bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Purinoceptors Amplifies Glucose-Stimulated Insulin Release With Removal of its Pulsatility
Authors: Salehi Albert , Qader Saleem S. , Grapengiesser Eva , Hellman Bo
Published: March 2007

External ATP has been proposed to be an autocrine regulator of glucose-stimulated insulin secretion and responsible for the synchronization of the Ca2+ rhythmicity in the β-cells required for a pulsatile release of insulin from the pancreas. The importance of external ATP for glucose-stimulated insulin release was evaluated in rats with the aid of 2-deoxy-N-methyladenosine-3,5-bisphosphate (MRS 2179), an inhibitor of the purinoceptors known to affect the Ca2+ signaling in β-cells. The concentration of cytoplasmic Ca2+ was measured in single β-cells and small aggregates with ratiometric fura-2 technique and the release of insulin recorded from isolated islets and the perfused pancreas. Addition of 1 μmol/l ATP induced premature cytoplasmic Ca2+ concentration ([Ca2+]i) oscillations similar to those found in β-cells exposed to 20 mmol/l glucose. In most experiments, the presence of 10 μmol/l MRS 2179 did not remove the glucose-induced [Ca2+]i rhythmicity in single β-cells or the synchronization seen in coupled cells. Nevertheless, the same concentration of MRS 2179 promptly interrupted the pulsatility (frequency 0.22 ± 0.01/min) of insulin secretion, raising the total amounts released from the pancreas. Prolonged exposure of islets to 1 and 10 μmol/l MRS 2179 enhanced insulin secretion at 20 mmol/l glucose 33% (P < 0.05) and 63% (P < 0.01), respectively, without affecting the release at 3 mmol/l glucose. The results support the idea that neural ATP signals entrain the islets into a common rhythm resulting in pulsatile release of insulin and that glucose stimulation of the secretory activity is counteracted by accumulation of inhibitory ATP around the β-cells.
Redox Control of Exocytosis
Authors: Ivarsson Rosita , Quintens Roel , Dejonghe Sandra , Tsukamoto Katsura , in ’t Veld Peter , Renström Erik , Schuit Frans C.
Published: March 2007

Cellular redox state is an important metabolic variable, influencing many aspects of cell function like growth, apoptosis, and reductive biosynthesis. In this report, we identify NADPH as a candidate signaling molecule for exocytosis in neuroendocrine cells. In pancreatic β-cells, glucose acutely raised the NADPH-to-NADP+ ratio and stimulated insulin release in parallel. Furthermore, intracellular addition of NADPH directly stimulated exocytosis of insulin granules. Effects of NADPH on exocytosis are proposed to be mediated by the redox proteins glutaredoxin (GRX) and thioredoxin (TRX) on the basis of the following evidence: 1) Expression of GRX mRNA is very high in β-cells compared with other studied tissues, and GRX protein expression is high in islets and in brain; 2) GRX and TRX are localized in distinct microdomains in the cytosol of β-cells; and 3) microinjection of recombinant GRX potentiated effects of NADPH on exocytosis, whereas TRX antagonized the NADPH effect. We propose that the NADPH/GRX/TRX redox regulation mediates a novel signaling pathway of nutrient-induced insulin secretion.
Rosiglitazone Improves Glomerular Hyperfiltration, Renal Endothelial Dysfunction, and Microalbuminuria of Incipient Diabetic Nephropathy in Patients
Authors: Pistrosch Frank , Herbrig Kay , Kindel Beate , Passauer Jens , Fischer Sabine , Gross Peter
Published: March 2007

Microalbuminuria, an early feature of diabetic nephropathy, indicates intrarenal endothelial damage. In type 2 diabetes, microalbuminuria is strongly related to insulin resistance. We therefore investigated whether rosiglitazone, an insulin-sensitizing drug that is known to improve endothelial dysfunction, was able to improve intrarenal endothelial dysfunction and microalbuminuria. Nineteen type 2 diabetic patients participated in this double-blind cross-over trial. Nine patients with newly diagnosed disease without microalbuminuria were randomized to a treatment with rosiglitazone or nateglinide, each for 12 weeks. Ten patients with microalbuminuria were randomized to rosiglitazone or placebo, each for 12 weeks in addition to their previous antidiabetic medication. After each treatment, glomerular filtration rate (GFR), renal plasma flow, and filtration fraction were measured before and after blockade of nitric oxide (NO) by intravenous administration of N-monomethyl-l-arginine-acetate (l-NMMA). Ten healthy subjects served as control subjects. Type 2 diabetic patients at baseline showed glomerular hyperfiltration compared with healthy control subjects. Rosiglitazone reduced elevated GFR and filtration fraction toward control primarily in patients with microalbuminuria (GFR: 133.4 ± 9.8 vs. 119.6 ± 8.7 ml/min; filtration fraction: 23.2 ± 1.7 vs. 20.5 ± 1.6% before and after rosiglitazone, respectively; control subjects: GFR 111.7 ± 8.6 ml/min, filtration fraction 20.4 ± 1.5%). Rosiglitazone improved intrarenal NO bioavailability in type 2 diabetes toward control as shown by infusion of l-NMMA. Rosiglitazone reduced albumin excretion in type 2 diabetes with microalbuminuria from 116.5 ± 31 to 40.4 ± 12 mg/day. Rosiglitazone ameliorated glomerular hyperfiltration in early type 2 diabetes, improved NO bioavailability, and lessened renal end-organ damage in type 2 diabetes with microalbuminuria.
Mechanisms of Compensatory β-Cell Growth in Insulin-Resistant Rats
Authors: Jetton Thomas L. , Lausier James , LaRock Kyla , Trotman Winifred E. , Larmie Brooke , Habibovic Aida , Peshavaria Mina , Leahy Jack L.
Published: March 2007

The physiological mechanisms underlying the compensatory growth of β-cell mass in insulin-resistant states are poorly understood. Using the insulin-resistant Zucker fatty (fa/fa) (ZF) rat and the corresponding Zucker lean control (ZLC) rat, we investigated the factors contributing to the age-/obesity-related enhancement of β-cell mass. A 3.8-fold β-cell mass increase was observed in ZF rats as early as 5 weeks of age, an age that precedes severe insulin resistance by several weeks. Closer investigation showed that ZF rat pups were not born with heightened β-cell mass but developed a modest increase over ZLC rats by 20 days that preceded weight gain or hyperinsulinemia that first developed at 24 days of age. In these ZF pups, an augmented survival potential of β-cells of ZF pups was observed by enhanced activated (phospho-) Akt, phospho-BAD, and Bcl-2 immunoreactivity in the postweaning period. However, increased β-cell proliferation in the ZF rats was only detected at 31 days of age, a period preceding massive β-cell growth. During this phase, we also detected an increase in the numbers of small β-cell clusters among ducts and acini, increased duct pancreatic/duodenal homeobox-1 (PDX-1) immunoreactivity, and an increase in islet number in the ZF rats suggesting duct- and acini-mediated heightened β-cell neogenesis. Interestingly, in young ZF rats, specific cells associated with ducts, acini, and islets exhibited an increased frequency of PDX-1+/phospho-Akt+ staining, indicating a potential role for Akt in β-cell differentiation. Thus, several adaptive mechanisms account for the compensatory growth of β-cells in ZF rats, a combination of enhanced survival and neogenesis with a transient rise in proliferation before 5 weeks of age, with Akt serving as a potential mediator in these processes.
Physiological Increases in Uncoupling Protein 3 Augment Fatty Acid Oxidation and Decrease Reactive Oxygen Species Production Without Uncoupling Respiration in Muscle Cells
Authors: MacLellan J. Darcy , Gerrits Martin F. , Gowing Adrienne , Smith Peter J.S. , Wheeler Michael B. , Harper Mary-Ellen
Published: March 2007

Decreased uncoupling protein (UCP)3 is associated with insulin resistance in muscle of pre-diabetic and diabetic individuals, but the function of UCP3 remains unclear. Our goal was to elucidate mechanisms underlying the negative correlation between UCP3 and insulin resistance in muscle. We determined effects of physiologic UCP3 overexpression on glucose and fatty acid oxidation and on mitochondrial uncoupling and reactive oxygen species (ROS) production in L6 muscle cells. An adenoviral construct caused a 2.2- to 2.5-fold increase in UCP3 protein. Palmitate oxidation was increased in muscle cells incubated under normoglycemic or hyperglycemic conditions, whereas adenoviral green fluorescent protein infection or chronic low doses of the uncoupler dinitrophenol had no effect. Increased UCP3 did not affect glucose oxidation, whereas dinitrophenol and insulin treatments caused increases. Basal oxygen consumption, assessed in situ using self-referencing microelectrodes, was not significantly affected, whereas dinitrophenol caused increases. Mitochondrial membrane potential was decreased by dinitrophenol but was not affected by increased UCP3 expression. Finally, mitochondrial ROS production decreased significantly with increased UCP3 expression. Results are consistent with UCP3 functioning to facilitate fatty acid oxidation and minimize ROS production. As impaired fatty acid metabolism and ROS handling are important precursors in muscular insulin resistance, UCP3 is an important therapeutic target in type 2 diabetes.
Increased p85/55/50 Expression and Decreased Phosphotidylinositol 3-Kinase Activity in Insulin-Resistant Human Skeletal Muscle
Authors: Bandyopadhyay Gautam K. , Yu Joseph G. , Ofrecio Jachelle , Olefsky Jerold M.
Published: March 2007

Insulin resistance is predominantly characterized by decreased insulin-stimulated glucose uptake into skeletal muscle. In the current study, we have assessed various aspects of the phosphatidylinositol (PI) 3-kinase pathway in skeletal muscle biopsies obtained from normal, obese nondiabetic, and type 2 diabetic subjects, before and after a 5-h insulin infusion. We found a highly significant inverse correlation between in vivo insulin sensitivity (as measured by the glucose infusion rate) and increased protein expression of p85/55/50, protein kinase C (PKC)-θ activity, levels of pSer307 insulin receptor substrate (IRS)-1 and p-Jun NH2-terminal kinase (JNK)-1, and myosin heavy chain IIx fibers. Increased basal phosphorylation of Ser307 IRS-1 in the obese and type 2 diabetic subjects corresponds with decrease in insulin-stimulated IRS-1 tyrosine phosphorylation, PI 3-kinase activity, and insulin-induced activation of Akt and, more prominently, PKC-ζ/λ. In summary, increased expression of the PI 3-kinase adaptor subunits p85/55/50, as well as increased activity of the proinflammatory kinases JNK-1, PKC-θ, and, to a lesser extent, inhibitor of κB kinase-β, are associated with increased basal Ser307 IRS-1 phosphorylation and decreased PI 3-kinase activity and may follow a common pathway to attenuate in vivo insulin sensitivity in insulin-resistant subjects. These findings demonstrate interacting mechanisms that can lead to impaired insulin-stimulated PI 3-kinase activity in skeletal muscle from obese and type 2 diabetic subjects.
Preconditioning the Diabetic Heart
Authors: Tsang Andrew , Hausenloy Derek J. , Mocanu Mihaela M. , Carr Richard D. , Yellon Derek M.
Published: March 2007

Conflicting evidence exists whether diabetic myocardium can be protected by ischemic preconditioning (IPC). The phosphatidylinositol 3-kinase (PI3K)-Akt pathway is important in IPC. However, components of this cascade have been found to be defective in diabetes. We hypothesize that IPC in diabetic hearts depends on intact signaling through the PI3K-Akt pathway to reduce myocardial injury. Isolated perfused Wistar (normal) and Goto-Kakizaki (diabetic) rat hearts were subjected to 1) 35 min of regional ischemia and 120 min of reperfusion with infarct size determined; 2) preconditioning (IPC) using 5 min of global ischemia followed by 10 min of reperfusion performed one, two, or three times before prolonged ischemia; or 3) determination of Akt phosphorylation after stabilization or after one and three cycles of IPC. In Wistar rats, one, two, and three cycles of IPC reduced infarct size 44.7 ± 3.8% (P < 0.05), 31.4 ± 4.9% (P < 0.01), and 34.3 ± 6.1% (P < 0.01), respectively, compared with controls (60.7 ± 4.5%). However, in diabetic rats only three cycles of IPC significantly reduced infarction to 20.8 ± 2.6% from 46.6 ± 5.2% in controls (P < 0.01), commensurate with significant Akt phosphorylation after three cycles of IPC. To protect the diabetic myocardium, it appears necessary to increase the IPC stimulus to achieve the threshold for cardioprotection and a critical level of Akt phosphorylation to mediate myocardial protection.
Insulin and Inositol 1,4,5-Trisphosphate Trigger Abnormal Cytosolic Ca2+ Transients and Reveal Mitochondrial Ca2+ Handling Defects in Cardiomyocytes of ob/ob Mice
Authors: Fauconnier Jérémy , Lanner Johanna T. , Zhang Shi-Jin , Tavi Pasi , Bruton Joseph D. , Katz Abram , Westerblad Håkan
Published: March 2007

Obesity, insulin resistance, and type 2 diabetes are leading causes of heart failure, and defective cellular Ca2+ handling seems to be a fundamental problem in diabetes. Therefore, we studied the effect of insulin on Ca2+ homeostasis in normal, freshly isolated mouse ventricular cardiomyocytes and whether Ca2+ handling was changed in an animal model of obesity and type 2 diabetes, ob/ob mice. Electrically evoked Ca2+ transients were smaller and slower in ob/ob compared with wild-type cardiomyocytes. Application of insulin (6 or 60 nmol/l) increased the amplitude of Ca2+ transients in wild-type cells by ∼30%, whereas it broadened the transients and triggered extra Ca2+ transients in ob/ob cells. The effects of insulin in ob/ob cells could be reproduced by application of a membrane-permeant inositol trisphosphate (IP3) analog and blocked by a frequently used IP3 receptor inhibitor, 2-aminoethoxydiphenyl borate. In ob/ob cardiomyocytes, insulin increased the IP3 concentration and mitochondrial Ca2+ handling was impaired. In conclusion, we propose a model where insulin increases IP3 in ob/ob cardiomyocytes, which prolongs the electrically evoked Ca2+ release. This, together with an impaired mitochondrial Ca2+ handling, results in insulin-mediated extra Ca2+ transients in ob/ob cardiomyocytes that may predispose for arrhythmias in vivo.
Platelet Function Profiles in Patients With Type 2 Diabetes and Coronary Artery Disease on Combined Aspirin and Clopidogrel Treatment
Authors: Angiolillo Dominick J. , Fernandez-Ortiz Antonio , Bernardo Esther , Ramírez Celia , Sabaté Manel , Jimenez-Quevedo Pilar , Hernández Rosana , Moreno Raul , Escaned Javier , Alfonso Fernando , Bañuelos Camino , Costa Marco A. , Bass Theodore A. , Macaya Carlos
Published: March 2007

To assess platelet function profiles in diabetic and nondiabetic patients on aspirin and clopidogrel therapy, two patient populations were included to investigate the 1) acute effects of a 300-mg clopidogrel loading dose (group 1, n = 52) and 2) long-term effects of clopidogrel (group 2, n = 120) on platelet function in diabetic compared with nondiabetic patients already on aspirin treatment. Patients were stratified according to the presence of type 2 diabetes. Platelet aggregation was assessed using light transmittance aggregometry (groups 1 and 2). Platelet activation (P-selectin expression and PAC-1 binding) was determined using whole-blood flow cytometry (group 2). Clopidogrel response was also assessed. In group 1, platelet aggregation was significantly increased in diabetic (n = 16) compared with nondiabetic (n = 36) patients at baseline and up to 24 h following a 300-mg loading dose (P = 0.005). In group 2, platelet aggregation and activation were increased in diabetic (n = 60) compared with nondiabetic (n = 60) subjects (P < 0.05 for all platelet function assays). Diabetic subjects had a higher number of clopidogrel nonresponders (P = 0.04). Diabetic patients have increased platelet reactivity compared with nondiabetic subjects on combined aspirin and clopidogrel treatment. Reduced sensitivity to antiplatelet drugs may contribute to the increased atherothombotic risk in diabetic patients.
Therapeutic Roles of Peroxisome Proliferator–Activated Receptor Agonists
Authors: Staels Bart , Fruchart Jean-Charles
Published: March 2007

Peroxisome proliferator–activated receptors (PPARs) play key roles in the regulation of energy homeostasis and inflammation, and agonists of PPARα and -γ are currently used therapeutically. Fibrates, first used in the 1970s for their lipid-modifying properties, were later shown to activate PPARα. These agents lower plasma triglycerides and VLDL particles and increase HDL cholesterol, effects that are associated with cardiovascular benefit. Thiazolidinediones, acting via PPARγ, influence free fatty acid flux and thus reduce insulin resistance and blood glucose levels. PPARγ agonists are therefore used to treat type 2 diabetes. PPARα and -γ agonists also affect inflammation, vascular function, and vascular remodeling. As knowledge of the pleiotropic effects of these agents advances, further potential indications are being revealed, including roles in the management of cardiovascular disease (CVD) and the metabolic syndrome. Dual PPARα/γ agonists (currently in development) look set to combine the properties of thiazolidinediones and fibrates, and they hold considerable promise for improving the management of type 2 diabetes and providing an effective therapeutic option for treating the multifactorial components of CVD and the metabolic syndrome. The functions of a third PPAR isoform, PPARδ, and its potential as a therapeutic target are currently under investigation.
Association Between Common Polymorphisms of the Proopiomelanocortin Gene and Body Fat Distribution
Authors: Baker Michelle , Gaukrodger Nicole , Mayosi Bongani M. , Imrie Helen , Farrall Martin , Watkins Hugh , Connell John M.C. , Avery Peter J. , Keavney Bernard
Published: March 2007

Rare mutations in the proopiomelanocortin (POMC) gene cause severe early-onset childhood obesity. However, it is unknown whether common variants in POMC are responsible for variation in body weight or fat distribution within the commonly observed range in the population. We tested for association between three polymorphisms spanning the POMC gene and obesity phenotypes in 1,428 members of 248 families. There was significant association between genotypes at the C8246T (P < 0.0001) and C1032G (P = 0.003) polymorphisms and waist-to-hip ratio (WHR) corrected for age, sex, smoking, exercise, and alcohol consumption. Each T allele at C8246T (or G allele at C1032G) was associated with a 0.2-SD–higher WHR in a codominant fashion. When WHR was additionally corrected for BMI, thus providing a measure of body fat distribution throughout the range of BMI, there remained significant evidence for association with both markers that was of similar magnitude and statistical significance. There was no association between genotype at any polymorphism and BMI or plasma leptin level. These data show that genetic variants at the POMC locus influence body fat distribution within the normal range, suggesting a novel role for POMC in metabolic regulation.
The X-Linked Inhibitor of Apoptosis Protein Enhances Survival of Murine Islet Allografts
Authors: Plesner Annette , Liston Peter , Tan Rusung , Korneluk Robert G. , Verchere C. Bruce
Published: March 2007

Allotransplantation of pancreatic islets represents a promising approach to treat type 1 diabetes. Destruction of β-cells in islet allografts involves multiple immune mechanisms that lead to activation of caspases and apoptotic cell death. The X-linked inhibitor of apoptosis (XIAP) inhibits apoptosis induced by a variety of triggers, primarily by preventing the activation of caspases. To determine whether XIAP would protect β-cells from apoptosis, we used a recombinant adenovirus to overexpress XIAP in transformed murine β-cells and in freshly isolated islets. In vitro cytokine-induced β-cell death was decreased to baseline levels in XIAP-transduced MIN-6 and NIT-1 cell lines compared with controls. To evaluate the potential of XIAP overexpression to prevent in vivo allogeneic graft rejection, we transduced Balb/c islets ex vivo with XIAP before transplantation into CBA mice with streptozotocin-induced diabetes. We observed that almost all mice receiving allografts of XIAP-expressing islets maintained normoglycemia until the experiment was terminated (45–72 days posttransplant), whereas control mice receiving islets transduced with adenovirus expressing LacZ were hyperglycemic by ∼17 days posttransplantation due to graft rejection. Immunohistochemistry revealed preservation of β-cells and clearance of infiltrating immune cells in the XIAP-expressing islet grafts. The in vitro allogeneic response of splenocytes isolated from recipients of XIAP-expressing grafts 8 weeks posttransplant was similar to that seen in nonprimed allogeneic mice, suggesting that XIAP overexpression may lead to the acceptance of islet allografts in diabetic recipients. Long-term protection of islet allografts by XIAP overexpression may enhance the survival of islet transplants in diabetes.
XIAP Overexpression in Human Islets Prevents Early Posttransplant Apoptosis and Reduces the Islet Mass Needed to Treat Diabetes
Authors: Emamaullee Juliet A. , Rajotte Ray V. , Liston Peter , Korneluk Robert G. , Lakey Jonathan R.T. , Shapiro A.M. James , Elliott John F.
Published: March 2007

The Edmonton Protocol for treatment of type 1 diabetes requires islets from two or more donors to achieve euglycemia in a single recipient, primarily because soon after portal infusion, the majority of the transplanted cells undergo apoptosis due to hypoxia and hypoxia reperfusion injury. X-linked inhibitor of apoptosis protein (XIAP) is a potent endogenous inhibitor of apoptosis that is capable of blocking the activation of multiple downstream caspases, and XIAP overexpression has previously been shown to enhance engraftment of a murine β-cell line. In this study, human islets transduced with a XIAP-expressing recombinant adenovirus were resistant to apoptosis and functionally recovered following in vitro stresses of hypoxia and hypoxia with reoxygenation (models reperfusion injury). Furthermore Ad-XIAP transduction dramatically reduced the number of human islets required to reverse hyperglycemia in chemically diabetic immunodeficient mice. These results suggest that by transiently overexpressing XIAP in the immediate posttransplant period, human islets from a single donor might be used to effectively treat two diabetic recipients.
Interferon-α as a Mediator of Polyinosinic:Polycytidylic Acid–Induced Type 1 Diabetes
Authors: Devendra Devasenan , Jasinski Jean , Melanitou Evie , Nakayama Maki , Li Marcella , Hensley Brooke , Paronen Johanna , Moriyama Hiroaki , Miao Dongmei , Eisenbarth George S. , Liu Edwin
Published: March 2007

A number of studies and clinical case reports have implicated interferon (IFN)-α as a potential mediator of type 1 diabetes pathogenesis. Administration of polyinosinic:polycytidylic acid (poly I:C), a mimic of viral double-stranded RNA, induces diabetes in C57BL/6 mice expressing the B7.1 costimulatory molecule in islets. We investigated the potential role of IFN-α in this disease model. The quantitative correlation between IFN-α levels and time to diabetes, diabetes prevention with anti–IFN-α antibody, and ability of IFN-α itself to induce diabetes are consistent with the hypothesis that poly I:C in this model acts by induction of IFN-α in a genetically susceptible host. Numerous recent studies highlight the importance of the innate immune system and toll receptors in determining adaptive immune responses, and we speculate that for type 1 diabetes, viral and other environmental factors may act through induction of IFNs.
Conditional Expression Demonstrates the Role of the Homeodomain Transcription Factor Pdx1 in Maintenance and Regeneration of β-Cells in the Adult Pancreas
Authors: Holland Andrew M. , Góñez L. Jorge , Naselli Gaetano , MacDonald Raymond J. , Harrison Leonard C.
Published: March 2007

The homeodomain transcription factor Pdx1 is essential for pancreas development. To investigate the role of Pdx1 in the adult pancreas, we employed a mouse model in which transcription of Pdx1 could be reversibly repressed by administration of doxycycline. Repression of Pdx1 in adult mice impaired expression of insulin and glucagon, leading to diabetes within 14 days. Pdx1 repression was associated with increased cell proliferation predominantly in the exocrine pancreas and upregulation of genes implicated in pancreas regeneration. Following withdrawal of doxycycline and derepression of Pdx1, normoglycemia was restored within 28 days; during this period, Pdx1+/Ins+ and Pdx+/Ins− cells were observed in association with the duct epithelia. These findings confirm that Pdx1 is required for β-cell function in the adult pancreas and indicate that in the absence of Pdx1 expression, a regenerative program is initiated with the potential for Pdx1-dependent β-cell neogenesis.
Exposure to Chronic High Glucose Induces β-Cell Apoptosis Through Decreased Interaction of Glucokinase With Mitochondria
Authors: Kim Won-Ho , Lee June Woo , Suh Young Ho , Hong Shin Hee , Choi Joo Sun , Lim Joo Hyun , Song Ji Hyun , Gao Bin , Jung Myeong Ho
Published: March 2007

Chronic hyperglycemia is toxic to pancreatic β-cells, impairing cellular functioning as observed in type 2 diabetes; however, the mechanism underlying β-cell dysfunction and the resulting apoptosis via glucose toxicity are not fully characterized. Here, using MIN6N8 cells, a mouse pancreatic β-cell line, we show that chronic exposure to high glucose increases cell death mediated by Bax oligomerization, cytochrome C release, and caspase-3 activation. During apoptosis, glucokinase (GCK) expression decreases in high-glucose–treated cells, concomitant with a decrease in cellular ATP production and insulin secretion. Moreover, exposure to a chronically high dose of glucose decreases interactions between GCK and mitochondria with an increase in Bax binding to mitochondria and cytochrome C release. These events are prevented by GCK overexpression, and phosphorylation of proapoptotic Bad proteins in GCK-overexpressing cells is prolonged compared with Neo-transfected cells. Similar results are obtained using primary islet cells. Collectively, these data demonstrate that β-cell apoptosis from exposure to chronic high glucose occurs in relation to lowered GCK expression and reduced association with mitochondria. Our results show that this may be one mechanism by which glucose is toxic to β-cells and suggests a novel approach to prevent and treat diabetes by manipulating Bax- and GCK-controlled signaling to promote apoptosis or proliferation.
Overactivation of S6 Kinase 1 as a Cause of Human Insulin Resistance During Increased Amino Acid Availability
Authors: Tremblay Frédéric , Krebs Michael , Dombrowski Luce , Brehm Attila , Bernroider Elisabeth , Roth Erich , Nowotny Peter , Waldhäusl Werner , Marette André , Roden Michael
Published: March 2007

To examine the molecular mechanisms by which plasma amino acid elevation impairs insulin action, we studied seven healthy men twice in random order during infusion of an amino acid mixture or saline (total plasma amino acid ∼6 vs. ∼2 mmol/l). Somatostatin-insulin-glucose clamps created conditions of low peripheral hyperinsulinemia (∼100 pmol/l, 0–180 min) and prandial-like peripheral hyperinsulinemia (∼430 pmol/l, 180–360 min). At low peripheral hyperinsulinemia, endogenous glucose production (EGP) did not change during amino acid infusion but decreased by ∼70% during saline infusion (EGP150–180 min 11 ± 1 vs. 3 ± 1 μmol · kg−1 · min−1, P = 0.001). Prandial-like peripheral hyperinsulinemia completely suppressed EGP during both protocols, whereas whole-body rate of glucose disappearance (Rd) was ∼33% lower during amino acid infusion (Rd 330–360 min 50 ± 4 vs. 75 ± 6 μmol · kg−1 · min−1, P = 0.002) indicating insulin resistance. In skeletal muscle biopsies taken before and after prandial-like peripheral hyperinsulinemia, plasma amino acid elevation markedly increased the ability of insulin to activate S6 kinase 1 compared with saline infusion (∼3.7- vs. ∼1.9-fold over baseline). Furthermore, amino acid infusion increased the inhibitory insulin receptor substrate-1 phosphorylation at Ser312 and Ser636/639 and decreased insulin-induced phosphoinositide 3-kinase activity. However, plasma amino acid elevation failed to reduce insulin-induced Akt/protein kinase B and glycogen synthase kinase 3α phosphorylation. In conclusion, amino acids impair 1) insulin-mediated suppression of glucose production and 2) insulin-stimulated glucose disposal in skeletal muscle. Our results suggest that overactivation of the mammalian target of rapamycin/S6 kinase 1 pathway and inhibitory serine phosphorylation of insulin receptor substrate-1 underlie the impairment of insulin action in amino acid–infused humans.
Increased Dietary Substrate Delivery Alters Hepatic Fatty Acid Recycling in Healthy Men
Authors: Timlin Maureen T. , Barrows Brian R. , Parks Elizabeth J.
Published: March 2007

Sources of fatty acids flowing to the liver may be used for triacylglycerol (TAG) synthesis. Our objective was to quantify contributions of nonesterified fatty acids (NEFAs), de novo lipogenesis, and dietary fatty acids to VLDL-TAG in the fed state after meal feeding in healthy subjects (n = 6). The effect of substrate delivery rate was also determined by comparison with data obtained under a continuous-feeding regimen. A liquid diet was administered by mouth or via feeding tube. Contributions of NEFAs, de novo lipogenesis, and dietary fatty acids to VLDL-TAG were quantified using stable isotopes and gas chromatography-mass spectrometry. Contribution of NEFAs to VLDL-TAG was similar under meal feeding and continuous feeding, although insulin area under the curve (AUC) was greater under meal feeding (1,597 ± 455 vs. 471 ± 484 pmol · h · l−1, P < 0.004). Lipogenesis achieved a higher AUC with meal feeding versus continuous feeding (88.7 ± 84.4 vs. 1.9 ± 19.3 μmol · h · l−1, P = 0.03) supporting greater stimulation of de novo lipogenesis from increased glucose delivery rate. The contribution of dietary fatty acids to VLDL-TAG was also greater with meal feeding. These data demonstrate for the first time in humans the well-coordinated use of fatty acids by the liver during the transition from fasted to fed states and highlight the dominant role of NEFAs for VLDL-TAG synthesis in both states.
Acute Bidirectional Manipulation of Muscle Glucose Uptake by In Vivo Electrotransfer of Constructs Targeting Glucose Transporter Genes
Authors: Cleasby Mark E. , Davey Jonathan R. , Reinten Tracie A. , Graham Michael W. , James David E. , Kraegen Edward W. , Cooney Gregory J.
Published: March 2007

Analysis of conventional germ-line or tissue-specific gene manipulation in vivo is potentially confounded by developmental adaptation of animal physiology. We aimed to adapt the technique of in vivo electrotransfer (IVE) to alter local gene expression in skeletal muscle of rodents as a means of investigating the role of specific proteins in glucose metabolism in vivo. We utilized a square-wave electroporator to induce intracellular electrotransfer of DNA constructs injected into rat or mouse muscles and investigated the downstream effects. In initial studies, expression of green fluorescent protein reporter was induced in 53 ± 10% of muscle fibers peaking at 7 days, and importantly, the electrotransfer procedure itself did not impact upon the expression of stress proteins or our ability to detect a reduction in 2-deoxyglucose tracer uptake by electroporated muscle of high-fat-fed rats during hyperinsulinemic-euglycemic clamp. To demonstrate functional effects of electrotransfer of constructs targeting glucose transporters, we administered vectors encoding GLUT-1 cDNA and GLUT-4 short hairpin RNAs (shRNAs) to rodent muscles. IVE of the GLUT-1 gene resulted in a 57% increase in GLUT-1 protein, accompanied by a proportionate increase in basal 2-deoxyglucose tracer uptake into muscles of starved rats. IVE of vectors expressing two shRNAs for GLUT-4 demonstrated to reduce specific protein expression and 2-deoxyglucose tracer uptake in 3T3-L1 adipocytes into mouse muscle caused a 51% reduction in GLUT-4 protein, associated with attenuated clearance of tracer to muscle after a glucose load. These results confirm that glucose transporter expression is largely rate limiting for glucose uptake in vivo and highlight the utility of IVE for the acute manipulation of muscle gene expression in the study of the role of specific proteins in glucose metabolism.
Changes of Adiponectin Oligomer Composition by Moderate Weight Reduction
Authors: Bobbert Thomas , Rochlitz Helmut , Wegewitz Uta , Akpulat Suzan , Mai Knut , Weickert Martin O. , Möhlig Matthias , Pfeiffer Andreas F.H. , Spranger Joachim
Published: March 2007

Adiponectin affects lipid metabolism and insulin sensitivity. However, adiponectin circulates in three different oligomers that may also have distinct biological functions. We aimed to analyze the role of these oligomers in obesity and lipid metabolism after weight reduction. A total of 17 obese volunteers (15 women and 2 men) participated in a weight reduction program. Individuals were characterized before and after 6 months of a balanced diet. Adiponectin was determined by enzyme-linked immunosorbent assay, and oligomers were detected by nondenaturating Western blot. BMI decreased (35.1 ± 1.2 to 32.8 ± 1.1 kg/m2, P < 0.001), which was associated with an improved metabolite profile. Total adiponectin increased from 5.3 ± 0.5 to 6.1 ± 0.6 μg/ml (P = 0.076). High (HMW) and medium molecular weight (MMW) adiponectin oligomers significantly increased during weight reduction (HMW: 0.37 ± 0.07 to 0.4 ± 0.08 μg/ml, P = 0.042; MMW: 2.3 ± 0.2 to 2.9 ± 0.3 μg/ml, P = 0.007), while low molecular weight (LMW) did not significantly change. Body weight inversely correlated with HMW (r = −0.695, P = 0.002) and positively with LMW (r = 0.579, P = 0.015). Interestingly, HDL cholesterol and HMW were strongly correlated (r = 0.665, P = 0.007). Indeed, HMW and free fatty acids before weight reduction predicted ∼60% of HDL changes during intervention. In conclusion, weight reduction results in a relative increase of HMW/MMW adiponectin and a reduction of LMW adiponectin. Total adiponectin and especially HMW adiponectin are related to circulating HDL cholesterol.
Glucagon-Like Peptide 1 Regulates Sequential and Compound Exocytosis in Pancreatic Islet β-Cells
Authors: Kwan Edwin P. , Gaisano Herbert Y.
Published: March 2007

Glucagon-like peptide 1 (GLP-1) has been postulated to potentiate insulin secretion by cAMP-mediated enhancement of mobilization and priming of secretory granules, but the precise exocytic events are unknown. We used epi-fluorescent microscopy of the fluorescent dye FM1-43, which incorporates into the plasma membrane and the exocytosing secretory granules (appearing as plasma membrane hotspots). KCl evoked exocytosis of 1.8 ± 0.5 hotspots/rat β-cell at the cell periphery, 82% of which are single transient increases of low amplitudes (151 ± 7%), suggesting single secretory granule exocytosis; and the remaining 18% are stepwise increases in plasma membrane hotspots with higher amplitudes (170 ± 9%), suggesting sequential secretory granule to secretory granule exocytic fusions. Addition of GLP-1 increased the hotspots to 6.0 ± 0.7/β-cell and exhibited a larger number of stepwise (41%) than transient (10%) increases with higher amplitudes of 259 ± 19 and 278 ± 23%, respectively. More interestingly, GLP-1 also evoked a robust and sustained pattern (49%) with even higher amplitudes of 354 ± 18%, which are likely accelerated sequential secretory granule–secretory granule fusions. Electron microscopy studies collaborated with these imaging results, showing that GLP-1 increased the number of docked secretory granules at the plasma membrane and also increased the number of events showing direct contact of oncoming secretory granules with secretory granules undergoing exocytosis. We conclude that the potentiation of insulin secretion by GLP-1 is contributed by the mobilization of more insulin secretory granules to dock at the plasma membrane and the acceleration of sequential secretory granule–secretory granule fusions.
Transgenic Mouse Overexpressing Syntaxin-1A as a Diabetes Model
Authors: Lam Patrick P.L. , Leung Yuk-Man , Sheu Laura , Ellis James , Tsushima Robert G. , Osborne Lucy R. , Gaisano Herbert Y.
Published: March 2007

Soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) protein syntaxin-1A (STX-1A) plays a role not only in exocytosis, but also binds and regulates Ca2+ and K+ (voltage-gated K+ and ATP-sensitive K+ channels) to influence the sequence of events leading to secretion. Islet levels of STX-1A and cognate SNARE proteins are reduced in type 2 diabetic rodents, suggesting their role in dysregulated insulin secretion contributing to the abnormal glucose homeostasis. We investigated the specific role of STX-1A in pancreatic β-cells by generating transgenic mice, which express a moderately increased level (∼30% higher) of STX-1A in pancreatic islets (hereafter called STX-1A mice). The STX-1A mice displayed fasting hyperglycemia and a more sustained elevation of plasma glucose levels after an intraperitoneal glucose tolerance test, with correspondingly reduced plasma insulin levels. Surprisingly, β-cells from the STX-1A male mice also exhibited abnormal insulin tolerance. To unequivocally determine the β-cell secretory defects, we used single-cell analyses of exocytosis by patch clamp membrane capacitance measurements and ion channel recordings. Depolarization-evoked membrane capacitance increases were reduced in the STX-1A mouse islet β-cells. The STX-1A mouse also exhibited reduced currents through the Ca2+ channels but little change in the voltage-gated K+ channel or ATP-sensitive K+ channel. These results suggest that fluctuation of islet STX-1A levels in diabetes could influence the pathological and differential regulation of β-cell ion channels and the exocytotic machinery, collectively contributing to the impaired insulin secretion.
Chronic Hyperglycemia, Independent of Plasma Lipid Levels, Is Sufficient for the Loss of β-Cell Differentiation and Secretory Function in the db/db Mouse Model of Diabetes
Authors: Kjørholt Cecilie , Åkerfeldt Mia C. , Biden Trevor J. , Laybutt D. Ross
Published: March 2007

The β-cell is a highly specialized cell with a unique differentiation that optimizes glucose-induced insulin secretion (GIIS). Here, we evaluated changes in gene expression that accompany β-cell dysfunction in the db/db mouse model of type 2 diabetes. In db/db islets, mRNA levels of many genes implicated in β-cell glucose sensing were progressively reduced with time, as were several transcription factors important for the maintenance of β-cell differentiation. Conversely, genes normally suppressed in β-cells, such as a variety of stress response mediators and inhibitor of differentiation/DNA binding 1, a gene capable of inhibiting differentiation, were markedly increased. We assessed whether this global alteration in the pattern of β-cell gene expression was related more to chronic hyperglycemia or hyperlipidemia; db/db mice were treated with phlorizin, which selectively lowered plasma glucose, or bezafibrate, which selectively lowered plasma lipids. GIIS as well as the majority of the changes in gene expression were completely normalized by lowering glucose but were unaffected by lowering lipids. However, the restoration of GIIS was not accompanied by normalized uncoupling protein 2 or peroxisome proliferator–activated receptor γ mRNA levels, which were upregulated in db/db islets. These studies demonstrate that hyperglycemia, independent of plasma lipid levels, is sufficient for the loss of β-cell differentiation and secretory function in db/db mice.
Activation of Peripheral Blood CD14+ Monocytes Occurs in Diabetes
Authors: Cipolletta Christine , Ryan Kathryn E. , Hanna Elinor V. , Trimble Elisabeth R.
Published: March 2007

Blood levels of inflammatory markers associated with endothelial dysfunction and atherosclerosis are increased in diabetic patients; the highest levels occur in poorly controlled diabetes. We investigated the activation state of peripheral blood monocytes in diabetes with respect to scavenger receptor (CD36) expression and monocyte chemoattractant protein-1, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and peroxisome proliferator–activated receptors mRNA expression. CD14+ monocytes were isolated from peripheral blood of type 1 and type 2 diabetic patients with good (HbA1c <7.0%) or poor (>9.4%) glycemic control and a group of nondiabetic subjects. Monocytes from diabetic subjects displayed increased CD36 cell surface expression (P < 0.0005) and increased uptake of oxidized LDL (P < 0.05). Monocyte chemoattractant protein-1 gene expression was increased in monocytes from both groups of diabetic subjects (P < 0.05). Both CD68 and peroxisome proliferator–activated receptor-γ gene expression were increased in the poorly controlled diabetic group (P < 0.05 for each), whose monocytes also displayed increased attachment to endothelial monolayers (P < 0.0005 vs. nondiabetic control subjects). In poorly controlled diabetes, CD14+ monocytes are functionally activated and show some of the differentiation markers associated with macrophages. These monocytes also demonstrate an increased ability for attachment to normal endothelial cells, one of the early stages in atherogenesis.
Interleukin-6 and Diabetes
Authors: Kristiansen Ole P. , Mandrup-Poulsen Thomas
Published: March 2007

Inflammatory mechanisms play a key role in the pathogenesis of type 1 diabetes. Individuals who progress to type 2 diabetes display features of low-grade inflammation years in advance of disease onset. This low-grade inflammation has been proposed to be involved in the pathogenetic processes causing type 2 diabetes. Mediators of inflammation such as tumor necrosis factor-α, interleukin (IL)-1β, the IL-6 family of cytokines, IL-18, and certain chemokines have been proposed to be involved in the events causing both forms of diabetes. IL-6 has in addition to its immunoregulatory actions been proposed to affect glucose homeostasis and metabolism directly and indirectly by action on skeletal muscle cells, adipocytes, hepatocytes, pancreatic β-cells, and neuroendocrine cells. Here we argue that IL-6 action—in part regulated by variance in the IL-6 and IL-6α receptor genes—contributes to, but is probably neither necessary nor sufficient for, the development of both type 1 and type 2 diabetes. Thus, the two types of diabetes are also in this respect less apart than apparent. However, the mechanisms are not clear, and we therefore propose future directions for studies in this field.
Psammomys Obesus, a Model for Environment-Gene Interactions in Type 2 Diabetes
Authors: Kaiser Nurit , Nesher Rafael , Donath Marc Y. , Fraenkel Merav , Behar Vered , Magnan Christophe , Ktorza Alain , Cerasi Erol , Leibowitz Gil
Published: March 2007

Type 2 diabetes is characterized by insulin resistance and progressive β-cell failure. Deficient insulin secretion, with increased proportions of insulin precursor molecules, is a common feature of type 2 diabetes; this could result from inappropriate β-cell function and/or reduced β-cell mass. Most studies using tissues from diabetic patients are retrospective, providing only limited information on the relative contribution of β-cell dysfunction versus decreased β-cell mass to the “β-cell failure” of type 2 diabetes. The gerbil Psammomys obesus is a good model to address questions related to the role of insulin resistance and β-cell failure in nutritionally induced diabetes. Upon a change from its natural low-calorie diet to the calorie-rich laboratory food, P. obesus develops moderate obesity associated with postprandial hyperglycemia. Continued dietary load, superimposed on its innate insulin resistance, results in depletion of pancreatic insulin stores, with increased proportions of insulin precursor molecules in the pancreas and the blood. Inadequate response of the preproinsulin gene to the increased insulin needs is an important cause of diabetes progression. Changes in β-cell mass do not correlate with pancreatic insulin stores and are unlikely to play a role in disease initiation and progression. The major culprit is the inappropriate insulin production with depletion of insulin stores as a consequence. Similar mechanisms could operate during the evolution of type 2 diabetes in humans.
Characteristics of Autoimmunity in Type 1 Diabetes and Type 1.5 Overlap With Type 2 Diabetes
Authors: McDevitt Hugh O.
Published: March 2007

This presentation is an overview of mechanisms for developing and maintaining self-tolerance in mammalian organisms. Because this meeting is focused on type 1 diabetes and its mechanisms, the discussion deals primarily with mechanisms of T-cell tolerance, since type 1 diabetes in both effector and initiator phases is primarily a T-cell–mediated autoimmune disease. Emphasis is placed on more recently discovered mechanisms of maintaining self-tolerance (autoimmune regulator [AIRE]) and a new defect in T-cell negative selection. The emerging picture is that of a polygenic disease with various combinations of different alleles of many genes with important roles in the normal immune response or normal immune responses.
Type 1 and Type 2 Diabetes
Authors: Tuomi Tiinamaija
Published: March 2007

Type 1 and type 2 diabetes frequently co-occur in the same families, suggesting common genetic susceptibility. Such mixed family history is associated with an intermediate phenotype of diabetes: insulin resistance and cardiovascular complications in type 1 diabetic patients and lower BMI and less cardiovascular complications as well as lower C-peptide concentrations in type 2 diabetic patients. GAD antibody positivity is more common in type 2 diabetic patients from mixed families than from common type 2 diabetes families. The mixed family history is associated with more type 1–like genetic (HLA and insulin gene) and phenotypic characteristics in type 2 diabetic patients, especially in the GAD antibody–positive subgroup. Leaving out the extreme ends of diabetes phenotypes, young children progressing rapidly to total insulin deficiency and strongly insulin-resistant subjects mostly with non-Europid ethnic origin, a large proportion of diabetic patients may have both type 1 and type 2 processes contributing to their diabetic phenotype.
Dense Core Vesicle Proteins IA-2 and IA-2β
Authors: Kubosaki Atsutaka , Nakamura Shinichiro , Notkins Abner Louis
Published: March 2007

IA-2 and IA-2β are members of the transmembrane protein tyrosine phosphatase family located in dense core vesicles of neuroendocrine cells, including the β-cells of pancreatic islets. In the present study, by mating C57BL/6Nci IA-2+/− with IA-2β+/− mice, we generated double knockout mice (IA-2−/−/IA-2β−/−) to study the effect of the combined deletion of these two proteins on insulin secretion and blood glucose levels. The double knockout mice appeared healthy at birth and showed normal growth and development. Histological examination and immunostaining for insulin, glucagon, somatostatin, and pancreatic polypeptide revealed no difference between the double knockout and wild-type mice. Nonfasting blood glucose and insulin levels also were within the normal range. However, compared with the wild-type mice, the double knockout mice showed glucose intolerance and an absent first-phase insulin release curve. No evidence of insulin resistance was observed nor were there alterations in fasting blood glucose, insulin, or leptin levels in the double knockout mice maintained on a high-fat diet compared with the wild-type mice maintained on the same diet. In addition, to determine whether the combined deletion of IA-2 and IA-2β played any role in the development of diabetes in NOD mice, we generated double knockout mice on the NOD/LtJ background. The incidence of diabetes in these mice was not significantly different than that in the wild-type mice. Taken together, our experiments show that the dense core vesicle proteins IA-2 and IA-2β, alone or in combination, are involved in insulin secretion, but neither alone nor in combination are they required for the development of diabetes in NOD mice.