bluuebunny/crossref_metadata_2025 huggingface dataset:
Troglitazone Not Only Increases GLUT4 but Also Induces Its Translocation in Rat Adipocytes
Authors: Shintani Mitsuyo , Nishimura Haruo , Yonemitsu Shin , Ogawa Yoshihiro , Hayashi Tatsuya , Hosoda Kiminori , Inoue Gen , Nakao Kazuwa
Published: March 2007

Thiazolidinediones, insulin-sensitizing agents, have been reported to increase glucose uptake along with the expression of glucose transporters in adipocytes and cardiomyocytes. Recently, we have further suggested that the translocation of GLUT4 is stimulated by thiazolidinediones in L6 myocytes. However, the direct effects of thiazolidinediones on translocation of glucose transporters have not yet been determined. In this study, using hemagglutinin epitope-tagged GLUT4 (GLUT4-HA), we provide direct evidence of the effect of troglitazone on the translocation of GLUT4 in rat epididymal adipocytes. Primary cultures of rat adipocytes were transiently transfected with GLUT4-HA and overexpressed eightfold compared with endogenous GLUT4 in transfected cells. A total of 24 h of treatment with troglitazone (10−4 mol/l) increased the cell surface level of GLUT4-HA by 1.5 ± 0.03–fold (P < 0.01) without changing the total amount of GLUT4-HA, whereas it increased the protein level of endogenous GLUT4 (1.4-fold) without changing that of GLUT1. Thus, the direct effect on the translocation can be detected apart from the increase in endogenous GLUT4 content using GLUT4-HA. Troglitazone not only increased the translocation of GLUT4-HA on the cell surface in the basal state but also caused a leftward shift in the dose-response relations between GLUT4-HA translocation and insulin concentration in the medium (ED50: from ∼0.1 to 0.03 nmol/l). These effects may partly contribute to the antidiabetic activity of troglitazone in patients with obesity and type 2 diabetes.
Direct Thiazolidinedione Action on Isolated Rat Skeletal Muscle Fuel Handling Is Independent of Peroxisome Proliferator–Activated Receptor-γ−Mediated Changes in Gene Expression
Authors: Brunmair Barbara , Gras Florian , Neschen Susanne , Roden Michael , Wagner Ludwig , Waldhäusl Werner , Fürnsinn Clemens
Published: March 2007

Thiazolidinediones (TZDs) are believed to induce insulin sensitization by modulating gene expression via agonistic stimulation of the nuclear peroxisome proliferator–activated receptor-γ (PPAR-γ). We have shown earlier that the TZD troglitazone inhibits mitochondrial fuel oxidation in isolated rat skeletal muscle. In the present study, rat soleus muscle strips were exposed to TZDs to examine whether the inhibition of fuel oxidation is mediated by PPAR-γ activation. Our findings consistently indicated direct, acute, and PPAR-γ−independent TZD action on skeletal muscle fuel metabolism. Rapid stimulation of lactate release by 20 μmol/l troglitazone within 30 min suggested that direct TZD action on skeletal muscle in vitro does not rely on changes in gene expression rates (12.6 ± 0.6 [control] vs. 16.0 ± 0.8 μmol · g−1 · h−1 [troglitazone]; P < 0.01). This conclusion was supported by the failure of actinomycin D and cycloheximide to block the effects of troglitazone. Mitochondrial fuel oxidation was consistently inhibited by six different TZDs (percent inhibition of CO2 production from palmitate after 25 h: troglitazone, −61 ± 2%; pioglitazone, −43 ± 7% ; rosiglitazone, −22 ± 6%; BM13.1258, −47 ± 9%; BM15.2054, −51 ± 4%; and T-174, −59 ± 4% [P < 0.005 each]), but not by PPAR-γ agonistic compounds not belonging to the TZD class (JTT-501, −5 ± 7% [NS]; prostaglandin J2, 17 ± 7% [P < 0.05]), which further argues against dependence on PPAR-γ activation. In summary, our findings provided good evidence that direct inhibition of mitochondrial fuel oxidation in isolated skeletal muscle is a group-specific effect of TZDs and is independent of PPAR-γ−mediated gene expression.
Hyperglycemia Activates p53 and p53-Regulated Genes Leading to Myocyte Cell Death
Authors: Fiordaliso Fabio , Leri Annarosa , Cesselli Daniela , Limana Federica , Safai Bijan , Nadal-Ginard Bernardo , Anversa Piero , Kajstura Jan
Published: March 2007

To determine whether enzymatic p53 glycosylation leads to angiotensin II formation followed by p53 phosphorylation, prolonged activation of the renin-angiotensin system, and apoptosis, ventricular myocytes were exposed to levels of glucose mimicking diabetic hyperglycemia. At a high glucose concentration, O-glycosylation of p53 occurred between 10 and 20 min, reached its peak at 1 h, and then decreased with time. Angiotensin II synthesis increased at 45 min and 1 h, resulting in p38 mitogen-activated protein (MAP) kinase–driven p53 phosphorylation at Ser 390. p53 phosphorylation was absent at the early time points, becoming evident at 1 h, and increasing progressively from 3 h to 4 days. Phosphorylated p53 at Ser 18 and activated c-Jun NH2-terminal kinases were identified with hyperglycemia, whereas extracellular signal-regulated kinase was not phosphorylated. Upregulation of p53 was associated with an accumulation of angiotensinogen and AT1 and enhanced production of angiotensin II. Bax quantity also increased. These multiple adaptations paralleled the concentrations of glucose in the medium and the duration of the culture. Myocyte death by apoptosis directly correlated with glucose and angiotensin II levels. Inhibition of O-glycosylation prevented the initial synthesis of angiotensin II, p53, and p38-MAP kinase (MAPK) phosphorylation and apoptosis. AT1 blockade had no influence on O-glycosylation of p53, but it interfered with p53 phosphorylation; losartan also prevented phosphorylation of p38-MAPK by angiotensin II. Inhibition of p38-MAPK mimicked at a more distal level the consequences of losartan. In conclusion, these in vitro results support the notion that hyperglycemia with diabetes promotes myocyte apoptosis mediated by activation of p53 and effector responses involving the local renin-angiotensin system.
A Prospective Study of Drinking Patterns in Relation to Risk of Type 2 Diabetes Among Men
Authors: Conigrave Katherine M. , Hu B. Frank , Camargo Carlos A. , Stampfer Meir J. , Willett Walter C. , Rimm Eric B.
Published: March 2007

Using data from a 12-year prospective study, we determined the importance of the pattern of alcohol consumption as a risk factor for type 2 diabetes in a cohort of 46,892 U.S. male health professionals who completed biennial postal questionnaires. Overall, 1,571 new cases of type 2 diabetes were documented. Compared with zero alcohol consumption, consumption of 15–29 g/day of alcohol was associated with a 36% lower risk of diabetes (RR = 0.64; 95% CI 0.53–0.77). This inverse association between moderate consumption and diabetes remained if light drinkers rather than abstainers were used as the reference group (RR = 0.60, CI 0.50–0.73). There were few heavy drinkers, but the inverse association persisted to those drinking ≥50 g/day of alcohol (RR = 0.60, CI 0.43–0.84). Frequency of consumption was inversely associated with diabetes. Consumption of alcohol on at least 5 days/week provided the greatest protection, even when less than one drink per drinking day was consumed (RR = 0.48, CI 0.27–0.86). Compared with infrequent drinkers, for each additional day per week that alcohol was consumed, risk was reduced by 7% (95% CI 3–10%) after controlling for average daily consumption. There were similar and independent inverse associations for beer, liquor, and white wine. Our findings suggested that frequent alcohol consumption conveys the greatest protection against type 2 diabetes, even if the level of consumption per drinking day is low. Beverage choice did not alter risk.
Orexin A Preferentially Excites Glucose-Sensitive Neurons in the Lateral Hypothalamus of the Rat In Vitro
Authors: Liu Xiao Hui , Morris Richard , Spiller David , White Michael , Williams Gareth
Published: March 2007

Falls in blood glucose induce hunger and initiate feeding. The lateral hypothalamic area (LHA) contains glucose-sensitive neurons (GSNs) and orexin neurons, both of which are stimulated by falling blood glucose and are implicated in hypoglycemia-induced feeding. We combined intracellular electrophysiological recording with fluorescein labeling of GSNs to determine their neuroanatomic and functional relationships with orexin neurons. Orexin A (1 μmol/l) caused a 500% increase (P < 0.01) in spontaneous firing rate and rapid and lasting depolarization that was tetrodotoxin-resistant and thus a direct postsynaptic effect. Orexin A altered the intrinsic neuronal properties of GSNs, consistent with increased excitability. Confocal microscopy showed that GSNs were intimately related to orexin neurons: orexin-immunoreactive axons were frequently entwined around GSN dendrites, establishing close and putatively synaptic contacts. Orexin-cell axons also passed in close proximity to glucose-responsive neurons, which are inhibited by low glucose, but orexin A caused smaller depolarization than on GSNs and only a 200% increase in spontaneous firing rate (P < 0.05 vs. GSN). We conclude that GSNs are specific target neurons for orexin A and suggest that they may mediate, at least in part, the acute appetite-stimulating effect of orexin A. Orexin neurons may regulate GSNs so as to control the onset and termination of hypoglycemia-induced feeding.
Longitudinal Study on Pubertal Insulin Resistance
Authors: Goran Michael I. , Gower Barbara A.
Published: March 2007

Previous cross-sectional studies show that puberty is associated with a reduction in insulin sensitivity (SI), but no longitudinal studies have examined this change in detail. This study is a longitudinal study in 60 children (33 male and 27 female subjects; 32 Caucasian and 28 African-American) examined at Tanner stage I (age 9.2 ± 1.4 years) and after 2.0 ± 0.6 years of follow-up, by which time 29 children remained at Tanner stage I and 31 had progressed to Tanner stage III or IV. Tanner stage was assessed by physical examination. SI, the acute insulin response (AIR), and the disposition index (DI) were determined by the tolbutamide-modified intravenous glucose tolerance test and minimal modeling, body fat mass was assessed by dual-energy X-ray absorptiometry, visceral fat was determined by computed tomography, and fasting blood was analyzed for hormone levels. In children progressing to Tanner stage III, SI fell significantly by 32% (4.4 ± 3.0 to 3.0 ± 1.7 × 10−4min−1/[μIU/ml]), AIR increased by 30%, DI fell by 27%, and there was a significant increase in fasting glucose (93.5 ± 5.0 to 97.0 ± 4.1 mg/dl) and insulin (14.3 ± 8.1 to 18.6 ± 11.0 μIU/ml). In children remaining at Tanner stage I, there was a slight increase in SI (6.4 ± 3.1 to 7.4 ± 3.5 × 10−4min−1/[μIU/ml]) with no significant change in AIR or fasting glucose and insulin. The pubertal fall in SI was more consistent in African-Americans; remained significant after controlling for age, sex, and change in fat mass, visceral fat, and fat-free mass; and was similar in children at low, medium, and high body fat. Change in SI was not significantly related to change in fasting hormone levels, but change in AIR was significantly related to change in androstendione (r = 0.39; P = 0.04). Pubertal transition from Tanner stage I to Tanner stage III was associated with a 32% reduction in SI, and increases in fasting glucose, insulin, and AIR. These changes were similar across sex, ethnicity, and obesity. The significant fall in DI suggests conservation in β-cell function or an inadequate β-cell response to the fall in SI. The fall in SI was not associated with changes in body fat, visceral fat, IGF-I, androgens, or estradiol.
Reduced Insulinotropic Effect of Gastric Inhibitory Polypeptide in First-Degree Relatives of Patients With Type 2 Diabetes
Authors: Meier Juris J. , Hücking Katrin , Holst Jens J. , Deacon Carolyn F. , Schmiegel Wolff H. , Nauck Michael A.
Published: March 2007

In patients with type 2 diabetes, gastric inhibitory polypeptide (GIP) has lost much of its insulinotropic activity. Whether this is similar in first-degree relatives of patients with type 2 diabetes is unknown. A total of 21 first-degree relatives, 10 patients with type 2 diabetes, and 10 control subjects (normal oral glucose tolerance) were examined. During a hyperglycemic “clamp” (140 mg/dl for 120 min), synthetic human GIP (2 pmol · kg−1 · min−1) was infused intravenously (30–90 min). With exogenous GIP, patients with type 2 diabetes responded with a lower increment (Δ) in insulin (P = 0.0003) and C-peptide concentrations (P < 0.0001) than control subjects. The GIP effects in first-degree relatives were diminished compared with control subjects (Δ insulin: P = 0.04; Δ C-peptide: P = 0.016) but significantly higher than in patients with type 2 diabetes (P ≤ 0.05). The responses over the time course were below the 95% CI derived from control subjects in 7 (insulin) and 11 (C-peptide) of 21 first-degree relatives of patients with type 2 diabetes. In conclusion, a reduced insulinotropic activity of GIP is typical for a substantial subgroup of normoglycemic first-degree relatives of patients with type 2 diabetes, pointing to an early, possibly genetic defect.
Ghrelin Causes Hyperphagia and Obesity in Rats
Authors: Wren Alison M. , Small Caroline J. , Abbott Caroline R. , Dhillo Waljit S. , Seal Leighton J. , Cohen Mark A. , Batterham Rachel L. , Taheri Shahrad , Stanley Sarah A. , Ghatei Mohammad A. , Bloom Stephen R.
Published: March 2007

Ghrelin, a circulating growth hormone–releasing peptide derived from the stomach, stimulates food intake. The lowest systemically effective orexigenic dose of ghrelin was investigated and the resulting plasma ghrelin concentration was compared with that during fasting. The lowest dose of ghrelin that produced a significant stimulation of feeding after intraperitoneal injection was 1 nmol. The plasma ghrelin concentration after intraperitoneal injection of 1 nmol of ghrelin (2.83 ± 0.13 pmol/ml at 60 min postinjection) was not significantly different from that occurring after a 24-h fast (2.79 ± 0.32 pmol/ml). After microinjection into defined hypothalamic sites, ghrelin (30 pmol) stimulated food intake most markedly in the arcuate nucleus (Arc) (0–1 h food intake, 427 ± 43% of control; P < 0.001 vs. control, P < 0.01 vs. all other nuclei), which is potentially accessible to the circulation. After chronic systemic or intracerebroventricular (ICV) administration of ghrelin for 7 days, cumulative food intake was increased (intraperitoneal ghrelin 13.6 ± 3.4 g greater than saline-treated, P < 0.01; ICV ghrelin 19.6 ± 5.5 g greater than saline-treated, P < 0.05). This was associated with excess weight gain (intraperitoneal ghrelin 21.7 ± 1.4 g vs. saline 10.6 ± 1.9 g, P < 0.001; ICV ghrelin 15.3 ± 4.3 g vs. saline 2.2 ± 3.8 g, P < 0.05) and adiposity. These data provide evidence that ghrelin is important in long-term control of food intake and body weight and that circulating ghrelin at fasting concentrations may stimulate food intake.
Plasma Free Fatty Acid Uptake and Oxidation Are Already Diminished in Subjects at High Risk for Developing Type 2 Diabetes
Authors: Mensink Marco , Blaak Ellen E. , van Baak Marleen A. , Wagenmakers Anton J.M. , Saris Wim H.M.
Published: March 2007

The objective of this study was to investigate to what extent disturbances in fatty acid metabolism found in type 2 diabetes are already present in subjects at high risk for developing diabetes (i.e., impaired glucose tolerance [IGT]). Components of fatty acid metabolism were measured in male subjects with IGT during postabsorptive conditions and during 60 min of exercise (50% Vo2max) with the use of the stable isotope tracer [U-13C]palmitate in combination with indirect calorimetry, and those values were compared with previously published findings in male type 2 diabetic and male obese subjects. No differences were found between groups in energy expenditure and in total fat and carbohydrate oxidation. Rate of appearance and rate of disappearance of plasma free fatty acid (FFA) were lower in subjects with IGT and type 2 diabetes compared with obese subjects (P < 0.05). Plasma FFA oxidation was lower in subjects with IGT and type 2 diabetes compared with obese subjects at rest and tended to be lower during exercise (rest: 3.7 ± 0.3, 4.4 ± 0.6, and 6.9 ± 1.0 μmol · kg fat-free mass [FFM]−1 · min−1, P < 0.01; exercise: 15.0 ± 1.7, 14.1 ± 1.9, and 19.6 ± 1.5 μmol · kg FFM−1 · min−1 for IGT, type 2 diabetic, and obese subjects, respectively, P = 0.07). Triglyceride-derived fatty acid oxidation, however, was elevated in subjects with IGT and type 2 diabetes during exercise (3.6 ± 1.4, 1.4 ± 1.4, and –4.0 ± 2.0 μmol · kg FFM−1 · min−1 for IGT, type 2 diabetic, and obese subjects, respectively; P < 0.05). These data demonstrate that male subjects with a prediabetic condition (IGT) have the same defects in fatty acid utilization as subjects with type 2 diabetes, suggesting that these disturbances may play an important role in the progression from IGT to type 2 diabetes.
Ceramide Mediates Insulin Resistance by Tumor Necrosis Factor-α in Brown Adipocytes by Maintaining Akt in an Inactive Dephosphorylated State
Authors: Teruel Teresa , Hernandez Rosario , Lorenzo Margarita
Published: March 2007

Tumor necrosis factor (TNF)-α causes insulin resistance on glucose uptake in fetal brown adipocytes. We explored the hypothesis that some effects of TNF-α could be mediated by the generation of ceramide, given that TNF-α treatment induced the production of ceramide in these primary cells. A short-chain ceramide analog, C2-ceramide, completely precluded insulin-stimulated glucose uptake and insulin-induced GLUT4 translocation to plasma membrane, as determined by Western blot or immunofluorescent localization of GLUT4. These effects were not produced in the presence of a biologically inactive ceramide analog, C2-dihydroceramide. Analysis of the phosphatidylinositol (PI) 3-kinase signaling pathway indicated that C2-ceramide precluded insulin stimulation of Akt kinase activity, but not of PI-3 kinase or protein kinase C-ζ activity. C2-ceramide completely abolished insulin-stimulated Akt/protein kinase B phosphorylation on regulatory residues Thr 308 and Ser 473, as did TNF-α, and inhibited insulin-induced mobility shift in Akt1 and Akt2 separated in PAGE. Moreover, C2-ceramide seemed to activate a protein phosphatase (PP) involved in dephosphorylating Akt because 1) PP2A activity was increased in C2-ceramide− and TNF-α−treated cells, 2) treatment with okadaic acid concomitantly with C2-ceramide completely restored Akt phosphorylation by insulin, and 3) transient transfection of a constitutively active form of Akt did not restore Akt activity. Our results indicate that ceramide produced by TNF-α induces insulin resistance in brown adipocytes by maintaining Akt in an inactive dephosphorylated state.
Modulation of Incipient Glomerular Lesions in Experimental Diabetic Nephropathy by Hypotensive and Subhypotensive Dosages of an ACE Inhibitor
Authors: Fabris Bruno , Candido Riccardo , Carraro Michele , Fior Francesco , Artero Mary , Zennaro Cristina , Cattin Maria Rosa , Fiorotto Angela , Bortoletto Monica , Millevoi Cristina , Bardelli Moreno , Faccini Luigi , Carretta Renzo
Published: March 2007

A glomerular permeability defect occurs early in the course of type 1 diabetes and precedes the onset of microalbuminuria and renal morphological changes. Recently, ACE inhibitors have been shown to prevent loss of glomerular membrane permselective function, but the mechanism of this nephroprotective effect is still being debated. The objective of the present study was to evaluate the effects of hypotensive and subhypotensive dosages of the ACE inhibitor quinapril ex vivo and of its active metabolite quinaprilat in vitro on the glomerular albumin permeability (Palb) defect in the early phases of experimental diabetes. For the ex vivo study, six groups of male Wistar rats were evaluated for 4 weeks. One group served as a nondiabetic control (C); the other five groups were rendered diabetic and included untreated diabetic rats (D) and diabetic rats receiving quinapril at the dosages of 5 (DQ1), 2.5 (DQ2), 1.25 (DQ3), and 0.625 (DQ4) mg · kg−1 · day−1. Dosage-dependent effects of quinapril on systolic blood pressure and the glomerular filtration rate were observed. In contrast, control of Palb in isolated glomeruli exposed to oncotic gradients, proteinuria, and glomerular and tubular hypertrophy was obtained with subhypotensive dosages (DQ3 and DQ4 groups) of the ACE inhibitor. In the in vitro study, quinaprilat reduced Palb significantly in concentration ranges from 10−6 to 10−14 mol/l compared with results in control glomeruli. The effect on Palb may have occurred by mechanisms different from kidney ACE inhibitor. These study results indicated that ACE inhibitor treatment prevents the early onset of the Palb defect in experimental diabetes. This effect seemed to occur independently of systemic or glomerular hemodynamic changes and, at least partially, from kidney ACE inhibition.
Physiologic Hyperinsulinemia Enhances Human Skeletal Muscle Perfusion by Capillary Recruitment
Authors: Coggins Matthew , Lindner Jonathan , Rattigan Steve , Jahn Linda , Fasy Elizabeth , Kaul Sanjiv , Barrett Eugene
Published: March 2007

Despite intensive study, the relation between insulin’s action on blood flow and glucose metabolism remains unclear. Insulin-induced changes in microvascular perfusion, independent from effects on total blood flow, could be an important variable contributing to insulin’s metabolic action. We hypothesized that modest, physiologic increments in plasma insulin concentration alter microvascular perfusion in human skeletal muscle and that these changes can be assessed using contrast-enhanced ultrasound (CEU), a validated method for quantifying flow by measurement of microvascular blood volume (MBV) and microvascular flow velocity (MFV). In the first protocol, 10 healthy, fasting adults received insulin (0.05 mU · kg−1 · min−1) via a brachial artery for 4 h under euglycemic conditions. At baseline and after insulin infusion, MBV and MFV were measured by CEU during continuous intravenous infusion of albumin microbubbles with intermittent harmonic ultrasound imaging of the forearm deep flexor muscles. In the second protocol, 17 healthy, fasting adults received a 4-h infusion of either insulin (0.1 mU · kg−1 · min−1, n = 9) or saline (n = 8) via a brachial artery. Microvascular volume was assessed in these subjects by an alternate CEU technique using an intra-arterial bolus injection of albumin microbubbles at baseline and after the 4-h infusion. With both protocols, muscle glucose uptake, plasma insulin concentration, and total blood flow to the forearm were measured at each stage. In protocol 2 subjects, tissue extraction of 1-methylxanthine (1-MX) was measured as an index of perfused capillary volume. Caffeine, which produces 1-MX as a metabolite, was administered to these subjects before the study to raise plasma 1-MX levels.  In protocol 1 subjects, insulin increased muscle glucose uptake (180%, P < 0.05) and MBV (54%, P < 0.01) and decreased MFV (−42%, P = 0.07) in the absence of significant changes in total forearm blood flow. In protocol 2 subjects, insulin increased glucose uptake (220%, P < 0.01) and microvascular volume (45%, P < 0.05) with an associated moderate increase in total forearm blood flow (P < 0.05). Using forearm 1-MX extraction, we observed a trend, though not significant, toward increasing capillary volume in the insulin-treated subjects. In conclusion, modest physiologic increments in plasma insulin concentration increased microvascular blood volume, indicating altered microvascular perfusion consistent with a mechanism of capillary recruitment. The increases in microvascular (capillary) volume (despite unchanged total blood flow) indicate that the relation between insulin’s vascular and metabolic actions cannot be fully understood using measurements of bulk blood flow alone.
Intrinsic Defects in the T-Cell Lineage Results in Natural Killer T-Cell Deficiency and the Development of Diabetes in the Nonobese Diabetic Mouse
Authors: Yang Yang , Bao Min , Yoon Ji-Won
Published: March 2007

T-cell–mediated autoimmune diabetes in nonobese diabetic (NOD) mice is closely associated with natural killer T (NKT)-cell deficiency. To determine whether intrinsic defects of the T-cell lineage contribute to the pathogenesis of the disease and NKT cell deficiency, we reconstituted the T-cell compartment in NOD.scid or BALB.scid mice with T-cells from NOD, nonobese diabetes-resistant (NOR), or AKR thymic precursor cells and examined the development of the NKT cell population. NKT cells developed well from AKR thymic precursor cells but not from other precursor cells in both recipient strains. Insulitis and diabetes developed only in the NOD.scid recipients of NOD or NOR precursor cells. When thymic precursor cells of β2-microglobulin gene–deficient AKR mice, which have a deficient NKT population, were introduced into NOD.scid recipients, both CD4+ and CD8+ T-cell populations developed and the recipient mice developed insulitis and diabetes. We conclude that NKT cells originate from a T-cell–committed thymic precursor population and that the deficiency in the NKT cell population in NOD mice results from intrinsic defects within the T-cell lineage and plays a major role in the development of autoimmune diabetes in the presence of both the NOD thymus and antigen-presenting cells.
Protection From Type 1 Diabetes in the Face of High Levels of Activated Autoaggressive Lymphocytes in a Viral Transgenic Mouse Model Crossed to the SV129 Strain
Authors: von Herrath Matthias G. , Wolfe Tom , Möhrle Ursula , Coon Bryan , Hughes Anna
Published: March 2007

In comparing the incidence of virally induced type 1 diabetes in F1 crosses of RIP-LCMV mice to three different mouse strains identical at the major histocompatibility complex H-2Db locus, we surprisingly found that disease development was reduced by 80% in F1 crosses to the SV129 genetic background and by 60% after eight backcrosses to the original C57BL/6 RIP-LCMV mice. In this model, diabetes is strongly dependent on a virally induced H-2Db–restricted cytotoxic T-cell (CTL) response. Importantly, numbers and effector functions of autoaggressive CD4 and CD8 lymphocytes were not decreased in the protected mice, and CTLs were still able to kill syngeneic islet cells in vitro with equal efficacy compared with CTLs from the original RIP-LCMV strain. Furthermore, CTLs were able to extravasate into islets in vivo, and no evidence for induction of regulatory cells was observed. However, regeneration of β-cells in islets under “attack” occurred only in the protected SV129-crossed animals, whereas it was not evident at any time in any mice that developed diabetes. Thus, genetic factors can “override” the diabetogenic potential of high numbers of autoaggressive lymphocytes through, for example, increased islet regeneration. This finding has important implications for interpreting numbers and pathogenicity of autoreactive lymphocytes in prediabetic patients of genetically diverse backgrounds.
p57KIP2 Expression in Normal Islet Cells and in Hyperinsulinism of Infancy
Authors: Kassem S. A. , Ariel I. , Thornton P. S. , Hussain K. , Smith V. , Lindley K. J. , Aynsley-Green A. , Glaser B.
Published: March 2007

Most cases of hyperinsulinism of infancy (HI) are caused by mutations in either the sulfonylurea receptor-1 (SUR1) or the inward rectifying K+ channel Kir6.2, two subunits of the β-cell ATP-sensitive K+ channel (KATP channel). Histologically, HI can be divided into two major subtypes. The diffuse form is recessively inherited and involves all β-cells within the pancreas. Focal HI consists of adenomatous hyperplasia within a limited region of the pancreas, and it is caused by somatic loss of heterozygosity (LOH), including maternal Ch11p15-ter in a β-cell precursor carrying a germ-line mutation in the paternal allele of SUR1 or Kir6.2. Several imprinted genes are located within this chromosomal region, some of which, including p57KIP2 and IGF-II, have been associated with the regulation of cell proliferation. Using double immunostaining, we examined p57KIP2 expression in different islet cell types, in control pancreases from different developmental stages (n = 15), and in pancreases from patients with both diffuse (n = 4) and focal HI (n = 9). Using immunofluorescence and computerized image analysis, we quantified IGF-II expression in β-cells from patients with focal HI (n = 8). Within the pancreas, p57KIP2 was specifically localized to the endocrine portion. β-Cells demonstrated the highest frequency of expression (34.9 ± 2.7%) compared with ∼1–3% in other cell types. The fraction of β-cells expressing p57KIP2 did not vary significantly during development. β-Cells within the focal lesions did not express p57KIP2, whereas IGF-II staining inside focal lesions was mildly increased compared with unaffected surrounding tissue. In conclusion, we demonstrate that p57KIP2 is expressed and is paternally imprinted in human pancreatic β-cells. Loss of expression in focal HI is caused by LOH and is associated with increased proliferation and increased IGF-II expression. Manipulation of p57KIP2 expression in β-cells may provide a mechanism by which proliferation can be modulated, and thus this gene is a potential therapeutic target for reversing the β-cell failure observed in diabetes.
Insulin Signal Transduction in Skeletal Muscle From Glucose-Intolerant Relatives With Type 2 Diabetes
Authors: Storgaard Heidi , Song Xiao Mei , Jensen Christine B. , Madsbad Sten , Björnholm Marie , Vaag Allan , Zierath Juleen R.
Published: March 2007

To determine whether defects in the insulin signal transduction cascade are present in skeletal muscle from prediabetic individuals, we excised biopsies from eight glucose-intolerant male first-degree relatives of patients with type 2 diabetes (IGT relatives) and nine matched control subjects before and during a euglycemic-hyperinsulinemic clamp. IGT relatives were insulin-resistant in oxidative and nonoxidative pathways for glucose metabolism. In vivo insulin infusion increased skeletal muscle insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation (P = 0.01) and phosphatidylinositide 3-kinase (PI 3-kinsase) activity (phosphotyrosine and IRS-1 associated) in control subjects (P < 0.02) but not in IGT relatives (NS). The incremental increase in insulin action on IRS-1 tyrosine phosphorylation was lower in IGT relatives versus control subjects (P < 0.05). The incremental defects in signal transduction noted for IRS-1 and PI 3-kinase may be attributed to elevated basal phosphorylation/activity of these parameters, because absolute phosphorylation/activity under insulin-stimulated conditions was similar between IGT relatives and control subjects. Insulin increased Akt serine phosphorylation in control subjects and IGT relatives, with a tendency for reduced phosphorylation in IGT relatives (P = 0.12). In conclusion, aberrant phosphorylation/activity of IRS-1, PI 3-kinase, and Akt is observed in skeletal muscle from relatives of patients with type 2 diabetes with IGT. However, the elevated basal activity of these signaling intermediates and the lack of a strong correlation between these parameters to glucose metabolism suggests that other defects of insulin signal transduction and/or downstream components of glucose metabolism may play a greater role in the development of insulin resistance in skeletal muscle from relatives of patients with type 2 diabetes.
Overfeeding Rapidly Induces Leptin and Insulin Resistance
Authors: Wang Jiali , Obici Silvana , Morgan Kimyata , Barzilai Nir , Feng Zhaohui , Rossetti Luciano
Published: March 2007

In common forms of obesity, hyperphagia, hyperinsulinemia, and hyperleptinemia coexist. Here, we demonstrate rapid induction of insulin and leptin resistance by short-term overfeeding. After 3 and 7 days on the assigned diet regimen, rats were tested for their biological responses to acute elevations in plasma insulin and leptin concentrations. Severe resistance to the metabolic effects of both leptin and insulin ensued after just 3 days of overfeeding. During the insulin clamp studies, glucose production was decreased by ∼70% in control rats and 28–53% in overfed rats. Similarly, leptin infusion doubled the contribution of gluconeogenesis to glucose output in control rats but failed to modify gluconeogenesis in overfed animals. These findings demonstrate a paradoxical and rapid collapse of the leptin system in response to nutrient excess. This partial failure is tightly coupled with the onset of insulin resistance.
Identification of Novel Genes Differentially Expressed in Omental Fat of Obese Subjects and Obese Type 2 Diabetic Patients
Authors: Corominola Helena , Conner Laura J. , Beavers Lisa S. , Gadski Robert A. , Johnson Dwayne , Caro Jose F. , Rafaeloff-Phail Ronit
Published: March 2007

Obesity is associated with an increased risk for developing type 2 diabetes, insulin resistance, hypertension, dyslipidemia, cardiovascular disease, respiratory dysfunction, and certain forms of cancer. Insulin resistance in many type 2 diabetic patients is the result of increased visceral adiposity. To identify novel genes implicated in type 2 diabetes and/or obesity and to elucidate the molecular mechanisms underlying both diseases, we analyzed gene expression in omental fat from lean and obese nondiabetic subjects and obese type 2 diabetic patients using mRNA differential display and subtracted library techniques. After screening over 13,800 subtracted cDNA clones and 6,912 cDNA amplification products, we identified 2,078 cDNAs that showed potential differential expression in the omental fat of lean versus obese nondiabetic subjects versus obese type 2 diabetic patients. Data analysis showed that 70.7% of these clones corresponded to unknown genes (26.7% matched express sequence tags [ESTs]) and 29.3% corresponded to known genes. Reverse Northern and classic Northern analyses further confirmed that the expression of five of these cDNA clones was elevated in obese nondiabetic subjects and obese type 2 diabetic patients. Four candidate genes were further evaluated for tissue distribution, which showed expression primarily in adipose and skeletal muscle tissue, and chromosomal localization. We concluded that both mRNA differential display and subtracted cDNA libraries are powerful tools for identifying novel genes implicated in the pathogenesis of obesity and type 2 diabetes.
Effects of Insulin and Antioxidant on Plasma 8-Hydroxyguanine and Tissue 8-Hydroxydeoxyguanosine in Streptozotocin-Induced Diabetic Rats
Authors: Park Kyong Soo , Kim Jae Hyeon , Kim Min Seon , Kim Jeong Mi , Kim Suk Kyeong , Choi Jeong Yun , Chung Myung Hee , Han Boghee , Kim Seong Yeon , Lee Hong Kye
Published: March 2007

Cumulating evidence suggests that enhanced oxidative stress may contribute to diabetic angiopathy. The levels of 8-hydroxydeoxyguanosine (8-OHdG) and 8-hydroxyguanine (8-OHG), indicators of oxidative DNA damage, in tissue or body fluid are increased in diabetic patients. However, it is unclear whether plasma 8-OHG correlates with tissue 8-OHdG and whether insulin or antioxidant treatment reduces plasma 8-OHG in diabetic state. In this study, we measured the 8-OHG levels in plasma as well as the 8-OHdG levels in liver and kidney in streptozotocin-induced diabetic rats (DR) treated with insulin (DR+I), insulin and probucol (DR+I/P), or insulin and vitamin E (DR+I/E). There was a correlation between plasma 8-OHG levels and tissue 8-OHdG levels (plasma 8-OHG vs. liver 8-OHdG: r = 0.64, P < 0.001; plasma 8-OHG vs. kidney 8-OHdG: r = 0.38, P = 0.06). DR had levels of plasma 8-OHG that were three times higher than control rats (CR), whereas they had levels of tissue 8-OHdG that were ∼1.5–2 times higher. Plasma 8-OHG levels in DR were almost normalized by insulin treatment, although insulin partially corrected hyperglycemia (plasma 8-OHG: CR 3.3 ± 2.7 pmol/ml; DR 10.4 ± 2.3 pmol/ml, P < 0.05 vs. CR; DR with insulin 3.6 ± 1.0 pmol/ml, P < 0.05 vs. DR). However, tissue 8-OHdG levels in DR were significantly decreased by combined treatment with insulin and antioxidant (probucol or vitamin E), but not by insulin treatment alone. This data suggests that plasma 8-OHG could be a useful biomarker of oxidative DNA damage in diabetic subjects. The mechanism of differential response of plasma 8-OHG and tissue 8-OHdG to insulin and antioxidant treatment remains to be elucidated.
A Missense Mutation of Pax4 Gene (R121W) Is Associated With Type 2 Diabetes in Japanese
Authors: Shimajiri Yoshinori , Sanke Tokio , Furuta Hiroto , Hanabusa Tadashi , Nakagawa Takayuki , Fujitani Yoshio , Kajimoto Yoshitaka , Takasu Nobuyuki , Nanjo Kishio
Published: March 2007

Pax4 is one of the transcription factors that play an important role in the differentiation of islet β-cells. We scanned the Pax4 gene in 200 unrelated Japanese type 2 diabetic patients and found a missense mutation (R121W) in 6 heterozygous patients and 1 homozygous patient (mutant allele frequency 2.0%). The mutation was not found in 161 nondiabetic subjects. The R121W mutation was located in the paired domain and was thought to affect its transcription activity through lack of DNA binding. Six of seven patients had family history of diabetes or impaired glucose tolerance, and four of seven had transient insulin therapy at the onset. One of them, a homozygous carrier, had relatively early onset diabetes and slowly fell into an insulin-dependent state without an autoimmune-mediated process. This is the first report of a Pax4 gene mutation that exhibits loss of function and seems to be associated with type 2 diabetes. This work provides significant implications for the Pax4 gene as one of the predisposing genes for type 2 diabetes in the Japanese.
Further Mapping of the Idd5.1 Locus for Autoimmune Diabetes in NOD Mice
Authors: Lamhamedi-Cherradi Salah-Eddine , Boulard Olivier , Gonzalez Cristina , Kassis Najiby , Damotte Diane , Eloy Laure , Fluteau Guy , Lévi-Strauss Matthieu , Garchon Henri-Jean
Published: March 2007

The Idd5 locus for autoimmune diabetes in nonobese diabetic (NOD) mice has been mapped to the proximal half of chromosome 1 and appears to include two loci, Idd5.1 and Idd5.2, Idd5.1 being a candidate homolog of the human IDDM12 locus. Using new recombinant congenic lines, we have reduced the Idd5.1 interval to 5 cM at most, between D1Mit279 and D1Mit19 (not included). This interval now excludes the Casp8 and Cflar (Flip) candidate genes. It still retains Cd28 and Ctla4 and also includes Icos (inducible costimulator). The previously reported differential expression of Ctla4, which is induced at a lower level in NOD than in B6-activated T-cells, was found independent of Idd5.1 itself because Ctla4 expression was induced at a low level in T-cells from Idd5.1-congenic mice. The Idd5.1 locus protected against both spontaneous and cyclophosphamide-induced diabetes, but it did not prevent inflammatory infiltration of the islets of Langerhans. Furthermore, diabetogenic precursor spleen cells from prediabetic NOD and Idd5.1-congenic mice were equally capable of transferring diabetes to immunodeficient NOD.scid/scid recipient mice. The Idd5.1 locus might affect a late event of disease development, subsequent to the onset of insulitis and possibly taking place in the islets of Langerhans.
Effects of Glucose and Amino Acids on Free ADP in βHC9 Insulin-Secreting Cells
Authors: Ronner Peter , Naumann C. Maik , Friel Edward
Published: March 2007

Stimulation of insulin release by glucose is widely thought to be coupled to a decrease in the activity of ATP-sensitive K+ channels(KATP channels) that is caused by a decreased concentration of free ADP. To date, most other investigators have reported only on total cellular ADP concentrations, even though only a small fraction of all ADP is free and only the free ADP affects KATP channels. We tested the hypothesis that amino acids elicit insulin release via a decrease in the activity of KATP channels owing to a decrease in the level of free ADP. We estimated the concentration of free ADP in βHC9 hyperplastic insulin-secreting cells based on the cell diameter and on luminometric measurements of ATP, phosphocreatine, and total creatine. The concentration of free ADP fell exponentially as the concentration of glucose increased. A physiological mixture of amino acids greatly stimulated insulin release at 0-30 mmol/l glucose but affected the concentration of free ADP only to a minor degree and significantly so only at ≤2 mmol/l glucose. In the presence of 2-deoxyglucose and NaN3, amino acids were unable to stimulate insulin release. When KATP channels were held open with diazoxide(and the plasma membrane partially depolarized with high extracellular KCl),amino acids still stimulated insulin release. We conclude that amino acid—induced insulin release depends on two components: a yet-unknown amino acid sensor and KATP channels, which serve to attenuate hormone release when cellular energy stores are low. We propose that glucose-induced insulin release may be regulated similarly by two components:glucokinase and KATP channels.
Metabolic Regulation by Leucine of Translation Initiation Through the mTOR-Signaling Pathway by Pancreatic β-Cells
Authors: Xu Guang , Kwon Guim , Cruz Wilhelm S. , Marshall Connie A. , McDaniel Michael L.
Published: March 2007

Recent findings have demonstrated that the branched-chain amino acid leucine can activate the translational regulators, phosphorylated heat- and acid-stable protein regulated by insulin (PHAS-I) and p70 S6 kinase(p70s6k), in an insulin-independent and rapamycin-sensitive manner through mammalian target of rapamycin (mTOR), although the mechanism for this activation is undefined. It has been previously established that leucine-induced insulin secretion by β-cells involves increased mitochondrial metabolism by oxidative decarboxylation and allosteric activation of glutamate dehydrogenase (GDH). We now show that these same intramitochondrial events that generate signals for leucine-induced insulin exocytosis are required to activate the mTOR mitogenic signaling pathway byβ-cells. Thus, a minimal model consisting of leucine and glutamine as substrates for oxidative decarboxylation and an activator of GDH,respectively, confirmed the requirement for these two metabolic components and mimicked closely the synergistic interactions achieved by a complete complement of amino acids to activate p70s6k in a rapamycin-sensitive manner. Studies using various leucine analogs also confirmed the close association of mitochondrial metabolism and the ability of leucine analogs to activate p70s6k. Furthermore, selective inhibitors of mitochondrial function blocked this activation in a reversible manner, which was not associated with a global reduction in ATP levels. These findings indicate that leucine at physiological concentrations stimulates p70s6k phosphorylation via the mTOR pathway, in part, by serving both as a mitochondrial fuel and an allosteric activator of GDH. Leucine-mediated activation of protein translation through mTOR may contribute to enhanced β-cell function by stimulating growth-related protein synthesis and proliferation associated with the maintenance of β-cell mass.
Expression and Distribution of Lactate/Monocarboxylate Transporter Isoforms in Pancreatic Islets and the Exocrine Pancreas
Authors: Zhao Chao , Wilson Marieangela C. , Schuit Franz , Halestrap Andrew P. , Rutter Guy A.
Published: March 2007

Transport of lactate across the plasma membrane of pancreatic isletβ-cells is slow, as described by Sekine et al. (J Biol Chem269:4895-4902, 1994), which is a feature that may be important for normal nutrient-induced insulin secretion. Although eight members of the monocarboxylate transporter (MCT) family have now been identified, the expression of these isoforms within the exocrine and endocrine pancreas has not been explored in detail. Using immunocytochemical analysis of pancreatic sections fixed in situ, we demonstrated three phenomena. First,immunoreactivity of the commonly expressed lactate transporter isoform MCT1 is near zero in both α- and β-cells but is abundant in the pancreatic acinar cell plasma membrane. No MCT2 or MCT4 was detected in any pancreatic cell type. Second, Western analysis of purified β- and non—β-cell membranes revealed undetectable levels of MCT1 and MCT4. In derived β-cell lines, MCT1 was absent from MIN6 cells and present in low amounts in INS-1 cell membranes and at high levels in RINm5F cells. MCT4 was weakly expressed in MIN6 β-cells. Third, CD147, an MCT-associated chaperone protein, which is closely colocalized with MCT1 on acinar cell membranes, was absent from islet cell membranes. CD147 was also largely absent from MIN6 and INS-1 cells but abundant in RINm5F cells. Low expression of MCT1, MCT2, and MCT4 contributes to the enzymatic configuration ofβ-cells, which is poised to ensure glucose oxidation and the generation of metabolic signals and may also be important for glucose sensing in islet non—β-cells. MCT overexpression throughout the islet could contribute to deranged hormone secretion in some forms of type 2 diabetes.
α- and β-Cell Responses to Small Changes in Plasma Glucose in the Conscious Dog
Authors: Flattem Nancy , Igawa Kayano , Shiota Masakazu , Emshwiller Maya G. , Neal Doss W. , Cherrington Alan D.
Published: March 2007

The responses of the pancreatic α- and β-cells to small changes in glucose were examined in overnight-fasted conscious dogs. Each study consisted of an equilibration (-140 to -40 min), a control (-40 to 0 min), and a test period (0 to 180 min), during which BAY R3401 (10 mg/kg), a glycogen phosphorylase inhibitor, was administered orally, either alone to create mild hypoglycemia or with peripheral glucose infusion to maintain euglycemia or create mild hyperglycemia. Drug administration in the hypoglycemic group decreased net hepatic glucose output (NHGO) from 8.9 ± 1.7 (basal) to 6.0 ± 1.7 and 5.8 ± 1.0 μmol · kg-1· min-1 by 30 and 90 min. As a result, the arterial plasma glucose level decreased from 5.8 ± 0.2 (basal) to 5.2 ± 0.3 and 4.4 ± 0.3 mmol/l by 30 and 90 min, respectively (P < 0.01). Arterial plasma insulin levels and the hepatic portal-arterial difference in plasma insulin decreased (P < 0.01) from 78 ± 18 and 90± 24 to 24 ± 6 and 12 ± 12 pmol/l over the first 30 min of the test period and decreased to 18 ± 6 and 0 pmol/l by 90 min,respectively. The arterial glucagon levels and the hepatic portal-arterial difference in plasma glucagon increased from 43 ± 5 and 4 ± 2 to 51 ± 5 and 10 ± 5 ng/l by 30 min (P < 0.05) and to 79 ± 16 and 31 ± 15 ng/l by 90 min (P < 0.05),respectively. In euglycemic dogs, the arterial plasma glucose level remained at 5.9 ± 0.1 mmol/l, and the NHGO decreased from 10 ± 0.6 to-3.3 ± 0.6 μmol · kg-1 · min-1(180 min). The insulin and glucagon levels and the hepatic portal-arterial differences remained constant. In hyperglycemic dogs, the arterial plasma glucose level increased from 5.9 ± 0.2 to 6.2 ± 0.2 mmol/l by 30 min, and the NHGO decreased from 10 ± 1.7 to 0 μmol ·kg-1 · min-1 by 30 min. The arterial plasma insulin levels and the hepatic portal-arterial difference in plasma insulin increased from 60 ± 18 and 78 ± 24 to 126 ± 30 and 192± 42 pmol/l by 30 min, after which they averaged 138 ± 24 and 282 ± 30 pmol/l, respectively. The arterial plasma glucagon levels and the hepatic portal-arterial difference in plasma glucagon decreased slightly from 41 ± 7 and 4 ± 3 to 34 ± 7 and 3 ± 2 ng/l during the test period. These data show that the α- and β-cells of the pancreas respond as a coupled unit to very small decreases in the plasma glucose level.