bluuebunny/crossref_metadata_2025 huggingface dataset:
Normalization of Skeletal Muscle Glycogen Synthesis and Glycolysis in Rosiglitazone-Treated Zucker Fatty Rats
Authors: Jucker Beat M. , Schaeffer Thomas R. , Haimbach Robin E. , McIntosh Thomas S. , Chun Daniel , Mayer Matthew , Ohlstein Derek H. , Davis Hugh M. , Smith Stephen A. , Cobitz Alexander R. , Sarkar Susanta K.
Published: March 2007

The aim of this study was to characterize insulin-stimulated skeletal muscle glucose metabolism in Zucker fatty rats and to provide insight into the therapeutic mechanism by which rosiglitazone increases insulin-stimulated glucose disposal in these rats. Metabolic parameters were measured using combined in vivo 13C nuclear magnetic resonance (NMR) spectroscopy to measure skeletal muscle glucose uptake and its distributed fluxes (glycogen synthesis and glycolysis), and 31P NMR was used to measure simultaneous changes in glucose-6-phosphate (G-6-P) during a euglycemic-hyperinsulinemic clamp in awake Zucker fatty rats. Three groups of Zucker fatty rats (fatty rosiglitazone [FRSG], fatty control [FC], lean control [LC]) were treated for 7 days before the experiment (3 mg/kg rosiglitazone or vehicle via oral gavage). Rates of glycolysis and glycogen synthesis were assessed after treatment by monitoring 1,6-13C2 glucose label incorporation into 1-13C glycogen, 3-13C lactate, and 3-13C alanine during a euglycemic (∼7–8 mmol/l)-hyperinsulinemic (10 mU · kg−1 · min−1) clamp. The FRSG group exhibited a significant increase in insulin sensitivity, reflected by an increased whole-body glucose disposal rate during the clamp (24.4 ± 1.9 vs. 17.6 ± 1.4 and 33.2 ± 2.0 mg · kg−1 · min−1 in FRSG vs. FC [P < 0.05] and LC [P < 0.01] groups, respectively). The increased insulin-stimulated glucose disposal in the FRSG group was associated with a normalization of the glycolytic flux (52.9 ± 9.1) to LC (56.2 ± 16.6) versus FC (18.8 ± 8.6 nmol · g−1 · min−1, P < 0.02) and glycogen synthesis flux (56.3 ± 11.5) to LC (75.2 ± 15.3) versus FC (16.6 ± 12.8 nmol · g−1 · min−1, P < 0.05). [G-6-P] increased in the FRSG and LC groups versus baseline during the clamp (13.0 ± 11.1 and 16.9 ± 5.8%, respectively), whereas [G-6-P] in the FC group decreased (−23.3 ± 13.4%, P < 0.05). There were no differences between groups in intramyocellular glucose, as measured by biochemical assay. These data suggest that the increased insulin-stimulated glucose disposal in muscle after rosiglitazone treatment can be attributed to a normalization of glucose transport and metabolism.
Pancreatic Islet Transplantation Using the Nonhuman Primate (Rhesus) Model Predicts That the Portal Vein Is Superior to the Celiac Artery as the Islet Infusion Site
Authors: Hirshberg Boaz , Montgomery Sean , Wysoki Michael G. , Xu He , Tadaki Doug , Lee Janet , Hines Kenneth , Gaglia Jason , Patterson Noelle , Leconte John , Hale Douglas , Chang Richard , Kirk Alan D. , Harlan David M.
Published: March 2007

We’ve established a nonhuman primate islet allotransplant model to address questions such as whether transplanting islets into the gut’s arterial system would more safely and as effectively support long-term islet allograft survival compared with the traditional portal vein approach. We reasoned that islets make up <2% of pancreatic cell mass but consume an estimated 20% of arterial blood flow, suggesting an advantage for the arterial site. Access to the arterial system is also easier and safer than the portal system. Pancreatectomized rhesus macaques were transplanted with allogeneic islets infused into either the portal vein (n = 6) or the celiac artery (n = 4). To prevent rejection, primates were given daclizumab, tacrolimus, and rapamycin. In five of six portal vein experiments, animals achieved normoglycemia without exogenous insulin. In contrast, none of the animals given intra-arterial islets showed even transient insulin independence (P = 0.048). Two of the latter animals received a second islet transplant, this time to the portal system, and both achieved insulin independence. Thus, intraportal islet transplantation under conventional immunosuppression is feasible in primates and can result in long-term insulin independence when adequate immunosuppression is maintained. Arterial islet injection, however, does not appear to be a viable islet transplantation technique.
Elevated Spinal Cyclooxygenase and Prostaglandin Release During Hyperalgesia in Diabetic Rats
Authors: Freshwater Jason D. , Svensson Camilla I. , Malmberg Annika B. , Calcutt Nigel A.
Published: March 2007

Diabetic rats display exaggerated hyperalgesic behavior in response to noxious stimuli that may model aspects of painful diabetic neuropathy. This study examined the contribution of spinal prostaglandin production to this exaggerated hyperalgesic behavior. Rats were implanted with spinal dialysis probes and received noxious stimulation to the hind paw by subcutaneous injection of 0.5% formalin solution. Prostaglandin E2 (PGE2) was measured in dialysates of lumbar spinal cerebrospinal fluid concurrent with behavioral responses to formalin injection. In separate experiments, formalin-evoked behavioral responses were measured after intrathecal delivery of either a cyclooxygenase inhibitor or an EP1 receptor antagonist, and cyclooxygenase protein was measured in spinal cord homogenates. Diabetic rats exhibited exaggerated behavioral responses to paw formalin injection and a concurrent prolongation of formalin-evoked PGE2 release. Formalin-evoked behavioral responses were dose-dependently reduced in diabetic rats by spinal delivery of a cyclooxygenase inhibitor or an EP1 receptor antagonist. Protein levels of cyclooxygenase-2 were elevated in the spinal cord of diabetic rats, whereas cyclooxygenase-1 protein was reduced. Hyperalgesic behavior in diabetic rats is associated with both increased cyclooxygenase-2 protein and cyclooxygenase-mediated PGE2 release. Spinal delivery of selective inhibitors of cyclooxygenase-2 or antagonists of prostaglandin receptors may have therapeutic potential for treating painful diabetic neuropathy.
Hyperketonemia Increases Tumor Necrosis Factor-α Secretion in Cultured U937 Monocytes and Type 1 Diabetic Patients and Is Apparently Mediated by Oxidative Stress and cAMP Deficiency
Authors: Jain Sushil K. , Kannan Krishnaswamy , Lim Gideon , McVie Robert , Bocchini Joseph A.
Published: March 2007

An elevated blood level of tumor necrosis factor (TNF)-α is a validated marker of vascular inflammation, which can result in the development of vascular disease and atherosclerosis. This study examined the hypothesis that ketosis increases the TNF-α secretion, both in a cell culture model using U937 monocytes and in type 1 diabetic patients in vivo. U937 cells were cultured with ketone bodies (acetoacetate [AA] and β-hydroxybutyrate [BHB]) in the presence or absence of high levels of glucose in medium at 37°C for 24 h. This study demonstrates the following points. First, hyperketonemic diabetic patients have significantly higher levels of TNF-α than normoketonemic diabetic patients (P < 0.01) and normal control subjects (P < 0.01). There was a significant correlation (r = 0.36, P < 0.05; n = 34) between ketosis and oxidative stress as well as between oxidative stress and TNF-α levels (r = 0.47, P < 0.02; n = 34) in the blood of diabetic patients. Second, ketone body AA treatment increases TNF-α secretion, increases oxygen radicals production, and lowers cAMP levels in U937 cells. However, BHB did not have any effect on TNF-α secretion or oxygen radicals production in U937 cells. Third, exogenous addition of dibutyryl cAMP, endogenous stimulation of cAMP production by forskolin, and antioxidant N-acetylcysteine (NAC) prevented stimulation of TNF-α secretion caused by AA alone or with high glucose. Similarly, NAC prevented the elevation of TNF-α secretion and lowering of cAMP levels in H2O2-treated U937 cells. Fourth, the effect of AA on TNF-α secretion was inhibited by specific inhibitors of protein kinase A (H89), p38-mitogen-activated protein kinase (SB203580), and nuclear transcription factor (NF)κB (NFκB-SN50). This study demonstrates that hyperketonemia increases TNF-α secretion in cultured U937 monocytic cells and TNF-α levels in the blood of type 1 diabetic patients and is apparently mediated by AA-induced cellular oxidative stress and cAMP deficiency.
Association of Adiponectin Mutation With Type 2 Diabetes
Authors: Kondo Hidehiko , Shimomura Iichiro , Matsukawa Yuko , Kumada Masahiro , Takahashi Masahiko , Matsuda Morihiro , Ouchi Noriyuki , Kihara Shinji , Kawamoto Toshiharu , Sumitsuji Satoru , Funahashi Tohru , Matsuzawa Yuji
Published: March 2007

Adiponectin, also referred to as AdipoQ or ACRP30, is a plasma protein produced and secreted exclusively from adipose tissue. The protein contains a collagen-like domain and a C1q-like globular domain. A protease-generated globular segment enhances fatty acid oxidation in muscles, thereby modulating lipid and glucose metabolism. Plasma adiponectin levels are inversely correlated with the severity of insulin resistance. A recent genome-wide scan study mapped a susceptibility locus for type 2 diabetes and the metabolic syndrome to chromosome 3q27, where the adiponectin gene is located. Here, we screened Japanese patients with type 2 diabetes and age- and BMI-matched nondiabetic control subjects for mutations in adiponectin gene. We identified four missense mutations (R112C, I164T, R221S, and H241P) in the globular domain. Among these mutations, the frequency of I164T mutation was significantly higher in type 2 diabetic patients than in age- and BMI- matched control subjects (P < 0.01). Furthermore, plasma adiponectin concentrations of subjects carrying I164T mutation were lower than those of subjects without the mutation. All the subjects carrying I164T mutation showed some feature of metabolic syndrome, including hypertension, hyperlipidemia, diabetes, and atherosclerosis. Our findings suggest that I164T mutation is associated with low plasma adiponectin concentration and type 2 diabetes.
Protein Tyrosine Phosphatase 1B Reduction Regulates Adiposity and Expression of Genes Involved in Lipogenesis
Authors: Rondinone Cristina M. , Trevillyan James M. , Clampit Jill , Gum Rebecca J. , Berg Cathy , Kroeger Paul , Frost Leigh , Zinker Bradley A. , Reilly Regina , Ulrich Roger , Butler Madeline , Monia Brett P. , Jirousek Michael R. , Waring Jeffrey F.
Published: March 2007

Protein tyrosine phosphatase 1B (PTP1B) has been implicated as a negative regulator of insulin action. Overexpression of PTP1B protein has been observed in insulin-resistant states associated with obesity. Mice lacking a functional PTP1B gene exhibit increased insulin sensitivity and are resistant to weight gain. To investigate the role of PTP1B in adipose tissue from obese animals, hyperglycemic obese (ob/ob) mice were treated with PTP1B antisense oligonucleotide (ISIS-113715). A significant reduction in adiposity correlated with a decrease of PTP1B protein levels in fat. Antisense treatment also influenced the triglyceride content in adipocytes, correlating with a downregulation of genes encoding proteins involved in lipogenesis, such as sterol regulatory element-binding protein 1 and their downstream targets spot14 and fatty acid synthase, as well as other adipogenic genes, lipoprotein lipase, and peroxisome proliferator-activated receptor γ. In addition, an increase in insulin receptor substrate-2 protein and a differential regulation of the phosphatidylinositol 3-kinase regulatory subunit (p85α) isoforms expression were found in fat from antisense-treated animals, although increased insulin sensitivity measured by protein kinase B phosphorylation was not observed. These results demonstrate that PTP1B antisense treatment can modulate fat storage and lipogenesis in adipose tissue and might implicate PTP1B in the enlargement of adipocyte energy stores and development of obesity.
Reduced Expression of Th1-Associated Chemokine Receptors on Peripheral Blood Lymphocytes at Diagnosis of Type 1 Diabetes
Authors: Lohmann T. , Laue S. , Nietzschmann U. , Kapellen T.M. , Lehmann I. , Schroeder S. , Paschke R. , Kiess W.
Published: March 2007

We investigated the expression of Th1- and Th2-associated chemokine receptors on peripheral blood lymphocytes at diagnosis and in the first phase of type 1 diabetes. Peripheral blood mononuclear cells (PBMCs) of 25 patients with newly diagnosed type 1 diabetes, 10 patients with longstanding type 1 diabetes, and 35 healthy control subjects were examined for expression of the chemokine receptors CXCR4 (naive T-cells), CCR5 and CXCR3 (Th1 associated), and CCR3 and CCR4 (Th2 associated) on CD3+ lymphocytes. Furthermore, we analyzed chemokine serum levels (monocyte chemoattractant protein [MCP]-1, macrophage inflammatory protein [MIP]-1α, MIP-1β, and RANTES [regulated on activation, normal T-cell expressed and secreted]) and phytohemagglutinin (PHA)-stimulated cytokine secretion of Th1- (γ-interferon [IFN-γ] and tumor necrosis factor-α [TNF-α]) and Th2 (interleukin [IL]-4 and -10)-associated cytokines by PBMC. The patients with newly diagnosed type 1 diabetes were followed for these parameters at 6–12 months after diagnosis. The PBMCs of patients with newly diagnosed but not with longstanding type 1 diabetes showed reduced expression of the Th1-associated chemokine receptors CCR5 (P < 0.001 vs. control subjects) and CXCR3 (P < 0.002 vs. control subjects). This reduction correlated with reduced IFN-γ and TNF-α production of PBMCs after PHA stimulation and reversed 6–12 months after diagnosis to normal levels. CCR4 cells were reduced in both newly diagnosed and longstanding type 1 diabetic patients, which correlated to reduced PHA-stimulated IL-4 production. MIP-1α and MIP-1β levels were considerably elevated in a subgroup of patients with newly diagnosed diabetes. We assume that Th1-associated peripheral T-cells are reduced in a narrow time window at the time of diagnosis of diabetes, possibly due to extravasation in the inflamed pancreas. Thus, chemokine receptor expression of peripheral blood lymphocytes may be a useful surrogate marker for the immune activity of type 1 diabetes (e.g., in intervention trials).
Phagocytosis of Apoptotic Cells by Macrophages From NOD Mice Is Reduced
Authors: O’Brien Bronwyn A. , Huang Yongqian , Geng Xuan , Dutz Jan P. , Finegood Diane T.
Published: March 2007

Macrophages limit inflammatory responses by clearing apoptotic cells. Deficiencies in apoptotic cell phagocytosis have been linked to autoimmunity. In this study, we determined the efficiency with which macrophages from diabetes-prone NOD and diabetes-resistant NOR, Idd5, Balb/c, and C57BL/6 mice phagocytose apoptotic thymocytes and NIT-1 insulinoma cells. Peritoneal and bone marrow-derived macrophages from NOD mice engulfed fewer apoptotic thymocytes than macrophages from Balb/c mice (P < 0.05). Peritoneal macrophages from NOR and Idd5 NOD congenic mice were more proficient at engulfment than their NOD counterparts. Annexin V blockade diminished apoptotic thymocyte clearance and heat-labile serum factors augmented clearance. Binding of apoptotic thymocytes to NOD macrophages was also reduced, suggesting that the deficiency in phagocytosis may be partly attributable to a recognition defect. Peritoneal macrophages from female Balb/c and NOD mice were equally efficient in the engulfment of microspheres, suggesting that the phagocytic deficiency observed in NOD mice was specific for apoptotic cells. In summary, we have demonstrated a deficiency in phagocytic function of macrophages from NOD mice. Normal and diabetes-prone neonatal rodents have a wave of β-cell apoptosis coincident with the onset of target organ inflammation. A constitutive defect in the clearance of apoptotic β-cells may be contributory to the initiation of autoimmunity.
Succinate Is a Preferential Metabolic Stimulus-Coupling Signal for Glucose-Induced Proinsulin Biosynthesis Translation
Authors: Alarcon Cristina , Wicksteed Barton , Prentki Marc , Corkey Barbara E. , Rhodes Christopher J.
Published: March 2007

The secondary signals emanating from increased glucose metabolism, which lead to specific increases in proinsulin biosynthesis translation, remain elusive. It is known that signals for glucose-stimulated insulin secretion and proinsulin biosynthesis diverge downstream of glycolysis. Consequently, the mitochondrial products ATP, Krebs cycle intermediates, glutamate, and acetoacetate were investigated as candidate stimulus-coupling signals specific for glucose-induced proinsulin biosynthesis in rat islets. Decreasing ATP levels by oxidative phosphorylation inhibitors showed comparable effects on proinsulin biosynthesis and total protein synthesis. Although it is a cofactor, ATP is unlikely to be a metabolic stimulus-coupling signal specific for glucose-induced proinsulin biosynthesis. Neither glutamic acid methyl ester nor acetoacetic acid methyl ester showed a specific effect on glucose-stimulated proinsulin biosynthesis. Interestingly, among Krebs cycle intermediates, only succinic acid monomethyl ester specifically stimulated proinsulin biosynthesis. Malonic acid methyl ester, an inhibitor of succinate dehydrogenase, also specifically increased glucose-induced proinsulin biosynthesis without affecting islet ATP levels or insulin secretion. Glucose caused a 40% increase in islet intracellular succinate levels, but malonic acid methyl ester showed no further effect, probably due to efficient conversion of succinate to succinyl-CoA. In this regard, a GTP-dependent succinyl-CoA synthetase activity was found in cytosolic fractions of pancreatic islets. Thus, succinate and/or succinyl-CoA appear to be preferential metabolic stimulus-coupling factors for glucose-induced proinsulin biosynthesis translation.
Ouabain Suppresses Glucose-Induced Mitochondrial ATP Production and Insulin Release by Generating Reactive Oxygen Species in Pancreatic Islets
Authors: Kajikawa Mariko , Fujimoto Shimpei , Tsuura Yoshiyuki , Mukai Eri , Takeda Tomomi , Hamamoto Yoshiyuki , Takehiro Mihoko , Fujita Jun , Yamada Yuichiro , Seino Yutaka
Published: March 2007

We examined the effects of reduced Na+/K+-ATPase activity on mitochondrial ATP production and insulin release from rat islets. Ouabain, an inhibitor of Na+/K+-ATPase, augmented 16.7 mmol/l glucose–induced insulin release in the early period but suppressed it after a delay of 20–30 min. Unexpectedly, the ATP content in an islet decreases in the presence of 16.7 mmol/l glucose when Na+/K+-ATPase activity is diminished by ouabain, despite the reduced consumption of ATP by the enzyme. Ouabain also suppressed the increment of ATP content produced by glucose even in Ca2+-depleted or Na+-depleted conditions. That mitochondrial membrane hyperpolarization and O2 consumption in islets exposed to 16.7 mmol/l glucose were suppressed by ouabain indicates that the glycoside inhibits mitochondrial respiration but does not produce uncoupling. Ouabain induced mitochondrial reactive oxygen species (ROS) production that was blocked by myxothiazol, an inhibitor of site III of the mitochondrial respiratory chain. An antioxidant, α-tocopherol, also blocked ouabain-induced ROS production as well as the suppressive effect of ouabain on ATP production and insulin release. However, ouabain did not directly affect the mitochondrial ATP production originating from succinate and ADP. These results indicate that ouabain suppresses mitochondrial ATP production by generating ROS via transduction, independently of the intracellular cationic alternation that may account in part for the suppressive effect on insulin secretion.
Preservation of Human Islet Cell Functional Mass by Anti-Oxidative Action of a Novel SOD Mimic Compound
Authors: Bottino Rita , Balamurugan A.N. , Bertera Suzanne , Pietropaolo Massimo , Trucco Massimo , Piganelli Jon D.
Published: March 2007

The most commonly used technical approach to isolate human pancreatic islets intended for allotransplants generates a product that is hampered by mechanical and chemical insults, which dramatically reduce the mass of viable and functional transplantable cells. We tested a novel class of antioxidant chemical compounds (SOD mimics: AEOL10113 and AEOL10150) to protect human islets from oxidative stress in order to improve the preservation of the isolated tissue. Addition of SOD mimic in culture, after isolation, allowed for the survival of a significantly higher islet cell mass. Functional behavior and phenotypic cell characteristics of the SOD-treated islet preparations were preserved, as was the capacity to normalize diabetic mice, even when a marginal mass of islets was transplanted. The addition of SOD mimic during isolation, before culture, further reduced early cell loss. These results indicate that prompt interventions aimed at blocking oxidative stress can improve human islet survival, preserving a functional islet mass two- to threefold larger than the one usually obtained without adding any antioxidant compound. The ability to preserve functional islets without a dramatic loss represents a major advantage considering the scarce availability of islet tissue for clinical transplantation.
Maternal First-Trimester Enterovirus Infection and Future Risk of Type 1 Diabetes in the Exposed Fetus
Authors: Viskari Hanna R. , Roivainen Merja , Reunanen Antti , Pitkäniemi Janne , Sadeharju Karita , Koskela Pentti , Hovi Tapani , Leinikki Pauli , Vilja Pekka , Tuomilehto Jaakko , Hyöty Heikki
Published: March 2007

Previous studies have suggested that enterovirus infections during pregnancy may increase the risk of type 1 diabetes in the offspring. Our aim was to evaluate the role of first trimester enterovirus infections in a larger cohort of pregnant women. Two series of pregnant women were analyzed as follows: 948 women (series 1) and 680 women (series 2) whose child developed clinical diabetes before the ages of 15 or 7 years, respectively. An equal number of control women with a nondiabetic child was selected. Acute enterovirus infections were diagnosed by measuring IgM class antibodies against coxsackievirus B5 (series 1) and a mixture of coxsackievirus B3, coxsackievirus A16, and echovirus 11 antigens (series 2). In series 2, all sera were also analyzed for IgG class antibodies against an enterovirus peptide antigen. In addition, 152 randomly selected case-control pairs and all IgM-positive mothers’ sera were tested for enterovirus RNA (series 2). In series 1, 3.1% of case women had IgM antibodies against coxsackievirus B5 antigen compared with 4.1% of control women (NS). In series 2, 7.1% of case and 5.3% of control women had IgM against the mixture of enterovirus antigens (NS). IgG class enterovirus antibodies did not differ between the groups. Enterovirus RNA was found only in one case woman (0.3%) of the subgroup of samples and in 5.7% of 70 IgM-positive women. The results suggest that enterovirus infection during the first trimester of pregnancy is not associated with increased risk for type 1 diabetes in the child.
Association of the Pro12Ala Polymorphism in the PPAR-γ2 Gene With 3-Year Incidence of Type 2 Diabetes and Body Weight Change in the Finnish Diabetes Prevention Study
Authors: Lindi Virpi I. , Uusitupa Matti I.J. , Lindström Jaana , Louheranta Anne , Eriksson Johan G. , Valle Timo T. , Hämäläinen Helena , Ilanne-Parikka Pirjo , Keinänen-Kiukaanniemi Sirkka , Laakso Markku , Tuomilehto Jaakko ,   for the Finnish Diabetes Prevention Study Group
Published: March 2007

The association of the Pro12Ala polymorphism of the PPAR-γ2 gene with the incidence of type 2 diabetes was investigated in 522 subjects with impaired glucose tolerance (IGT) participating in the Finnish Diabetes Prevention Study. Subjects were randomized to either an intensive diet and exercise group or a control group. By 3 years of intervention, the odds ratio of the development of type 2 diabetes for subjects with the Ala12 allele was 2.11-fold compared with that for subjects with the Pro12Pro genotype (95% CI 1.20–3.72). The risk for type 2 diabetes increased also in subjects who gained weight or belonged to the control group. In the intervention group, subjects with the Ala12Ala genotype lost more weight during the follow-up than subjects with other genotypes (Pro12Pro vs. Ala12Ala P = 0.043), and none of subjects with the Ala12Ala genotype developed type 2 diabetes in this group. In conclusion, the Ala12 allele may predispose to the development of type 2 diabetes in obese subjects with IGT. However, beneficial changes in diet, increases in physical activity, and weight loss may reverse, to some extent, the diabetogenic impact of the Ala12 allele, possibly due to an improved insulin sensitivity.
Transplantation of Adipose Tissue Lacking Leptin Is Unable to Reverse the Metabolic Abnormalities Associated With Lipoatrophy
Authors: Colombo Carlo , Cutson Jaime J. , Yamauchi Toshimasa , Vinson Charles , Kadowaki Takashi , Gavrilova Oksana , Reitman Marc L.
Published: March 2007

Severe adipose tissue deficiency (lipoatrophy) causes insulin-resistant diabetes, elevated serum triglyceride and fatty acid levels, and massive triglyceride deposition in the liver. In lipoatrophic A-ZIP/F-1 mice, transplantation of normal adipose tissue greatly improved these parameters, whereas 1 week of leptin infusion had more modest effects. In contrast, leptin infusion was strikingly more effective in the aP2-n sterol response element binding protein 1 lipoatrophic mouse. Here we show that a longer duration of leptin infusion further improves the metabolic status of the A-ZIP/F-1 mice and that genetic background does not make a major contribution to the effect of leptin on glucose and insulin levels. Adipose transplantation using leptin-deficient ob/ob fat had no effect on the phenotype of the A-ZIP/F-1 mice. Moreover, the presence of ob/ob adipose tissue did not enhance the effects of leptin infusion. Serum adiponectin levels were 2% of control levels in the A-ZIP/F-1 mouse and increased only twofold with adipose transplantation and not at all after leptin infusion, suggesting that adiponectin deficiency is not a major contributor to the diabetic phenotype. Taken together, these results suggest that sequestration of triglycerides into fat may not be enough to restore a nondiabetic phenotype and that leptin deficiency plays a major role in causing the metabolic complications of lipoatrophy.
Delayed Transcapillary Transport of Insulin to Muscle Interstitial Fluid in Obese Subjects
Authors: Sjöstrand Mikaela , Gudbjörnsdottir Soffia , Holmäng Agneta , Lönn Lars , Strindberg Lena , Lönnroth Peter
Published: March 2007

Insulin-resistant subjects have a slow onset of insulin action, and the underlying mechanism has not been determined. To evaluate whether a delayed transcapillary transport is part of the peripheral insulin resistance, we followed the kinetics of infused insulin and inulin in plasma and muscle interstitial fluid in obese insulin-resistant patients and control subjects. A total of 10 lean and 10 obese men (BMI 24 ± 0.8 vs. 32 ± 0.8 kg/m2, P < 0.001) was evaluated during a hyperinsulinemic-euglycemic clamp (insulin infusion rate 120 mU · m−2 · min−1) combined with an inulin infusion. Measurements of insulin and inulin in plasma were taken by means of arterial-venous catheterization of the forearm and microdialysis in brachioradialis muscle combined with forearm blood flow measurements with vein occlusion pletysmography. The obese subjects had a significantly lower steady-state glucose infusion rate and, moreover, demonstrated a delayed appearance of insulin (time to achieve half-maximal concentration [T1/2] 72 ± 6 vs. 46 ± 6 min in control subjects, P < 0.05) as well as inulin (T1/2 83 ± 3 vs. 53 ± 7 min, P < 0.01) in the interstitial fluid. Also, the obese subjects had a delayed onset of insulin action (T1/2 70 ± 9 vs. 45 ± 5 min in control subjects, P < 0.05), and their forearm blood flow rate was significantly lower. These results demonstrate a delayed transcapillary transport of insulin and inulin from plasma to the muscle interstitial fluid and a delayed onset of insulin action in insulin-resistant obese subjects.
Glucose-Sensing in Glucagon-Like Peptide-1-Secreting Cells
Authors: Reimann Frank , Gribble Fiona M.
Published: March 2007

Glucagon-like peptide-1 (GLP-1) is released from intestinal L-cells in response to carbohydrate and fat in the diet. Despite the interest in GLP-1 as an antidiabetic agent, very little is known about the mechanism of stimulus-secretion coupling in L-cells. We investigated the electrophysiological events underlying glucose-induced GLP-1 release in the GLP-1-secreting cell line, GLUTag. Cells were studied using perforated-patch and standard whole-cell patch clamp recordings. GLUTag cells were largely quiescent and hyperpolarized in the absence of glucose. Increasing the glucose concentration between 0 and 20 mmol/l decreased the membrane conductance, caused membrane depolarization, and triggered the generation of action potentials. Action potentials were also triggered by tolbutamide (500 μmol/l) and were suppressed by diazoxide (340 μmol/l) or the metabolic inhibitor azide (3 mmol/l), suggesting an involvement of KATP channels. Large tolbutamide-sensitive washout currents developed in standard whole-cell recordings, confirming the presence of KATP channels. RT-PCR detected the KATP channel subunits Kir6.2 and SUR1 and glucokinase. GLP-1 secretion was also stimulated by glucose over the concentration range 0–25 mmol/l and by tolbutamide. Our results suggest that glucose triggers GLP-1 release through closure of KATP channels and action potential generation.
Plasma Membrane Ca2+-ATPase Overexpression Reduces Ca2+ Oscillations and Increases Insulin Release Induced by Glucose in Insulin-Secreting BRIN-BD11 Cells
Authors: Kamagate Adama , Herchuelz André , Van Eylen Françoise
Published: March 2007

In the mouse β-cell, glucose generates large amplitude oscillations of the cytosolic-free Ca2+ concentration ([Ca2+]i) that are synchronous to insulin release oscillations. To examine the role played by [ Ca2+]i oscillations in the process of insulin release, we examined the effect of plasma membrane Ca2+-ATPase (PMCA) overexpression on glucose-induced Ca2+ oscillations and insulin release in BRIN-BD11 cells. BRIN-BD11 cells were stably transfected with PMCA2wb. Overexpression could be assessed at the mRNA and protein level, with appropriate targeting to the plasma membrane assessed by immunofluorescence and the increase in PMCA activity. In response to K+, overexpressing cells showed a markedly reduced rise in [Ca2+]i. In response to glucose, control cells showed large amplitude [Ca2+]i oscillations, whereas overexpressing cells showed markedly reduced increases in [Ca2+]i without such large oscillations. Suppression of [Ca2+]i oscillations was accompanied by an increase in glucose metabolism and insulin release that remained oscillatory despite having a lower periodicity. Hence, [Ca2+] i oscillations appear unnecessary for glucose-induced insulin release and may even be less favorable than a stable increase in [ Ca2+]i for optimal hormone secretion. [Ca2+]i oscillations do not directly drive insulin release oscillations but may nevertheless intervene in the fine regulation of such oscillations.
Diacylglycerol Production and Protein Kinase C Activity Are Increased in a Mouse Model of Diabetic Embryopathy
Authors: Hiramatsu Yuji , Sekiguchi Naotaka , Hayashi Michio , Isshiki Keiji , Yokota Tamotsu , King George L. , Loeken Mary R.
Published: March 2007

Activation of the diacylglycerol-protein kinase C (DAG-PKC) cascade by excess glucose has been implicated in vascular complications of diabetes. Its involvement in diabetic embryopathy has not been established. We examined DAG production and PKC activities in embryos and decidua of streptozotocin (STZ)-diabetic or transiently hyperglycemic mice during neural tube formation. STZ diabetes significantly increased DAG and total PKC activity in decidua (1.5- and 1.4-fold, respectively) and embryos (1.7- and 1.3-fold, respectively) on day 9.5. Membrane-associated PKC α, βII, δ, and ζ were increased in decidua by 1.25- to 2.8-fold. Maternal hyperglycemia induced by glucose injection on day 7.5, the day before the onset of neural tube formation, also increased DAG, PKC activity, and PKC isoforms (1.1-, 1.6-, and 1.5-fold, respectively) in the embryo on day 9.5. Notably, membrane-associated PKC activity was increased 24-fold in embryos of diabetic mice with structural defects. These data indicate that hyperglycemia just before organogenesis activates the DAG-PKC cascade and is correlated with congenital defects.
Maternal Diabetes Increases the Risk of Caudal Regression Caused by Retinoic Acid
Authors: Chan Billy W.H. , Chan Kwok-siu , Koide Tsuyoshi , Yeung Sau-man , Leung Maran B.W. , Copp Andrew J. , Loeken Mary R. , Shiroishi Toshihiko , Shum Alisa S.W.
Published: March 2007

Maternal diabetes increases the risk of congenital malformations in the offspring of affected pregnancies. This increase arises from the teratogenic effect of the maternal diabetic milieu on the developing embryo, although the mechanism of this action is poorly understood. In the present study, we examined whether the vitamin A metabolite retinoic acid (RA), a common drug with well-known teratogenic properties, may interact with maternal diabetes to alter the incidence of congenital malformations in mice. Our results show that when treated with RA, embryos of diabetic mice are significantly more prone than embryos of nondiabetic mice to develop caudal regression, a defect that is highly associated with diabetic pregnancy in humans. By studying the vestigial tail (Wnt-3avt) mutant, we provide evidence that Wnt-3a, a gene that controls the development of the caudal region, is directly involved in the pathogenic pathway of RA-induced caudal regression. We further show that the molecular basis of the increased susceptibility of embryos of diabetic mice to RA involves enhanced downregulation of Wnt-3a expression. This positive interaction between RA and maternal diabetes may have implications for humans in suggesting increased susceptibility to environmental teratogens during diabetic pregnancy.
Peroxynitrite Induces Formation ofNε-(Carboxymethyl)Lysine by the Cleavage of Amadori Product and Generation of Glucosone and Glyoxal From Glucose
Authors: Nagai Ryoji , Unno Yuka , Hayashi Miki Cristina , Masuda Shuichi , Hayase Fumitaka , Kinae Naohide , Horiuchi Seikoh
Published: March 2007

Accumulation of advanced glycation end products (AGEs) on tissue proteins increases with pathogenesis of diabetic complications and atherosclerosis. Here we examined the effect of peroxynitrite (ONOO−) on the formation of Nε-(carboxymethyl)lysine (CML), a major AGE-structure. When glycated human serum albumin (HSA; Amadori-modified protein) was incubated with ONOO−, CML formation was detected by both enzyme-linked immunosorbent assay and high-performance liquid chromatography (HPLC) and increased with increasing ONOO− concentrations. CML was also formed when glucose, preincubated with ONOO−, was incubated with HSA but was completely inhibited by aminoguanidine, a trapping reagent for α-oxoaldehydes. For identifying the aldehydes that contributed to ONOO−-induced CML formation, glucose was incubated with ONOO− in the presence of 2,3-diaminonaphthalene. This experiment led to identification of glucosone and glyoxal by HPLC. Our results provide the first evidence that ONOO− can induce protein modification by oxidative cleavage of the Amadori product and also by generation of reactive α-oxoaldehydes from glucose.
The Prevalent Glu23Lys Polymorphism in the Potassium Inward Rectifier 6.2 (KIR6.2) Gene Is Associated With Impaired Glucagon Suppression in Response to Hyperglycemia
Authors: Tschritter Otto , Stumvoll Michael , Machicao Fausto , Holzwarth Martin , Weisser Melanie , Maerker Elke , Teigeler Anna , Häring Hans , Fritsche Andreas
Published: March 2007

Genetic factors play an important role in the pathogenesis of type 2 diabetes. The relevance to type 2 diabetes of the common polymorphism Glu23Lys in the potassium inward rectifier 6.2 (KIR6.2) gene is still controversial. The aim of this study was to assess whether this polymorphism influences β-cell function, α-cell function, or insulin action. We therefore studied 298 nondiabetic subjects using an oral glucose tolerance test (OGTT) and 75 nondiabetic subjects using a hyperglycemic clamp (10 mmol/l) with additional glucagon-like peptide (GLP)-1 and arginine stimulation. The prevalence of the Lys allele was ∼37%, and the Lys allele was associated with higher incremental plasma glucose during the OGTT (P = 0.03, ANOVA). Neither first- nor second-phase glucose-stimulated C-peptide secretion was affected by the presence of the polymorphism; nor were maximal glucose-, GLP-1-, or arginine-induced C-peptide secretion rates; nor was insulin sensitivity (all P > 0.7). However, the relative decrease in plasma glucagon concentrations during the 10 min after the glucose challenge was reduced in carriers of the Lys allele (10 ± 3% decrease from baseline in Lys/Lys, 18 ± 2% in Glu/Lys, and 20 ± 2% in Glu/Glu; P = 0.01, ANOVA). In conclusion, our findings suggest that the common Glu23Lys polymorphism in KIR6.2 is not necessarily associated with β-cell dysfunction or insulin resistance but with diminished suppression of glucagon secretion in response to hyperglycemia. Our findings thus confirm its functional relevance for glucose metabolism in humans.
Glucose Regulates the Release of Orexin-A From the Endocrine Pancreas
Authors: Ouedraogo Raogo , Näslund Erik , Kirchgessner Annette L.
Published: March 2007

Orexins (hypocretins) are novel neuropeptides that appear to play a role in the regulation of energy balances. Orexin-A (OXA) increases food intake in rodents, and fasting activates OXA neurons in both the lateral hypothalamic area and gut. OXA is also found in the endocrine pancreas; however, little is known about its release or functional significance. In this study, we show that depolarizing stimuli evoke the release of OXA from rat pancreatic islets in a calcium-dependent manner. Moreover, OXA release is stimulated by low glucose (2.8 mmol/l), similar to glucagon secretion, and inhibited by high glucose (16.7 mmol/l). Fasting increases plasma OXA, supporting the idea that orexin is released in response to hypoglycemia. Cells that secrete glucagon and insulin contain OXA and both cell types express orexin receptors. OXA increases glucagon secretion and decreases glucose-stimulated insulin release from isolated islets. OXA infusion increases plasma glucagon and glucose levels and decreases plasma insulin in fasted rats. We conclude that orexin-containing islet cells, like those in the brain and gut, are glucosensitive and part of a network of glucose “sensing” cells that becomes activated when blood glucose levels fall. OXA may modulate islet hormone secretion to maintain blood glucose levels during fasting.
Oxidative Injury and Apoptosis of Dorsal Root Ganglion Neurons in Chronic Experimental Diabetic Neuropathy
Authors: Schmeichel Ann M. , Schmelzer James D. , Low Phillip A.
Published: March 2007

We evaluated the effects of chronic hyperglycemia on L5 dorsal root ganglion (DRG) neurons using immunohistochemical and electrophysiologic techniques for evidence of oxidative injury. Experimental diabetic neuropathy was induced by streptozotocin. To evaluate the pathogenesis of the neuropathy, we studied peripheral nerve after 1, 3, and 12 months of diabetes. Electrophysiologic abnormalities were present from the first month and persisted over 12 months. 8-Hydroxy-2′-deoxyguanosine labeling was significantly increased at all time points in DRG neurons, indicating oxidative injury. Caspase-3 labeling was significantly increased at all three time points, indicating commitment to the efferent limb of the apoptotic pathway. Apoptosis was confirmed by a significant increase in the percentage of neurons undergoing apoptosis at 1 month (8%), 3 months (7%), and 12 months (11%). These findings support the concept that oxidative stress leads to oxidative injury of DRG neurons, with mitochondrium as a specific target, leading to impaired mitochondrial function and apoptosis, manifested clinically as a predominantly sensory neuropathy.
Impact of Two Common Polymorphisms in the PPARγ Gene on Glucose Tolerance and Plasma Insulin Profiles in Monozygotic and Dizygotic Twins
Authors: Poulsen Pernille , Andersen Gitte , Fenger Mogens , Hansen Torben , Echwald Søren M. , Vølund Aage , Beck-Nielsen Henning , Pedersen Oluf , Vaag Allan
Published: March 2007

The Pro12Ala polymorphism in the PPARγ2 gene has been associated with reduced risk of type 2 diabetes and insulin resistance. Recently, an association between dizygotic twinning and PPARγ gene polymorphisms has been proposed. We investigated the phenotypic appearance of the two polymorphisms (Pro12Ala and exon 6 C→T) in PPARγ among elderly twins (207 monozygotic [MZ] and 342 dizygotic [DZ]) and evaluated whether they could explain previously reported differences in plasma glucose and insulin profiles among MZ and DZ twins. We demonstrated a significant impact of the Pro12Ala polymorphism on glucose tolerance, diabetic status, homeostasis model assessment for insulin resistance, and plasma insulin profiles in twins. No impact of the silent exon 6 polymorphism on glucose homeostasis or plasma insulin profiles was found. Independent of the polymorphisms, we observed a significant impact of zygosity status per se on the plasma insulin profile after oral glucose ingestion, with the MZ twins being more hyperinsulinemic, indicating insulin resistance, than the DZ twins. Nonsignificantly higher glucose concentrations were observed among MZ compared with DZ twins. We demonstrated an association between the Ala allele and reduced risk of diabetes and insulin resistance in twins. However, the differences in metabolic profiles among MZ and DZ twins were not explained by differences in frequencies of the genetic variants and may be due to intrauterine environmental factors operating in twins independent of genotype. Accordingly, our study simultaneously supports a role for both the intrauterine environment (thrifty phenotype) and for genetics (thrifty genotype) in the etiology of insulin resistance and perhaps glucose intolerance in twins.
The Derivation of Highly Germline-Competent Embryonic Stem Cells Containing NOD-Derived Genome
Authors: Brook Frances A. , Evans Edward P. , Lord Christopher J. , Lyons Paul A. , Rainbow Daniel B. , Howlett Sarah K. , Wicker Linda S. , Todd John A. , Gardner Richard L.
Published: March 2007

It would be extremely advantageous to the analysis of disease mechanisms in the spontaneous mouse model of type 1 diabetes, the nonobese diabetic (NOD) strain, if genes in this strain could be modified in vivo using embryonic stem (ES) cells and homologous recombination. However, a NOD ES cell line with adequate germline transmission has not yet been reported. We report the development of highly germline-competent ES cell lines from the F1 hybrid of NOD and 129 for use in NOD gene targeting. Consequently, we developed ES cell lines derived from (NOD × 129)F1 × 129 backcross 1 mice, which were intercrossed to select for homozygosity of particular regions of NOD genome known to contain disease loci.
Role for VPAC2 Receptor-Mediated Signals in Pancreas Development
Authors: Rachdi Latif , Marie Jean-Claude , Scharfmann Raphael
Published: March 2007

Mature pancreatic cells develop from progenitors that proliferate and differentiate into endocrine and exocrine cells. This development is thought to be controlled by secreted soluble factors acting on their target cells after binding to membrane receptors. Here, we analyzed the impact on embryonic pancreatic development of ligands that bind to protein G-coupled receptors and increase cAMP accumulation. We found that embryonic pancreatic epithelial cells were sensitive to vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide. These factors generate signals after binding to the VPAC2 receptor, which is expressed by immature pancreatic epithelial cells between embryonic days 12 and 16. Finally, in vitro, VIP exposure increased the survival and proliferation of immature pancreatic cells, leading to an increase in the number of endocrine cells that will develop.
Improvement of the Mitochondrial Antioxidant Defense Status Prevents Cytokine-Induced Nuclear Factor-κB Activation in Insulin-Producing Cells
Authors: Azevedo-Martins Anna Karenina , Lortz Stephan , Lenzen Sigurd , Curi Rui , Eizirik Decio L. , Tiedge Markus
Published: March 2007

Proinflammatory cytokines (interleukin-1β [IL-1β], tumor necrosis factor-α [TNF-α], and γ-interferon [IFN-γ]) initiate a variety of signal cascades in pancreatic β-cells that affect the expression level of genes involved in both the destruction and the protection of the β-cell. The generation of nitric oxide (NO) via the inducible NO synthase (iNOS) and oxygen free radicals play a key role in cytokine-mediated β-cell destruction. Within these signal cascades, the activation of the transcription factor nuclear factor-κB (NF-κB) is crucial, and many cytokine-sensitive genes contain binding sites for this transcription factor in their promoter regions. The aim of this study was to characterize the cytokine-mediated activation of NF-κB and the subsequent expression of iNOS protein in insulin-producing RINm5F cells with an improved antioxidant defense status by overexpression of the cytoprotective enzymes catalase (Cat), glutathione peroxidase (Gpx), and the cytoplasmic Cu/Zn superoxide dismutase (Cu/ZnSOD). RINm5F cells with diverse mitochondrial antioxidative defense status were generated by stable overexpression of MnSOD constructs in sense (MnSOD sense) and antisense orientation (MnSOD antisense). Cytokine-induced (IL-1β or cytokine mix consisting of IL-1β + TNF-α + IFN-γ) activation of NF-κB in RINm5F cells was reduced by >80% through overexpression of MnSOD. The activity of the iNOS promoter remained at basal levels in cytokine-stimulated MnSOD sense cells. In contrast, the suppression of MnSOD gene expression in cytokine-stimulated MnSOD antisense cells resulted in a threefold higher activation of NF-κB and a twofold higher activation of the iNOS promoter as compared with control cells. The iNOS protein expression was significantly reduced after a 6- and 8-h cytokine incubation of MnSOD sense cells. The low activity level of MnSOD in RINm5F MnSOD antisense cells increased the iNOS protein expression in particular during the early phase of cytokine-mediated toxicity. Cat, Gpx, and the cytoplasmic Cu/ZnSOD did not affect the activation of NF-κB and the iNOS promoter. In conclusion, the overexpression of MnSOD, which inactivates specifically mitochondrially derived oxygen free radicals, significantly reduced the activation of NF-κB in insulin-producing cells. As a consequence of this protective effect in the early cytokine signaling pathways, the induction of iNOS, an important event in the β-cell destruction process, was also significantly reduced. The results provide evidence that mitochondrially derived reactive oxygen species (ROS) play a critical role in the activation of the cytokine-sensitive transcription factor NF-κB. Overexpression of MnSOD may thus be beneficial for β-cell survival through suppression of oxygen free radical formation, prevention of NF-κB activation, and iNOS expression.