl) were included. On separate occasions, an oral glucose tolerance test (75 g), an intravenous infusion of GLP-1 (0.5 pmol · kg−1 · min−1 over 30 min), and an intravenous infusion of GIP (1.0 pmol · kg−1 · min−1 over 30 min) were performed. Venous blood samples were drawn for the determination of glucose (glucose oxidase), insulin, C-peptide, GLP-1 (total and intact), and GIP (total and intact; specific immunoassays). Plasma levels of GIP (3–42) and GLP-1 (9–36 amide) were calculated. Statistics were performed using repeated-measures and one-way ANOVA. After the oral glucose load, plasma concentrations of intact GLP-1 and intact GIP reached similar levels in both groups (P = 0.31 and P = 0.87, respectively). The concentrations of GIP (3–42) and GLP-1 (9–36 amide) were significantly higher in the patients than in the control subjects (P = 0.0021 and P = 0.027, respectively). During and after the exogenous infusion, GLP-1 (9–36 amide) and GIP (3–42) reached higher plasma concentrations in the CRI patients than in the control subjects (P < 0.001 and P = 0.0033, respectively), whereas the plasma levels of intact GLP-1 and GIP were not different between the groups (P = 0.29 and P = 0.27, respectively). Plasma half-lives were 3.4 ± 0.6 and 2.3 ± 0.4 min for intact GLP-1 (P = 0.13) and 5.3 ± 0.8 and 3.3 ± 0.4 min for the GLP-1 metabolite (P = 0.029) for CRI patients vs. healthy control subjects, respectively. Plasma half-lives of intact GIP were 6.9 ± 1.4 and 5.0 ± 1.2 min (P = 0.31) and 38.1 ± 6.0 and 22.4 ± 3.0 min for the GIP metabolite (P = 0.032) for CRI patients vs. healthy control subjects, respectively. Insulin concentrations tended to be lower in the patients during all experiments, whereas C-peptide levels tended to be elevated. These data underline the importance of the kidneys for the final elimination of GIP and GLP-1. The initial dipeptidyl peptidase IV-mediated degradation of both hormones is almost unaffected by impairments in renal function. Delayed elimination of GLP-1 and GIP in renal insufficiency may influence the pharmacokinetics and pharmacodynamics of dipeptidyl peptidase IV-resistant incretin derivatives to be used for the treatment of patients with type 2 diabetes.
In Vitro Transcriptional Induction of the Human Apolipoprotein A-II Gene by Glucose
Authors: Sauvaget Dominique , Chauffeton Valérie , Dugué-Pujol Sonia , Kalopissis Athina-Despina , Guillet-Deniau Isabelle , Foufelle Fabienne , Chambaz Jean , Leturque Armelle , Cardot Philippe , Ribeiro Agnès
Published: March 2007

Type 2 diabetic patients present high triglyceride and low HDL levels, significant determinants for the risk of atherosclerosis. Transgenic mice overproducing human apolipoprotein (apo)A-II, one of the two major apos of HDLs, display the same lipid disorders. Here, we investigated the possible regulation of apoA-II gene expression by glucose. In primary rat hepatocytes and in HepG2 cells, the transcription of the human apoA-II gene was upregulated by glucose. This response was mediated by a hormone-responsive element within the enhancer of the apoA-II promoter and was dependent on hepatocyte nuclear factor-4α. Accordingly, in transgenic mice, the human apoA-II gene is stimulated by a high-carbohydrate diet after fasting and at weaning. By contrast, the apoA-II mRNA level is not modified in streptozotocin-induced diabetic rats. In transgenic mice overexpressing the human apoA-II gene, plasma human apoA-II concentration was positively correlated with blood glucose levels. These mice displayed a marked delay in plasma glucose tolerance as compared with control mice. We hypothesize that the following pathogenic pathway might occur in the course of type 2 diabetes: increased apoA-II level causes a rise in plasma triglyceride level and glucose intolerance, resulting in hyperglycemia, which in turn might further increase apoA-II gene transcription.
Role of Poly(ADP-Ribose) Polymerase Activation in Diabetic Neuropathy
Authors: Obrosova Irina G. , Li Fei , Abatan Omorodola I. , Forsell Mark A. , Komjáti Katalin , Pacher Pal , Szabó Csaba , Stevens Martin J.
Published: March 2007

Oxidative and nitrosative stress play a key role in the pathogenesis of diabetic neuropathy, but the mechanisms remain unidentified. Here we provide evidence that poly(ADP-ribose) polymerase (PARP) activation, a downstream effector of oxidant-induced DNA damage, is an obligatory step in functional and metabolic changes in the diabetic nerve. PARP-deficient (PARP−/−) mice were protected from both diabetic and galactose-induced motor and sensory nerve conduction slowing and nerve energy failure that were clearly manifest in the wild-type (PARP+/+) diabetic or galactose-fed mice. Two structurally unrelated PARP inhibitors, 3-aminobenzamide and 1,5-isoquinolinediol, reversed established nerve blood flow and conduction deficits and energy failure in streptozotocin-induced diabetic rats. Sciatic nerve immunohistochemistry revealed enhanced poly(ADP-ribosyl)ation in all experimental groups manifesting neuropathic changes. Poly(ADP-ribose) accumulation was localized in both endothelial and Schwann cells. Thus, the current work identifies PARP activation as an important mechanism in diabetic neuropathy and provides the first evidence for the potential therapeutic value of PARP inhibitors in this devastating complication of diabetes.
Hyperglycemia-Induced Defects in Angiogenesis in the Chicken Chorioallantoic Membrane Model
Authors: Larger Etienne , Marre Michel , Corvol Pierre , Gasc Jean-Marie
Published: March 2007

Diabetes is associated with abnormal angiogenesis. Increased angiogenesis contributes to severe forms of diabetic retinopathy, but angiogenesis is decreased in response to myocardial ischemia in diabetic patients. We evaluated the direct effect of hyperglycemia on angiogenesis in the chicken chorioallantoic membrane assay, a model of active neoangiogenesis. Hyperglycemia, lasting up to 7 days, was induced in 7-day-old chick embryos by a single intravitellus glucose injection. Control embryos received either water (volumic control) or mannitol (osmotic control). Hyperglycemia decreased angiogenesis in this model from the 5th day on. The pattern and expression level of the main vascular growth factors’ genes were not altered by hyperglycemia, as assessed by in situ hybridization and semiquantitative RT-PCR. As early as 2 days after hyperglycemia was induced, an increased apoptosis of endothelial cells and pericytes was detected by transferase-mediated deoxyuridine triphosphate nick-end labeling assay and electron microscopy. In the meantime, endothelial cell proliferation was decreased, as assessed by incorporation of bromo-deoxyuridine. Hyperglycemia can therefore impair angiogenesis without altering the expression level of vascular growth factors through induction of apoptosis and decreased proliferation of endothelial cells.
Molecular Mechanisms of High Glucose-Induced Cyclooxygenase-2 Expression in Monocytes
Authors: Shanmugam Narkunaraja , Gaw Gonzalo Irene T. , Natarajan Rama
Published: March 2007

The cyclooxygenase (COX)-2 enzyme has been implicated in the pathogenesis of several inflammatory diseases. However, its role in diabetic vascular disease is unclear. In this study, we evaluated the hypothesis that diabetic conditions can induce COX-2 in monocytes. High glucose treatment of THP-1 monocytic cells led to a significant three- to fivefold induction of COX-2 mRNA and protein expression but not COX-1 mRNA. High glucose-induced COX-2 mRNA was blocked by inhibitors of nuclear factor-κB (NF-κΒ), protein kinase C, and p38 mitogen-activated protein kinase. In addition, an antioxidant and inhibitors of mitochondrial superoxide, NADPH oxidase, and glucose metabolism to glucosamine also blocked high glucose-induced COX-2 expression to varying degrees. High glucose significantly increased transcription from a human COX-2 promoter-luciferase construct (twofold, P < 0.001). Promoter deletion analyses and inhibition of transcription by NF-κΒ superrepressor and cAMP-responsive element binding (CREB) mutants confirmed the involvement of NF-κΒ and CREB transcription factors in high glucose-induced COX-2 regulation. In addition, isolated peripheral blood monocytes from type 1 and type 2 diabetic patients had high levels of COX-2 mRNA, whereas those from normal volunteers showed no expression. These results show that high glucose and diabetes can augment inflammatory responses by upregulating COX-2 via multiple signaling pathways, leading to monocyte activation relevant to the pathogenesis of diabetes complications.
Association of the VEGF Gene With Proliferative Diabetic Retinopathy But Not Proteinuria in Diabetes
Authors: Ray David , Mishra Manoj , Ralph Shirley , Read Ian , Davies Robert , Brenchley Paul
Published: March 2007

Diabetic retinopathy and nephropathy cause significant morbidity in patients with diabetes. Vascular endothelial growth factor (VEGF) is a potent angiogenic and vascular permeability factor and is implicated in both of these diabetes complications. We previously reported transfection studies showing the VEGF −460 and VEGF +405 polymorphisms to increase basal VEGF promoter activity by 71% compared with the wild-type sequence. Therefore, we investigated the association of these VEGF polymorphisms with proliferative diabetic retinopathy and diabetic nephropathy. DNA was isolated from 267 U.K. Caucasians with diabetes, comprising 69 patients with proliferative retinopathy and 198 patients with other grades of retinopathy. The distribution of VEGF −460 genotype was significantly different between the proliferative retinopathy and nonproliferative retinopathy groups (P = 0.027); specifically, carriage of the VEGF −460C allele was associated with proliferative diabetic retinopathy (odds ratio 2.5 [95% CI 1.20–5.23]). The VEGF −460 genotype was predictive of retinopathy, even after controlling for blood pressure, glycemic control, duration of diabetes, and obesity (P = 0.02). The VEGF +405 genotype did not associate with proliferative retinopathy, and neither polymorphism was associated with diabetic nephropathy. The VEGF −460C polymorphism is a positive independent predictive factor for the development of proliferative diabetic retinopathy. Increased VEGF production from high-expressing haplotypes, including −460C, may promote neovascularization.
Hexosamine Pathway Is Responsible for Inhibition by Diabetes of Phenylephrine-Induced Inotropy
Authors: Pang Yi , Bounelis Pam , Chatham John C. , Marchase Richard B.
Published: March 2007

Hyperglycemia diminishes positive inotropic responses to agonists that activate phospholipase C (PLC) and generate inositol trisphosphate (1,4,5). The mechanisms underlying both the inotropic responses and hyperglycemia’s effects on them remain undetermined, but data from isolated cardiomyocytes suggest the involvement of capacitative Ca2+ entry (CCE), the influx of Ca2+ through plasma membrane channels activated in response to depletion of endoplasmic or sarcoplasmic reticulum Ca2+ stores. In neonatal rat cardiomyocytes, hyperglycemia decreased CCE induced by PLC-mediated agonists. The attenuation of CCE was also seen with glucosamine, and the inhibition by hyperglycemia was prevented by azaserine, thereby implicating hexosamine biosynthesis as the responsible metabolic pathway. In the current study, the importance of hexosamine metabolites to hyperglycemia’s effects on inotropic responses was examined in isolated perfused rat hearts. The inhibition by hyperglycemia of phenylephrine-induced inotropy was reversed with azaserine and mimicked by glucosamine. An independent inhibitor of CCE, SKF96365, was also effective in blunting inotropy. These treatments did not inhibit inotropy induced by activation of adenylate cyclase through β-adrenergic receptors. These data thus implicate CCE in responses to PLC-mediated agonists in the intact heart and point to the hexosamine pathway’s negative effect on CCE as being central to the inhibition seen with hyperglycemia.
Improved β-Cell Survival and Reduced Insulitis in a Type 1 Diabetic Rat Model After Treatment With a β-Cell–Selective KATP Channel Opener
Authors: Skak Kresten , Gotfredsen Carsten F. , Lundsgaard Dorthe , Hansen John Bondo , Sturis Jeppe , Markholst Helle
Published: March 2007

Treatment with ATP-sensitive K+ channel openers (KCOs) leads to inhibition of insulin secretion and metabolic “rest” in β-cells. It is hypothesized that in type 1 diabetes this may reduce β-cell death resulting from metabolic stress as well as reduce the immunogenicity of the β-cells during autoimmune β-cell destruction. We have investigated whether the β-cell−selective KCO compound, NN414, can be used to improve β-cell survival in DR-BB rats rendered diabetic by modulation of their immune system. The rats were treated three times daily on days 1–19 with NN414, diazoxide, or vehicle. On day 21, an intravenous glucose tolerance test was conducted to assess β-cell function. Postmortem histological analysis of rats’ pancreata assessed the degree of insulitis and β-cell volume. Among NN414-treated rats, 46% (16 of 35) were found to have a β-cell mass similar to that of nondiabetic controls and significant glucose-stimulated C-peptide values, whereas only 11% (4 of 36) of vehicle-treated rats possessed a normal β-cell mass and function (P < 0.002, by χ2 test). Furthermore, responsive NN414-treated rats were almost free of insulitis. Thus, this study demonstrated that treatment with KCO compounds can indeed lead to preservation of β-cell function and reduction of insulitis in a rat diabetes model.
Prophylactic Gene Therapy With Human Tissue Kallikrein Ameliorates Limb Ischemia Recovery in Type 1 Diabetic Mice
Authors: Emanueli Costanza , Graiani Galliam , Salis Maria B. , Gadau Sergio , Desortes Elisa , Madeddu Paolo
Published: March 2007

Diabetes macro- and microvascular disease causes tissue hypoperfusion. This deficit, together with a failure to mount an adequate angiogenic response, might explain why vascular occlusion evolves more severely among diabetic patients. The present study investigated whether prophylactic gene therapy with human tissue kallikrein (hTK) may protect diabetic limbs from the consequences of supervening ischemia. Vehicle (saline) or an adenovirus carrying the gene for either hTK (Ad.hTK) or luciferase (Ad.Luc) was injected into left adductor muscles of streptozotocin-induced type 1 diabetic mice 2 weeks before operative occlusion of the ipsilateral femoral artery. Saline-injected nondiabetic mice served as controls. Hindlimb blood flow recovery was analyzed sequentially over the 2 weeks after ischemia induction. At necroscopy, microvessel density and endothelial cell proliferation and apoptosis were quantified in skeletal muscles. We found that limb perfusion recovery of saline-injected type 1 diabetic mice is delayed because of insufficient reparative neovascularization and excessive activation of endothelial cell apoptosis. By contrast, prophylactic Ad.hTK renewed the ability to mount an appropriate neovascularization response to ischemia, suppressed apoptosis, and upregulated endothelial nitric oxide synthase expression. Ultimately, correction of diabetic endotheliopathy by Ad.hTK allowed proper perfusion recovery as seen in nondiabetic mice. These discoveries disclose new therapeutic options for the treatment of diabetic complications.
Modulation of Resistin Expression by Retinoic Acid and Vitamin A Status
Authors: Felipe Francisco , Bonet M. Luisa , Ribot Joan , Palou Andreu
Published: March 2007

This work identifies retinoic acid (RA), the acid form of vitamin A, as a signal that inhibits the expression of resistin, an adipocyte-secreted protein previously proposed to act as an inhibitor of adipocyte differentiation and as a systemic insulin resistance factor. Both 9-cis and all-trans RA reduced resistin mRNA levels in white and brown adipocyte cell model systems; the effect was time- and dose-dependent, was followed by a reduced secretion of resistin, and was reproduced by selective agonists of both RA receptors and rexinoid receptors. Association of CCAAT/enhancer-binding protein α (a positive regulator of the resistin gene) and its coactivators p300, cAMP response element-binding protein binding protein, and retinoblastoma protein with the resistin gene promoter was reduced in RA-treated adipocytes. RA administration to normal mice resulted in reduced resistin mRNA levels in brown and white adipose tissues, reduced circulating resistin levels, reduced body weight, and improved glucose tolerance. Resistin expression was also downregulated after dietary vitamin A supplementation in mice. The results raise the possibility that vitamin A status may contribute to modulate systemic functions through effects on the production of adipocyte-derived protein signals.
Restoration of Adiponectin Pulsatility in Severely Obese Subjects After Weight Loss
Authors: Calvani Menotti , Scarfone Antonio , Granato Luigi , Mora Elena Valera , Nanni Giuseppe , Castagneto Marco , Greco Aldo V. , Manco Melania , Mingrone Geltrude
Published: March 2007

Diurnal variations of adiponectin levels have been studied in normal-weight men and in diabetic and nondiabetic obese subjects, but no data have been reported in obese subjects after weight loss. We collected blood samples at 1-h intervals over 24 h from seven severely obese subjects before and after massive weight loss consequent to surgical operation (bilio-pancreatic diversion [BPD]) to measure adiponectin, insulin, glucose, and cortisol levels. Insulin sensitivity was assessed by euglycemic-hyperinsulinemic clamp (M value). Studies of diurnal variations and pulsatility of adiponectin, insulin, and cortisol were performed. The pulsatility index (PI) of adiponectin increased after BPD from 0.04 to 0.11 μg/min (P = 0.01). Insulin PI significantly increased after the operation (1.50 vs. 1.08 pmol · l–1 · min–1, P = 0.01), while cortisol PI did not significantly change. The adiponectin clearance rate changed from 0.001 ± 10−4 · min−1 before BPD to 0.004 ± 8 · 10−4 · min−1 after BPD (P = 0.03). Insulin clearance increased from 0.006 ± 6 · 10−4 · min−1 before BPD to 0.009 ± 4 · 10−4 · min−1 after BPD (P = 0.02). The M value doubled after surgery (27.08 ± 8.5 vs. 53.34 ± 9.3 μmol · kgFFM−1 · min−1; P < 0.001) becoming similar to the values currently reported for normal-weight subjects. In conclusion, in formerly severely obese subjects, weight loss paired with the reversibility of insulin resistance restores homeostatic control of the adiponectin secretion, contributing to the reduction of cardiovascular risk already described in these patients.
Metabotropic Glutamate Receptor Type 4 Is Involved in Autoinhibitory Cascade for Glucagon Secretion by α-Cells of Islet of Langerhans
Authors: Uehara Shunsuke , Muroyama Akiko , Echigo Noriko , Morimoto Riyo , Otsuka Masato , Yatsushiro Shouki , Moriyama Yoshinori
Published: March 2007

In islets of Langerhans, l-glutamate is stored in glucagon-containing secretory granules of α-cells and cosecreted with glucagon under low-glucose conditions. The l-glutamate triggers secretion of γ-aminobutyric acid (GABA) from β-cells, which in turn inhibits glucagon secretion from α-cells through the GABAA receptor. In the present study, we tested the working hypothesis that l-glutamate functions as an autocrine/paracrine modulator and inhibits glucagon secretion through a glutamate receptor(s) on α-cells. The addition of l-glutamate at 1 mmol/l; (R,S)-phosphonophenylglycine (PPG) and (S)-3,4-dicarboxyphenylglycine (DCPG), specific agonists for class III metabotropic glutamate receptor (mGluR), at 100 μmol/l; and (1S,3R,4S)-1-aminocyclopentane-1,3,4-tricarboxylic acid (ACPT-I) at 50 μmol/l inhibited the low-glucose–evoked glucagon secretion by 87, 81, 73, and 87%, respectively. This inhibition was dose dependent and was blocked by (R,S)-cyclopropyl-4-phosphonophenylglycine (CPPG), a specific antagonist of class III mGluR. Agonists of other glutamate receptors, including kainate and quisqualate, had little effectiveness. RT-PCR and immunological analyses indicated that mGluR4, a class III mGluR, was expressed and localized with α- and F cells, whereas no evidence for expression of other mGluRs, including mGluR8, was obtained. l-Glutamate, PPG, and ACPT-I decreased the cAMP content in isolated islets, which was blocked by CPPG. Dibutylyl-cAMP, a nonhydrolyzable cAMP analog, caused the recovery of secretion of glucagon. Pertussis toxin, which uncouples adenylate cyclase and inhibitory G-protein, caused the recovery of both the cAMP content and secretion of glucagon. These results indicate that α- and F cells express functional mGluR4, and its stimulation inhibits secretion of glucagon through an inhibitory cAMP cascade. Thus, l-glutamate may directly interact with α-cells and inhibit glucagon secretion.
Bifunctional Properties of Peroxisome Proliferator-Activated Receptor γ1 in KDR Gene Regulation Mediated via Interaction With Both Sp1 and Sp3
Authors: Sassa Yukio , Hata Yasuaki , Aiello Lloyd Paul , Taniguchi Yukio , Kohno Kimitoshi , Ishibashi Tatsuro
Published: March 2007

Vascular endothelial growth factor receptor 2 (KDR) plays a critical role in mediating a variety of vasculogenic and angiogenic processes, including diabetic retinopathy. We previously demonstrated that the promoter activity of the KDR gene in retinal capillary endothelial cells (RCECs) was regulated in part by the relative concentration of positive/negative transcription factors Sp1/Sp3. We also reported that the peroxisome proliferator-activated receptor (PPAR)γ ligand could inhibit intraocular angiogenesis. In the present study, the role of PPARγ1 in KDR gene regulation in RCECs was examined. PPARγ1 protein physically interacted with both Sp1 and Sp3. Transactivation and electrophoretic mobility shift assays clearly demonstrated novel findings that PPARγ1 increased KDR promoter activity by enhancing the interaction between Sp1, but not Sp3, and KDR promoter region without its ligand in RCECs. The ligand-binding site but not the DNA binding site of PPARγ1 enhanced the interaction between Sp1 and KDR promoter region. Conversely, PPARγ1 ligand 15-deoxy Δ (12,14)-prostaglandin J2 dose-dependently suppressed the binding of KDR promoter region with both Sp1 and Sp3, resulting an inhibition of KDR gene expression. In conclusion, PPARγ1 has bifunctional properties in the regulation of KDR gene expression mediated via interaction with both Sp1 and Sp3.
Effects of Diabetes and Insulin on the Expression of Galanin-Like Peptide in the Hypothalamus of the Rat
Authors: Fraley Gregory S. , Scarlett Jarrad M. , Shimada Issei , Teklemichael Dawit N. , Acohido Blake V. , Clifton Donald K. , Steiner Robert A.
Published: March 2007

Galanin-like peptide (GALP) is produced in a small population of neurons in the arcuate nucleus of the hypothalamus, and leptin stimulates the hypothalamic expression of GALP mRNA. Because insulin and leptin share common signaling pathways in the brain, we reasoned that GALP neurons might also be responsive to changes in circulating concentrations of insulin. To test this hypothesis, we first studied the effect of insulin deficiency on the expression of GALP by comparing levels of GALP mRNA between normal and diabetic animals. Streptozotocin-induced diabetes was associated with a significant reduction in the expression of GALP mRNA, which was reversed by treatment with either insulin or leptin. Second, we examined the effect of insulin administered directly into the brain on the expression of GALP mRNA in fasted rats. Hypothalamic levels of GALP mRNA were lower in animals after a 48-h fast, and central treatment with insulin reversed this effect. These results suggest that GALP neurons are direct targets for regulation by insulin and implicate these cells for a role in the metabolic and behavioral sequelae of type 1 diabetes.
From Blood Monocytes to Adipose Tissue-Resident Macrophages
Authors: Curat Cyrile A. , Miranville Alexandra , Sengenès Coralie , Diehl Michael , Tonus Carolin , Busse Rudi , Bouloumié Anne
Published: March 2007

Obesity has been suggested to be a low-grade systemic inflammatory state, therefore we studied the interaction between human adipocytes and monocytes via adipose tissue (AT)-derived capillary endothelium. Cells composing the stroma-vascular fraction (SVF) of human ATs were characterized by fluorescence-activated cell sorter (FACS) analysis and two cell subsets (resident macrophages and endothelial cells [ECs]) were isolated using antibody-coupled microbeads. Media conditioned by mature adipocytes maintained in fibrin gels were applied to AT-derived ECs. Thereafter, the expression of endothelial adhesion molecules was analyzed as well as the adhesion and transmigration of human monocytes. FACS analysis showed that 11% of the SVF is composed of CD14+/CD31+ cells, characterized as resident macrophages. A positive correlation was found between the BMI and the percentage of resident macrophages, suggesting that fat tissue growth is associated with a recruitment of blood monocytes. Incubation of AT-derived ECs with adipocyte-conditioned medium resulted in the upregulation of EC adhesion molecules and the increased chemotaxis of blood monocytes, an effect mimicked by recombinant human leptin. These results indicate that adipokines, such as leptin, activate ECs, leading to an enhanced diapedesis of blood monocytes, and suggesting that fat mass growth might be linked to inflammatory processes.
Catalase Protects Cardiomyocyte Function in Models of Type 1 and Type 2 Diabetes
Authors: Ye Gang , Metreveli Naira S. , Donthi Rajakumar V. , Xia Shen , Xu Ming , Carlson Edward C. , Epstein Paul N.
Published: March 2007

Many diabetic patients suffer from a cardiomyopathy that cannot be explained by poor coronary perfusion. Reactive oxygen species (ROS) have been proposed to contribute to this cardiomyopathy. Consistent with this we found evidence for induction of the antioxidant genes for catalase in diabetic OVE26 hearts. To determine whether increased antioxidant protection could reduce diabetic cardiomyopathy, we assessed cardiac morphology and contractility, Ca2+ handling, malondialdehyde (MDA)-modified proteins, and ROS levels in individual cardiomyocytes isolated from control hearts, OVE26 diabetic hearts, and diabetic hearts overexpressing the antioxidant protein catalase. Diabetic hearts showed damaged mitochondria and myofibrils, reduced myocyte contractility, slowed intracellular Ca2+ decay, and increased MDA-modified proteins compared with control myocytes. Overexpressing catalase preserved normal cardiac morphology, prevented the contractile defects, and reduced MDA protein modification but did not reverse the slowed Ca2+ decay induced by diabetes. Additionally, high glucose promoted significantly increased generation of ROS in diabetic cardiomyocytes. Chronic overexpression of catalase or acute in vitro treatment with rotenone, an inhibitor of mitochondrial complex I, or thenoyltrifluoroacetone, an inhibitor of mitochondrial complex II, eliminated excess ROS production in diabetic cardiomyocytes. The structural damage to diabetic mitochondria and the efficacy of mitochondrial inhibitors in reducing ROS suggest that mitochondria are a source of oxidative damage in diabetic cardiomyocytes. We also found that catalase overexpression protected cardiomyocyte contractility in the agouti model of type 2 diabetes. These data show that both type 1 and type 2 diabetes induce damage at the level of individual myocytes, and that this damage occurs through mechanisms utilizing ROS.
Complex Haplotypes of the PGC-1α Gene Are Associated With Carbohydrate Metabolism and Type 2 Diabetes
Authors: Oberkofler Hannes , Linnemayr Veronika , Weitgasser Raimund , Klein Kerstin , Xie Mingqiang , Iglseder Bernhard , Krempler Franz , Paulweber Bernhard , Patsch Wolfgang
Published: March 2007

Peroxisome proliferator–activated receptor coactivator-1α (PGC-1α) is a transcriptional coactivator implicated in transcriptional programs of hepatic gluconeogenesis, oxidative phosphorylation, and insulin release by β-cells. To study associations of the PGC-1α gene locus with carbohydrate metabolism and type 2 diabetes in humans, we identified several polymorphisms in the promoter region that were located in a haplotype block distinct from a second haplotype block containing part of intron 2 and extending beyond exon 13. Each block contained five common haplotypes. Oral glucose tolerance testing revealed associations of promoter haplotype combinations with 30- and 60-min postload plasma glucose levels, whereas haplotypes in both blocks were associated with indexes of β-cell function. The associations of promoter haplotypes are supported by functional studies showing that some polymorphisms are located in transcription factor binding sites and affect transactivation in an allele-specific manner. By comparing patients with type 2 diabetes and control subjects, we observed borderline significant differences of four-loci haplotype distributions in the downstream haplotype block. Moreover, the haplotype that was associated with the strongest insulin response to glucose conferred the lowest risk of type 2 diabetes (P < 0.01). Thus, the PGC-1α gene locus influences carbohydrate metabolism and contributes to type 2 diabetes in the population studied.
Regulation of α-Cell Function by the β-Cell in Isolated Human and Rat Islets Deprived of Glucose: the “Switch-off” Hypothesis
Authors: Hope Kristine M. , Tran Phuong Oanh T. , Zhou Huarong , Oseid Elizabeth , Leroy Eric , Robertson R. Paul
Published: March 2007

The “switch-off” hypothesis to explain β-cell regulation of α-cell function during hypoglycemia has not been assessed previously in isolated islets, largely because they characteristically do not respond to glucose deprivation by secreting glucagon. We examined this hypothesis using normal human and Wistar rat islets, as well as islets from streptozotocin (STZ)-administered β-cell-deficient Wistar rats. As expected, islets perifused with glucose and 3-isobutryl-1-methylxanthine did not respond to glucose deprivation by increasing glucagon secretion. However, if normal rat islets were first perifused with 16.7 mmol/l glucose to increase endogenous insulin secretion, followed by discontinuation of the glucose perifusate, a glucagon response to glucose deprivation was observed (peak change within 10 min after switch off = 61 ± 15 pg/ml [mean ± SE], n = 6, P < 0.01). A glucagon response from normal human islets using the same experimental design was also observed. A glucagon response (peak change within 7 min after switch off = 31 ± 1 pg/ml, n = 3, P < 0.01) was observed from β-cell-depleted, STZ-induced diabetic rats whose islets still secreted small amounts of insulin. However, when these islets were first perifused with both exogenous insulin and 16.7 mmol/l glucose, followed by switching off both the insulin and glucose perifusate, a significantly larger (P < 0.05) glucagon response was observed (peak change within 7 min after switch off = 71 ± 11 pg/ml, n = 4, P < 0.01). This response was not observed if the insulin perifusion was not switched off when the islets were deprived of glucose or when insulin was switched off without glucose deprivation. These data uniquely demonstrate that both normal, isolated islets and islets from STZ-administered rats can respond to glucose deprivation by releasing glucagon if they are first provided with increased endogenous or exogenous insulin. These results fully support the β-cell switch-off hypothesis as a key mechanism for the α-cell response to hypoglycemia.
Muscle Strength in Type 2 Diabetes
Authors: Andersen Henning , Nielsen Søren , Mogensen Carl E. , Jakobsen Johannes
Published: March 2007

Motor function in type 2 diabetes is largely unknown. In 36 type 2 diabetic patients and in 36 control subjects matched for sex, age, weight, height, and physical activity, strength of flexors and extensors at elbow, wrist, knee, and ankle was assessed at isokinetic dynamometry. The degree of neuropathy was determined by clinical scores, nerve conduction studies, and quantitative sensory testing. Eventually, all results were summed to obtain a neuropathy rank-sum score (NRSS). The degree of nephropathy and retinal condition were also evaluated. Diabetic patients had a 17 and 14% reduction of strength of ankle flexors (P < 0.02) and ankle extensors (P < 0.03), respectively. At the knee, strength of extensors and flexors was reduced by 7% (NS) and 14% (P < 0.05), respectively. At the elbow and wrist, muscle strength was preserved. The NRSS was related to the strength at the ankle (r = −0.45, P < 0.01) and knee (r = −0.42, P < 0.02). Following multiple regression analysis, the NRSS but not the degree of nephropathy or retinopathy was related to strength at the ankle and knee. In conclusion, type 2 diabetic patients may have muscle weakness at the ankle and knee related to presence and severity of peripheral neuropathy.
Differences in Insulin Resistance in Nondiabetic Subjects With Isolated Impaired Glucose Tolerance or Isolated Impaired Fasting Glucose
Authors: Festa Andreas , D’Agostino Ralph , Hanley Anthony J.G. , Karter Andrew J. , Saad Mohammed F. , Haffner Steven M.
Published: March 2007

Both impaired glucose tolerance (IGT) (as defined by the 1985 World Health Organization criteria) and impaired fasting glucose (IFG) (as defined by the 1997 American Diabetes Association criteria) represent intermediate metabolic states between normal and diabetic glucose homeostasis. Cardiovascular disease may be related to postglucose load rather than fasting glycemia, i.e., IGT rather than IFG. We hypothesized that subjects with IGT may be more insulin resistant and have higher levels of common cardiovascular risk factors than those with isolated IFG. In the Insulin Resistance Atherosclerosis Study (IRAS), we studied Si and first-phase insulin secretion (acute insulin response [AIR]), as derived from a frequently sampled intravenous glucose tolerance test, as well as common cardiovascular risk factors in four different glucose tolerance categories (NFG/NGT [n = 654], NFG/IGT [n = 255], IFG/NGT [n = 59], and IFG/IGT [n = 102]) among nondiabetic subjects. Subjects with isolated postchallenge hyperglycemia (NFG/IGT) had lower Si (means ± SE: 2.10 ± 0.04 vs. 2.59 ± 0.13 × 10−4 min−1 · μU−1 · ml−1; P = 0.005), lower proinsulin levels (34.4 ± 1.8 vs. 42.0 ± 4.5 pmol/l; P = 0.03), higher AIR (273.1 ± 18.1 vs. 215.9 ± 30.0 pmol/l; P = 0.04), higher C-reactive protein (2.49 ± 0.3 vs. 1.49 ± 0.5 mg/l; P = 0.0015), and higher triglyceride levels (137.7 ± 5.5 vs. 108.4 ± 8.9 mg/dl; P = 0.0025) than subjects with isolated fasting hyperglycemia (IFG/NGT). The relation of insulin resistance to glucose tolerance category was consistently seen in women and men and across the three ethnic groups of the IRAS (non-Hispanic whites, African Americans, and Hispanics). Nondiabetic individuals with isolated postchallenge hyperglycemia (IGT) are more insulin resistant than individuals with isolated fasting hyperglycemia (IFG). The risk factor pattern (including increased insulin resistance) seen in isolated IGT identifies a subgroup of nondiabetic individuals who are likely to benefit from early intervention.
A Novel Syndrome of Autosomal-Dominant Hyperinsulinemic Hypoglycemia Linked to a Mutation in the Human Insulin Receptor Gene
Authors: Højlund Kurt , Hansen Torben , Lajer Maria , Henriksen Jan Erik , Levin Klaus , Lindholm Jörgen , Pedersen Oluf , Beck-Nielsen Henning
Published: March 2007

Recently, various subtypes of familial hyperinsulinemic hypoglycemia with an autosomal-dominant inheritance have been etiologically characterized. In the present study, we have delineated the genetics and metabolic phenotype of a novel form of hypoglycemia in a large pedigree with an apparent autosomal-dominant transmission. After initial investigations of the proband, her mother, and a sister, the study was extended to 19 family members in three generations. Glucose tolerance was assessed by a 5-h oral glucose tolerance test (OGTT) and insulin sensitivity by euglycemic-hyperinsulinemic clamp in six affected family members and six control subjects. To identify the genetic cause of hypoglycemia, linkage analysis and mutation analysis of genomic DNA from all family members were performed. All affected family members were characterized by postprandial hypoglycemia, fasting hyperinsulinemia, and an elevated serum insulin-to-C-peptide ratio. The 5-h OGTT demonstrated hyperinsulinemic hypoglycemia, and the clamp studies showed reduced insulin sensitivity and clearance of serum insulin in affected family members compared with control subjects. Linkage analysis and subsequent mutation screening revealed a missense mutation (Arg1174Gln) in the tyrosine kinase domain of the insulin receptor gene that cosegregated with the disease phenotype (logarithm of odds [LOD] score 3.21). In conclusion, we report a novel syndrome of autosomal-dominant hyperinsulinemic hypoglycemia. The findings demonstrate the coexistence of severe postprandial hypoglycemia, insulin resistance, and impaired insulin clearance and suggest that hypoglycemia should be considered as a phenotype linked to heterozygote mutations in the insulin receptor gene.
Interleukin-6 Is a Novel Factor Mediating Glucose Homeostasis During Skeletal Muscle Contraction
Authors: Febbraio Mark A. , Hiscock Natalie , Sacchetti Massimo , Fischer Christian P. , Pedersen Bente K.
Published: March 2007

The mechanisms that mediate the tightly controlled production and clearance of glucose during muscular work are unclear, and it has been suggested that an unidentified “work factor” exists that influences the contraction-induced increase in endogenous glucose production (EGP). The cytokine interleukin (IL)-6 is released from skeletal muscle during contraction. Here we show that IL-6 contributes to the contraction-induced increase in EGP. Six men performed 2 h of bicycle exercise on three separate occasions, at a relatively high intensity (HI) or at a low intensity with (LO + IL-6) or without (LO) an infusion of recombinant human IL-6 that matched the circulating concentration of IL-6 seen in HI exercise. The stable isotope 6,6 2H2 glucose was infused to calculate EGP (rate of glucose appearance [Ra]), whole-body glucose disposal (rate of glucose disappearance [Rd]), and metabolic clearance rate (MCR) of glucose. Glucose Ra, Rd, and MCR were higher (P < 0.05) at HI than at LO. Throughout exercise at LO + IL-6, glucose Ra and Rd were higher (P < 0.05) than LO, even though the exercise intensity was identical. In addition, MCR was higher (P < 0.05) at LO + IL-6 than at LO at 90 min. Insulin, glucagon, epinephrine, norepinephrine, cortisol, and growth hormone were identical when comparing LO + IL-6 with LO. These data suggest that IL-6 influences glucose homeostasis during exercise. Our results provide potential new insights into factors that mediate glucose production and disposal and implicates IL-6 in the so-called “work factor.”
A Novel Variant of Ionotropic Glutamate Receptor Regulates Somatostatin Secretion From δ-Cells of Islets of Langerhans
Authors: Muroyama Akiko , Uehara Shunsuke , Yatsushiro Shouki , Echigo Noriko , Morimoto Riyo , Morita Mitsuhiro , Hayashi Mitsuko , Yamamoto Akitsugu , Koh Duk-Su , Moriyama Yoshinori
Published: March 2007

Many metabolic factors affect the secretion of insulin from β-cells and glucagon from α-cells of the islets of Langerhans to regulate blood glucose. Somatostatin from δ-cells, considered a local inhibitor of islet function, reduces insulin and glucagon secretion by activating somatostatin receptors in islet cells. Somatostatin secretion from δ-cells is increased by high glucose via glucose metabolism in a similar way to insulin secretion from β-cells. However, it is unknown how low glucose triggers somatostatin secretion. Because l-glutamate is cosecreted with glucagon from α-cells under low-glucose conditions and acts as a primary intercellular messenger, we hypothesized that glutamate signaling triggers the secretion of somatostatin. In this study, we showed that δ-cells express GluR4c-flip, a newly identified splicing variant of GluR4, an (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptor of rat. After treatment with l-glutamate, AMPA, or kainate, secretion of somatostatin from isolated islets was significantly stimulated under low-glucose conditions. The glutamate-dependent somatostatin secretion was Ca2+ dependent and blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. Somatostatin in turn inhibited the secretion of l-glutamate and glucagon from α-cells. These results indicate that l-glutamate triggers somatostatin secretion from δ-cells by way of the GluR4c-flip receptor under low-glucose conditions. The released somatostatin may complete the feedback inhibition of α-cells. Thus, α- and δ-cells may communicate with each other through l-glutamate and somatostatin signaling.
Metabolic and Inflammation Variable Clusters and Prediction of Type 2 Diabetes
Authors: Hanley Anthony J.G. , Festa Andreas , D’Agostino Ralph B. , Wagenknecht Lynne E. , Savage Peter J. , Tracy Russell P. , Saad Mohammed F. , Haffner Steven M.
Published: March 2007

Factor analysis, a multivariate correlation technique, has been used to provide insight into the underlying structure of the metabolic syndrome. The majority of previous factor analyses, however, have used only surrogate measures of insulin sensitivity; very few have included nontraditional cardiovascular disease (CVD) risk factors such as plasminogen activator inhibitor (PAI)-1, fibrinogen, and C-reactive protein (CRP); and only a limited number have assessed the ability of factors to predict type 2 diabetes. The objective of this study was to investigate, using factor analysis, the clustering of metabolic and inflammation variables using data from 1,087 nondiabetic participants in the Insulin Resistance Atherosclerosis Study (IRAS) and to determine the association of these clusters with risk of type 2 diabetes at follow-up. This study includes information on directly measured insulin sensitivity (Si) from the frequently sampled intravenous glucose tolerance test among African-American, Hispanic, and non-Hispanic white subjects aged 40–69 years. Principal factor analysis of data from nondiabetic subjects at baseline (1992–1994) identified three factors, which explained 28.4, 7.4, and 6% of the total variance in the dataset, respectively. Based on factor loadings of ≥ 0.40, these factors were interpreted as 1) a “metabolic” factor, with positive loadings of BMI, waist circumference, 2-h glucose, log triglyceride, and log PAI-1 and inverse loadings of log Si + 1 and HDL; 2) an “inflammation” factor, with positive loadings of BMI, waist circumference, fibrinogen, and log CRP and an inverse loading of log Si + 1; and 3) a “blood pressure” factor, with positive loadings of systolic and diastolic blood pressure. The results were similar within strata of ethnicity, and there were only subtle differences in sex-specific analyses. In a prospective analysis, each of the factors was a significant predictor of diabetes after a median follow-up period of 5.2 years, and each factor remained significant in a multivariate model that included all three factors, although this three-factor model was not significantly more predictive than models using either impaired glucose tolerance or conventional CVD risk factors. Factor analysis identified three underlying factors among a group of inflammation and metabolic syndrome variables, with insulin sensitivity loading on both the metabolic and inflammation variable clusters. Each factor significantly predicted diabetes in multivariate analysis. The findings support the emerging hypothesis that chronic subclinical inflammation is associated with insulin resistance and comprises a component of the metabolic syndrome.