bluuebunny/crossref_metadata_2025 huggingface dataset:
Marked Heterogeneity of Human Skeletal Muscle Lipolysis at Rest
Authors: Hagström-Toft Eva , Qvisth Veronica , Nennesmo Inger , Rydén Mikael , Bolinder Helene , Enoksson Staffan , Bolinder Jan , Arner Peter
Published: March 2007

In this study, variations in lipolysis among different muscle groups were examined by measuring local net glycerol release in vivo in healthy, normal-weight subjects (n = 11) during rested, postabsorptive conditions. Microdialysis of the gastrocnemius, deltoid, and vastus lateralis muscle regions revealed that extracellular glycerol concentrations of these three muscle regions were 84.7 ± 6.7, 59.7 + 7.3, and 56.4 ± 7.5 μmol/l, respectively, and the arterial plasma glycerol concentration was 44.8 ± 2.3 μmol/l (P = 0.0003–0.006, gastrocnemius vs. others). Local tissue blood flow, as measured by Xe clearance, did not differ among the regions. Net glycerol release was significantly higher in gastrocnemius muscle than in the two other regions. There were no regional differences in glycerol uptake when studied during glycerol infusion. Gastrocnemius muscle showed a dominance of type 1 fibers (70%), whereas the vastus lateralis muscle had equal distribution of fiber types (P = 0.02). No differences in intramuscular triaclyceride content, perimuscular fat, or the adipocyte-specific protein perilipin were observed among the muscle regions. Triglyceride turnover in the gastrocnemius muscle was 3.3 + 1.4% over 24 h, which is about 10 times more rapid than the turnover rate in subcutaneous adipose tissue (P < 0.01). Thus there were marked differences in lipolytic activity among skeletal muscle groups at rest, possibly reflecting variations in fiber type.
Y2 Receptor Deletion Attenuates the Type 2 Diabetic Syndrome of ob/ob Mice
Authors: Sainsbury Amanda , Schwarzer Christoph , Couzens Michelle , Herzog Herbert
Published: March 2007

Hypothalamic neuropeptide Y (NPY) is implicated in the regulation of a variety of physiological functions, notably energy homeostasis and reproduction. Chronically elevated NPY levels in the hypothalamus, as in genetically obese ob/ob mice, are associated with obesity, a syndrome of type 2 diabetes, and infertility. However, it is not known which of the five cloned Y receptors mediate these effects. Here, we show that crossing the Y2 receptor knockout mouse (Y2−/−) onto the ob/ob background attenuates the increased adiposity, hyperinsulinemia, hyperglycemia, and increased hypothalamo-pituitary-adrenal (HPA) axis activity of ob/ob mice. Compared with lean controls, ob/ob mice had elevated expression of NPY and agouti-related protein (AgRP) mRNA in the arcuate nucleus and decreased expression of proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) mRNA. Y2 deletion in ob/ob mice significantly increased the hypothalamic POMC mRNA expression, with no effect on NPY, AgRP, or CART expression. [Y2−/−ob/ob] mice were no different from ob/ob littermates with respect to food intake and body weight, and Y2 receptor deficiency had no beneficial effect on the infertility or the reduced hypothalamo-pituitary-gonadotropic function of ob/ob mice. These data demonstrate that Y2 receptors mediate the obese type 2 diabetes phenotype of ob/ob mice, possibly via alterations in melanocortin tonus in the arcuate nucleus and/or effects on the HPA axis.
Combined Effects of Genetic and Environmental Factors on Insulin Resistance Associated With Reduced Fetal Growth
Authors: Jaquet Delphine , Trégouët David A. , Godefroy Thierry , Nicaud Viviane , Chevenne Didi , Tiret Laurence , Czernichow Paul , Lévy-Marchal Claire
Published: March 2007

It has been suggested that the insulin resistance (IR) associated with reduced fetal growth results from interactions between genetic factors and an unfavorable fetal environment. In addition, the adipose tissue seems to play a key role in this association. We investigated whether polymorphisms in tumor necrosis factor (TNF)-α(G−308A), β3 adrenoreceptor (ADRB3)(G+250C), and peroxisome proliferator-activated receptor (PPAR)-γ2(Pro12Ala), key molecules of the adipose tissue, might affect the IR associated with reduced fetal growth. They were genotyped in 171 subjects who were born small for gestational age (SGA) and in 233 subjects who were born appropriate for gestational age (AGA) and underwent an oral glucose tolerance test (OGTT). The SGA group showed higher serum insulin concentrations than the AGA group at fasting (P = 0.03) and after stimulation (P = 0.0007), whereas no difference in serum glucose concentrations was observed. The frequencies of the alleles of these three polymorphisms were similar in both groups. In neither group did the polymorphisms affect glucose tolerance. In the SGA group, fasting insulin-to-glucose ratios were significantly higher in the TNF/−308A (P = 0.03), the PPAR/Ala12 (P = 0.01), and the ADRB3/+250G (P = 0.02) carriers than in the noncarriers. Results were comparable for fasting insulin concentration and insulin excursion under OGTT. No such amplification was observed in the AGA group. The effects of the PPAR/ProAla12 (P = 0.005) and the ADRB3/G+250G (P = 0.009) gene polymorphisms on IR indexes were significantly potentiated by BMI in the SGA group. In conclusion, our data exemplify the interaction between intrauterine environmental and genetic factors in the development of the IR associated with reduced fetal growth. They also point to the key role of adipose tissue in this association.
Mechanisms of the Triglyceride- and Cholesterol-Lowering Effect of Fenofibrate in Hyperlipidemic Type 2 Diabetic Patients
Authors: Forcheron Fabien , Cachefo Ana , Thevenon Sylvie , Pinteur Claudie , Beylot Michel
Published: March 2007

In humans, the precise mechanisms of the hypolipidemic action of fenofibrate, a peroxisome proliferator-activated receptor-α agonist, remain unclear. To gain insight on these mechanisms, we measured plasma lipids levels, lipids synthesis (hepatic de novo lipogenesis and cholesterol synthesis), and mRNA concentrations in circulating mononuclear cells (RT-PCR) of hydroxymethylglutaryl (HMG)-CoA reductase, LDL receptor, LDL receptor- related protein (LRP), scavenger receptor class B type I (SR-BI), ABCAI, and liver X receptor (LXR)-α in 10 control subjects and 9 hyperlipidemic type 2 diabetic patients. Type 2 diabetic subjects were studied before and after 4 months of fenofibrate administration. Fenofibrate decreased plasma triglycerides (P < 0.01) and total cholesterol (P < 0.05) concentrations and slightly increased HDL cholesterol (P < 0.05). Hepatic lipogenesis, largely enhanced in diabetic subjects (16.1 ± 2.1 vs. 7.5 ± 1.6% in control subjects, P < 0.01), was decreased by fenofibrate (9.8 ± 1.5%, P < 0.01). Fractional cholesterol synthesis was normal in diabetic subjects (3.5 ± 0.4 vs. 3.3 ± 0.5% in control subjects) and was unchanged by fenofibrate (3.5 ± 0.5%). Absolute cholesterol synthesis was, however, increased in diabetic subjects before and after fenofibrate (P < 0.05 vs. control subjects). HMG-CoA reductase, LDL receptor, LRP, and SR-BI mRNA concentrations were not different in type 2 diabetic and control subjects and were unchanged by fenofibrate. LXR-α mRNA levels were increased (P < 0.05) by fenofibrate. ABCAI mRNA concentrations, which were decreased in diabetic subjects (P < 0.05) before fenofibrate, were increased (P < 0.05) by fenofibrate to values comparable to those of control subjects. The plasma triglyceride-lowering effect of fenofibrate is explained in part by a decrease in hepatic lipogenesis, the moderate fall in total plasma cholesterol is not explained by a reduction of whole-body cholesterol synthesis, and the increase in LXR-α and ABCAI mRNA levels suggests that fenofibrate stimulated reverse cholesterol transport.
Inhibition of the JAK/STAT Signaling Pathway Prevents the High Glucose-Induced Increase in TGF-β and Fibronectin Synthesis in Mesangial Cells
Authors: Wang Xiaodan , Shaw Seán , Amiri Farhad , Eaton Douglas C. , Marrero Mario B.
Published: March 2007

High glucose (HG) causes glomerular mesangial cell (GMC) growth, production of transforming growth factor (TGF)-β, and increased synthesis of matrix proteins such as fibronectin, contributing to diabetic nephropathy. We recently found that exposure of cells to HG also activates the growth-promoting enzyme janus kinase 2 (JAK2) and its latent signal transducers and activators of transcription (STAT) transcription factors (STAT1, STAT3, and STAT5). Our purpose was to determine the effect that inhibition of JAK2 and these STAT transcription factors has on the HG-induced increase in TGF-β and fibronectin synthesis in GMC. Exposure of GMC to 25 mmol/l glucose caused the activation of JAK2, STAT1, STAT3, and STAT5 plus an increase in TGF-β and fibronectin synthesis, as compared with 5.5 mmol/l glucose. This HG-induced increase in synthesis of TGF-β and fibronectin was prevented by concomitant incubation with AG-490, a specific JAK2 inhibitor. The HG-induced JAK2, STAT1, and STAT3 tyrosine phosphorylations in GMC were also abolished by AG-490. Preincubation of GMC cultured in 25 mmol/l glucose with a specific JAK2 or STAT1 antisense oligonucleotide also prevented both TGF-β and fibronectin synthesis. These results provide direct evidence for linkages between JAK2, STAT1, and the glucose-induced overproduction of TGF-β and fibronectin in GMC.
All-trans Retinoic Acid Improves Structure and Function of Diabetic Rat Skin in Organ Culture
Authors: Varani James , Perone Patricia , Merfert Matthew G. , Moon Sang Eun , Larkin Dennis , Stevens Martin J.
Published: March 2007

Diabetes increases susceptibility to chronic ulceration. The cause of chronic wound formation in diabetic individuals is multifactorial but may be accelerated by changes in the structure and function of the skin secondary to impaired fibroblast proliferation, decreased collagen synthesis, and increased matrix metalloproteinase (MMP) expression. This study explored cellular and biochemical changes in organ cultures of skin from streptozotocin-diabetic (STZ-D) rats and the effects of all-trans retinoic acid (RA) on these changes. STZ-D rats were killed after 6 weeks. The skin was cut into 2-mm pieces and incubated in organ culture for 3 or 6 days in the absence or presence of 3 μmol/l RA. After organ culture incubation, control and RA-treated tissue was examined histologically after staining with hematoxylin and eosin. In parallel, organ culture-conditioned medium was assayed for MMPs. Additional organ cultures were examined for collagen synthesis using 3H-proline incorporation into trichloroacetic acid-precipitable material and for glycosaminoglycan production based on interaction with the cationic dye 1,9-dimethylmethylene blue and by staining of tissue sections with periodic acid Schiff reagents. Skin from 6-week STZ-D rats demonstrated features of dermal atrophy including thinning and disorganization of connective tissue bundles and increased space between bundles. The addition of RA resulted in cellular reactivation and partially reversed the histological features of dermal atrophy. Levels of latent and active MMP-9 and MMP-13 were elevated 4- and 10-fold, respectively, in STZ-D skin and reduced by 50–75% (P < 0.05) by RA. Collagen synthesis was increased by 30% (P < 0.05) by RA, whereas glycosaminoglycan expression was increased by only 9% (NS). RA also increased proliferation of STZ-D skin fibroblasts (approximately threefold over control; P < 0.05). Together, these data suggest that RA has the capacity to improve structure and function of diabetic skin.
Calcineurin Inhibitor–Free CD28 Blockade-Based Protocol Protects Allogeneic Islets in Nonhuman Primates
Authors: Adams Andrew B. , Shirasugi Nozomu , Durham Megan M. , Strobert Elizabeth , Anderson Dan , Rees Phyllis , Cowan Shannon , Xu Huaying , Blinder Yelena , Cheung Michael , Hollenbaugh Dianne , Kenyon Norma S. , Pearson Thomas C. , Larsen Christian P.
Published: March 2007

Recent success using a steroid-free immunosuppressive regimen has renewed enthusiasm for the use of islet transplantation to treat diabetes. Toxicities associated with the continued use of a calcineurin inhibitor may limit the wide-spread application of this therapy. Biological agents that block key T-cell costimulatory signals, in particular the CD28 pathway, have demonstrated extraordinary promise in animal models. LEA29Y (BMS-224818), a mutant CTLA4-Ig molecule with increased binding activity, was evaluated for its potential to replace tacrolimus and protect allogeneic islets in a preclinical primate model. Animals received either the base immunosuppression regimen (rapamycin and anti–IL-2R monoclonal antibody [mAb]) or the base immunosuppression and LEA29Y. Animals receiving the LEA29Y/rapamycin/anti–IL-2R regimen (n = 5) had significantly prolonged islet allograft survival (204, 190, 216, 56, and >220 days). In contrast, those animals receiving the base regimen alone (n = 2) quickly rejected the transplanted islets at 1 week (both at 7 days). The LEA29Y-based regimen prevented the priming of anti-donor T- and B-cell responses, as detected by interferon-γ enzyme-linked immunospot and allo-antibody production, respectively. The results of this study suggest that LEA29Y is a potent immunosuppressant that can effectively prevent rejection in a steroid-free immunosuppressive protocol and produce marked prolongation of islet allograft survival in a preclinical model.
Central Administration of Oleic Acid Inhibits Glucose Production and Food Intake
Authors: Obici Silvana , Feng Zhaohui , Morgan Kimyata , Stein Daniel , Karkanias George , Rossetti Luciano
Published: March 2007

The hypothalamus and other regions within the central nervous system (CNS) link the sensing of nutrients to the control of metabolism and feeding behavior. Here, we report that intracerebroventricular (ICV) administration of the long-chain fatty acid oleic acid markedly inhibits glucose production and food intake. The anorectic effect of oleic acid was independent of leptin and was accompanied by a decrease in the hypothalamic expression of neuropeptide Y. The short-chain fatty acid octanoic acid failed to reproduce the metabolic effects of oleic acid, and ICV coadministration of inhibitors of ATP-sensitive K+ channels blunted the effect of oleic acid on glucose production. This is the first demonstration that fatty acids can signal nutrient availability to the CNS, which in turn limits further delivery of nutrients to the circulation.
Glycogen-Dependent Effects of 5-Aminoimidazole-4-Carboxamide (AICA)-Riboside on AMP-Activated Protein Kinase and Glycogen Synthase Activities in RatSkeletal Muscle
Authors: Wojtaszewski Jørgen F.P. , Jørgensen Sebastian B. , Hellsten Ylva , Hardie D. Grahame , Richter Erik A.
Published: March 2007

5′-AMP-activated protein kinase (AMPK) functions as a metabolic switch in mammalian cells and can be artificially activated by 5-aminoimidazole-4-carboxamide (AICA)-riboside. AMPK activation during muscle contraction is dependent on muscle glycogen concentrations, but whether glycogen also modifies the activation of AMPK and its possible downstream effectors (glycogen synthase and glucose transport) by AICA-riboside in resting muscle is not known. Thus, we have altered muscle glycogen levels in rats by a combination of swimming exercise and diet and investigated the effects of AICA-riboside in the perfused rat hindlimb muscle. Two groups of rats, one with super-compensated muscle glycogen content (∼200–300% of normal; high glycogen [HG]) and one with moderately lowered muscle glycogen content (∼80% of normal; low glycogen [LG]), were generated. In both groups, the degree of activation of the α2 isoform of AMPK by AICA-riboside depended on muscle type (white gastrocnemius ≫ red gastrocnemius > soleus). Basal and AICA-riboside-induced α2-AMPK activity were markedly lowered in the HG group (∼50%) compared with the LG group. Muscle 2-deoxyglucose uptake was also increased and glycogen synthase activity decreased by AICA-riboside. Especially in white gastrocnemius, these effects, as well as the absolute activity levels of AMPK-α2, were markedly reduced in the HG group compared with the LG group. The inactivation of glycogen synthase by AICA-riboside was accompanied by decreased gel mobility and was eliminated by protein phosphatase treatment. We conclude that acute AICA-riboside treatment leads to phosphorylation and deactivation of glycogen synthase in skeletal muscle. Although the data do not exclude a role of other kinases/phosphatases, they suggest that glycogen synthase may be a target for AMPK in vivo. Both basal and AICA-riboside-induced AMPK-α2 and glycogen synthase activities, as well as glucose transport, are depressed when the glycogen stores are plentiful. Because the glycogen level did not affect adenine nucleotide concentrations, our data suggest that glycogen may directly affect the activation state of AMPK in skeletal muscle.
Interleukin-1 Plus γ-Interferon-Induced Pancreatic β-Cell Dysfunction Is Mediated by β-Cell Nitric Oxide Production
Authors: Thomas Helen E. , Darwiche Rima , Corbett John A. , Kay Thomas W.H.
Published: March 2007

Cytokines have been implicated in pancreatic β-cell destruction leading to type 1 diabetes. In vitro, a combination of γ-interferon (IFN-γ) and interleukin-1 (IL-1) stimulate inducible nitric oxide synthase (iNOS) expression in islets, and the resulting increased production of nitric oxide (NO) causes islet cell destruction. Islets contain macrophages, ductal cells, and endothelial cells that, when activated, may mediate islet cell damage by producing either NO themselves or cytokines that then stimulate NO production by β-cells. The aim of this study was to determine whether β-cell damage mediated by cytokine-induced NO production is dependent on β-cell production of NO, or whether NO produced by other cells in the islet is capable of destroying β-cells. To address this aim, we used transgenic mice expressing a dominant-negative IFN-γ receptor in β-cells (RIP-ΔγR). RIP-ΔγR islets are resistant to IL-1 + IFN-γ-induced inhibition of insulin secretion and DNA damage, indicating that β-cell IFN-γ responsiveness is required for IL-1 + IFN-γ-mediated β-cell damage. Although islets isolated from RIP-ΔγR mice are resistant to functional damage, these islets produce NO in response to IL-1 + IFN-γ, but at a lower concentration than that produced by wild-type islets. β-Cells appear to be the primary cellular source of IL-1 + IFN-γ-induced iNOS expression in wild-type islets. In contrast, IL-1 + IFN-γ fail to stimulate iNOS expression by insulin-expressing cells in islets isolated from RIP-ΔγR mice. IL-1 + IFN-γ-induced expression of iNOS was detected in non-β-cells in both wild-type and RIP-ΔγR islets. These findings support the hypothesis that NO must be produced by β-cells to induce damage.
Inhibition of Protein Kinase C δ Protects Rat INS-1 Cells Against Interleukin-1β and Streptozotocin-Induced Apoptosis
Authors: Carpenter Lee , Cordery Damien , Biden Trevor J.
Published: March 2007

Exposure of pancreatic β-cells to cytokines, such as interleukin-1β (IL-1β), is thought to contribute to the β-cell apoptosis that underlies the onset of type 1 diabetes. One important event triggered by IL-1β is induction of nitric oxide synthase (iNOS), an enzyme that catalyzes intracellular generation of the cytotoxic free radical NO. We recently described a novel requirement for the protein kinase C (PKC) isozyme PKCδ in this process. Our current aim, therefore, was to assess whether PKCδ also plays a role in β-cell apoptosis. As assessed by either annexin V staining or DNA fragmentation, IL-1β caused INS-1 cells to undergo apoptosis. This was completely blocked by adenoviral overexpression of a dominant-negative, kinase-dead (KD) PKCδ mutant. The corresponding PKCα virus was without effect. However, apoptosis caused by the cytotoxic agent streptozotocin (STZ), which acts independent of iNOS, was also inhibited by overexpression of PKCδKD. STZ was additionally shown to activate the proteolytic enzyme caspase-3, a key biochemical effector of end-stage apoptosis. Moreover, STZ caused a caspase-dependent cleavage of PKCδ, thereby releasing a COOH-terminal fragment corresponding to the kinase catalytic domain. Thus, proteolytic activation of PKCδ seems to be important in the distal apoptotic pathway induced by STZ. That IL-1β also activated caspase-3 and promoted PKCδ cleavage suggests that this distal pathway also contributes in the apoptotic response to the cytokine. These data therefore support a dual role for PKCδ in IL-1β-mediated cell death: it is required for efficient NO generation through regulation of iNOS levels but also contributes to apoptotic pathways downstream of caspase activation.
A Reg Family Protein Is Overexpressed in Islets From a Patient With New-Onset Type 1 Diabetes and Acts as T-Cell Autoantigen in NOD Mice
Authors: Gurr Werner , Yavari Reza , Wen Li , Shaw Margaret , Mora Conchi , Christa Laurence , Sherwin Robert S.
Published: March 2007

Genes overexpressed in pancreatic islets of patients with new-onset type 1 diabetes are potential candidates for novel disease-related autoantigens. RT-PCR-based subtractive hybridization was used on islets from a patient who died at the onset of type 1 diabetes, and it identified a type 1 diabetes-related cDNA encoding hepatocarcinoma-intestine-pancreas/pancreatic-associated protein (HIP/PAP). This protein belongs to the family of Reg proteins implicated in islet regeneration; its gene contains a putative interleukin-6 (IL-6) response element. Islets from healthy cadaveric human donors released HIP/PAP protein into the culture medium, and this release was enhanced by the addition of IL-6. The expression pattern of mouse homologues of HIP/PAP was determined in pancreata of prediabetic and diabetic NOD mice. Both groups showed positive immunostaining for HIP/PAP in islets and ductal epithelium. To test whether HIP/PAP is a target of islet-directed autoimmunity, we measured splenic T-cell responses against HIP/PAP in NOD mice. Spontaneous proliferation was detected after 4 weeks. Lymphocytes from islet infiltrates and pancreatic lymph nodes from 7- to 10-week-old NOD mice were used to establish an HIP/PAP-specific I-Ag7-restricted T-cell line, termed WY1, that also responded to mouse islets. WY1 cells homed to islets of NOD-SCID mice and adoptively transferred disease when coinjected with purified CD8+ cells from diabetic NOD mice. Our conclusion was that differential cloning of Reg from islets of a type 1 diabetic patient and the response of Reg to the cytokine IL-6 suggests that HIP/PAP becomes overexpressed in human diabetic islets because of the local inflammatory response. HIP/PAP acts as a T-cell autoantigen in NOD mice. Therefore, autoimmunity to HIP/PAP might create a vicious cycle, accelerating the immune process leading to diabetes.
A Metalloporphyrin-Based Superoxide Dismutase Mimic Inhibits Adoptive Transfer of Autoimmune Diabetes by a Diabetogenic T-Cell Clone
Authors: Piganelli Jon D. , Flores Sonia C. , Cruz Coral , Koepp Jeffrey , Batinic-Haberle Ines , Crapo James , Day Brian , Kachadourian Remy , Young Rebekah , Bradley Brenda , Haskins Kathryn
Published: March 2007

We present here the first report of a metalloporphyrin-based antioxidant that can prevent or delay the onset of autoimmune diabetes. Type 1 diabetes is an autoimmune process whereby T-cells recognize pancreatic β-cell antigens and initiate a leukocyte infiltrate that produces proinflammatory cytokines and reactive oxygen species (ROS), ultimately leading to β-cell destruction. Because islet β-cells have a reduced capacity to scavenge free radicals, they are very sensitive to ROS action. Metalloporphyrin-based superoxide dismutase (SOD) mimics scavenge ROS and protect cells from oxidative stress and apoptosis. To investigate the effect of SOD mimics and the role of oxidative stress in the development of autoimmune diabetes in vivo, we used a diabetogenic T-cell clone, BDC-2.5, to induce rapid onset of diabetes in young nonobese diabetic-severe combined immunodeficient mice (NOD.scid). Disease was significantly delayed or prevented altogether by treatment of recipient mice with an SOD mimic, AEOL-10113, before transfer of the BDC-2.5 clone. To investigate the mechanisms of protection, in vitro assays for T-cell proliferation and γ-interferon (IFN-γ) production were carried out using the T-cell clone BDC-2.5. We found that the SOD mimic significantly inhibited antigen-presenting cell-dependent T-cell proliferation and IFN-γ production in vitro. In addition, pretreatment of lipopolysaccharide (LPS)-stimulated peritoneal macrophages with SOD mimic inhibited the LPS-dependent increase in TNF-α as well as the NADPH oxidase-dependent release of superoxide. Finally, this compound protected NIT-1 insulinoma cells from interleukin-1β and alloxan cytotoxicity in vitro.
Overexpression of the Na/Ca Exchanger Shapes Stimulus-Induced Cytosolic Ca2+ Oscillations in Insulin-Producing BRIN-BD11 Cells
Authors: Van Eylen Françoise , Horta Oscar Diaz , Barez Aurore , Kamagate Adama , Flatt Peter R. , Macianskiene Regina , Mubagwa Kanigula , Herchuelz André
Published: March 2007

In response to glucose, mouse β-cells display slow oscillations of the membrane potential and cytosolic free Ca2+ concentration ([Ca2+]i), whereas rat β-cells display a staircase increase in these parameters. Mouse and rat islet cells differ also by their level of Na/Ca exchanger (NCX) activity. The view that the inward current generated by Na/Ca exchange shapes stimulus-induced electrical activity and [Ca2+]i oscillations in pancreatic β-cells was examined in insulin-producing BRIN-BD11 cells overexpressing the Na/Ca exchanger. BRIN-BD11 cells were stably transfected with NCX1.7, one of the exchanger isoforms identified in the β-cell. Overexpression could be assessed at the mRNA and protein level. Appropriate targeting to the plasma membrane could be assessed by microfluorescence and the increase in Na/Ca exchange activity. In response to K+, overexpressing cells showed a more rapid increase in [Ca2+]i on membrane depolarization as well as a more rapid decrease of [Ca2+]i on membrane repolarization. In response to glucose and tolbutamide, control BRIN cells showed large amplitude [Ca2+]i oscillations. In contrast, overexpressing cells showed a staircase increase in [Ca2+]i without such large oscillations. Diazoxide-induced membrane hyperpolarization restored large amplitude [Ca2+]i oscillations in overexpressing cells. The present data confirm that Na/Ca exchange plays a significant role in the rat β-cell [Ca2+]i homeostasis, the exchanger being a versatile system allowing both Ca2+ entry and outflow. Our data suggest that the current generated by the exchanger shapes stimulus-induced membrane potential and [Ca2+]i oscillations in insulin-secreting cells, with the difference in electrical activity and [Ca2+]i behavior seen in mouse and rat β-cells resulting in part from a difference in Na/Ca exchange activity between these two cells.
Inclusion of Low Amounts of Fructose With an Intraduodenal Glucose Load Markedly Reduces Postprandial Hyperglycemia and Hyperinsulinemia in the Conscious Dog
Authors: Shiota Masakazu , Moore Mary Courtney , Galassetti Pietro , Monohan Michael , Neal Doss W. , Shulman Gerald I. , Cherrington Alan D.
Published: March 2007

Intraportal infusion of small amounts of fructose markedly augmented net hepatic glucose uptake (NHGU) during hyperglycemic hyperinsulinemia in conscious dogs. In this study, we examined whether the inclusion of catalytic amounts of fructose with a glucose load reduces postprandial hyperglycemia and the pancreatic β-cell response to a glucose load in conscious 42-h-fasted dogs. Each study consisted of an equilibration (−140 to −40 min), control (−40 to 0 min), and test period (0–240 min). During the latter period, glucose (44.4 μmol · kg−1 · min−1) was continuously given intraduodenally with (2.22 μmol · kg−1 · min−1) or without fructose. The glucose appearance rate in portal vein blood was not significantly different with or without the inclusion of fructose (41.3 ± 2.7 vs. 37.3 ± 8.3 μmol · kg−1 · min−1, respectively). In response to glucose infusion without the inclusion of fructose, the net hepatic glucose balance switched from output to uptake (from 10 ± 2 to 11 ± 4 μmol · kg−1 · min−1) by 30 min and averaged 17 ± 6 μmol · kg−1 · min−1. The fractional extraction of glucose by the liver during the infusion period was 7 ± 2%. Net glycogen deposition was 2.44 mmol glucose equivalent/kg body wt; 49% of deposited glycogen was synthesized via the direct pathway. Net hepatic lactate production was 1.4 mmol/kg body wt. Arterial blood glucose rose from 4.1 ± 0.2 to 7.3 ± 0.4 mmol/l, and arterial plasma insulin rose from 42 ± 6 to 258 ± 66 pmol/l at 30 min, after which they decreased to 7.0 ± 0.5 mmol/l and 198 ± 66 pmol/l, respectively. Arterial plasma glucagon decreased from 54 ± 7 to 32 ± 3 ng/l. In response to intraduodenal glucose infusion in the presence of fructose, net hepatic glucose balance switched from 9 ± 1 μmol · kg−1 · min−1 output to 12 ± 3 and 28 ± 5 μmol · kg−1 · min−1 uptake by 15 and 30 min, respectively. The average NHGU (28 ± 5 μmol · kg−1 · min−1) and fractional extraction during infusion period (12 ± 2%), net glycogen deposition (3.68 mmol glucose equivalent/kg body wt), net hepatic lactate production (3.27 mmol/kg), and glycogen synthesis via the direct pathway (68%) were significantly higher (P < 0.05) compared to that in the absence of fructose. The increases in arterial blood glucose (from 4.4 ± 0.1 to 6.4 ± 0.2 mmol/l at 30 min) and arterial plasma insulin (from 48 ± 6 to 126 ± 30 pmol/l at 30 min) were significantly smaller (P < 0.05). In summary, the inclusion of small amounts of fructose with a glucose load augmented NHGU, increased hepatic glycogen synthesis via the direct pathway, and augmented hepatic glycolysis. As a result, postprandial hyperglycemia and insulin release by the pancreatic β-cell were reduced. In conclusion, catalytic amounts of fructose have the ability to improve glucose tolerance.
Reversibility of Glucose-Induced Changes in Mesangial Cell Extracellular Matrix Depends on the Genetic Background
Authors: Fornoni Alessia , Striker Liliane J. , Zheng Feng , Striker Gary E.
Published: March 2007

Adequate glycemic control protects most patients with diabetes from nephropathy, but a substantial fraction of patients develop progressive disease despite lowering glycemia. We isolated mesangial cells (MC) from the glomeruli of mouse strains that model these two outcomes in patients with diabetes, namely those that have the propensity (ROP) or resistance (B6) to develop progressive diabetic nephropathy. We determined the nature and reversibility of changes in selected extracellular matrix-related molecules after chronic exposure to elevated glucose concentration. MC were exposed to 25 mmol/l glucose for 5 weeks followed by 6 mmol/l glucose and 19 mmol/l mannitol for an additional 5 weeks. Matrix metalloproteinase-2 (MMP-2) and transforming growth factor-β1 (TGF-β1) levels increased in B6 MC exposed to 25 mmol/l glucose but returned to baseline levels when the glucose concentration was reduced to 6 mmol/l. MMP-2 and TGF-β1 were higher in ROP MC at baseline and increased in response to 25 mmol/l glucose, but remained elevated when glucose concentration was reduced. Type I collagen expression and accumulation increased in a reversible manner in B6 MC exposed to 25 mmol/l glucose. However, type I collagen expression was higher in ROP MC at baseline and remained unaffected by changes in glucose concentration. Thus, 25 mmol/l glucose induced reversible changes in MMP-2, TGF-β1, and type I collagen in MC of sclerosis-resistant mice but not in MC from sclerosis-prone mice. Therefore, progressive diabetic nephropathy may be secondary to stable alterations in the phenotype of MC as a result of the interplay between the genetic background and elevated glucose concentrations.
The Role of Poly(ADP-Ribose) Polymerase Activation in the Development of Myocardial and Endothelial Dysfunction in Diabetes
Authors: Pacher Pal , Liaudet Lucas , Soriano Francisco Garcia , Mabley Jon G. , Szabó Éva , Szabó Csaba
Published: March 2007

Patients with diabetes exhibit a high incidence of diabetic cardiomyopathy and vascular complications, which underlie the development of retinopathy, nephropathy, and neuropathy and increase the risk of hypertension, stroke, and myocardial infarction. There is emerging evidence that the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) importantly contributes to the development of endothelial dysfunction in a streptozotocin-induced model of diabetes. We investigated the role of PARP activation in the pathogenesis of cardiac dysfunction in streptozotocin-induced and genetic (nonobese diabetic) models of diabetes in rats and mice. Development of diabetes was accompanied by hyperglycemia, cardiac PARP activation, a selective loss of endothelium-dependent vasodilation in the thoracic aorta, and an early diastolic dysfunction of the heart. Treatment with a novel potent phenanthridinone-based PARP inhibitor, PJ34, starting 1 week after the onset of diabetes, restored normal vascular responsiveness and significantly improved cardiac dysfunction, despite the persistence of severe hyperglycemia. The beneficial effect of PARP inhibition persisted even after several weeks of discontinuation of the treatment. Thus, PARP activation plays a central role in the pathogenesis of diabetic cardiovascular (cardiac as well as endothelial) dysfunction. PARP inhibitors may exert beneficial effects against the development of cardiovascular complications in diabetes.
Vascular NADH Oxidase Is Involved in Impaired Endothelium-Dependent Vasodilation in OLETF Rats, a Model of Type 2 Diabetes
Authors: Kim Yong K. , Lee Mi-S. , Son Seok M. , Kim In J. , Lee Won S. , Rhim Byung Y. , Hong Ki W. , Kim Chi D.
Published: March 2007

Superoxide anion can modulate vascular smooth muscle tone and is potentially involved in diabetic vascular complications. The present study was undertaken to characterize both vascular production and the enzymatic source of superoxide anion in type 2 diabetic rats. In the thoracic aorta of OLETF rats, endothelium-dependent relaxation was markedly attenuated compared with that of control (LETO) rats in association with a significant increase in superoxide production (2,421.39 ± 407.01 nmol · min−1 · mg−1). The increased production of superoxide anion was significantly attenuated by diphenyleneiodonium (DPI; 10 μmol/l), an inhibitor of NAD(P)H oxidase. The production of superoxide anion in response to NADH as a substrate was markedly increased in the vascular homogenates, but NADPH, arachidonic acid, xanthine, and succinate produced only small increases in chemiluminescence. In line with these results, studies using various enzyme inhibitors, such as DPI, allopurinol, rotenone, NG-monomethyl-l-arginine, and indomethacin, suggest that the predominant source of superoxide anion in vascular particulate fraction is NADH-dependent membrane-bound oxidase. Furthermore, the expression of p22phox, a major component of vascular NAD(P)H oxidase, was markedly increased in the aorta from OLETF rats compared with that of LETO rats. These findings suggest that upregulated expression of p22phox mRNA and enhanced NADH oxidase activity contribute to the impaired endothelium-dependent vasodilation in OLETF rats.
A Genome-Wide Scan for Obesity in African-Americans
Authors: Zhu Xiaofeng , Cooper Richard S. , Luke Amy , Chen Guanjie , Wu Xiaodong , Kan Donghui , Chakravarti Aravinda , Weder Alan
Published: March 2007

A genome-wide scan using 387 short tandem repeat markers was conducted for obesity among 618 black individuals from 202 families residing in a suburb of Chicago. Evidence for linkage was evaluated with BMI and percent body fat (PBF) using a variance component analysis approach. Suggestive evidence for linkage was found for BMI on chromosome 5 (logarithm of odds [LOD] score = 1.9) and PBF on chromosome 6 (LOD score = 2.7). One additional region on chromosome 3 was linked to these phenotypes at a lower level of significance (LOD score = 1.8 and 0.95 for BMI and PBF, respectively); the linked marker on this chromosome lies in the same region implicated as harboring obesity genes in a previous study of a white population. The replication of linkage evidence using different ethnic groups reinforces the potential significance of this latter candidate region.
Asian-Specific HLA Haplotypes Reveal Heterogeneity of the Contribution of HLA-DR and -DQ Haplotypes to Susceptibility to Type 1 Diabetes.
Authors: Kawabata Yumiko , Ikegami Hiroshi , Kawaguchi Yoshihiko , Fujisawa Tomomi , Shintani Maki , Ono Masaya , Nishino Masanori , Uchigata Yasuko , Lee Inkyu , Ogihara Toshio
Published: March 2007

To assess the effect of Asian-specific HLA haplotypes on susceptibility to type 1 diabetes, we investigated the association of genotypic combinations of DRB1-DQB1 haplotypes with susceptibility to type 1 diabetes. We studied 132 Japanese patients with type 1 diabetes and 157 control subjects, along with 67 Korean patients and 109 control subjects. DRB1*0405-DQB1*0401 and DRB1*0901-DQB1*0303 were confirmed to be two major susceptible HLA haplotypes in the Japanese population. The frequencies of heterozygotes and homozygotes with DRB1*0405-DQB1*0401 were similarly higher in patients than in control subjects (homozygotes, 5.3% vs. 3.8%; heterozygotes, 48.5% vs. 26.1%). In contrast, homozygotes, but not heterozygotes, with DRB1*0901-DQB1*0303 were more frequent in patients with type 1 diabetes than in control subjects (homozygotes, 12.9% vs. 0.6%; heterozygotes, 22.0% vs. 24.8%). A similar tendency was also observed in the Korean population. In multiple logistic regression analysis, DRB1*0405-DQB1*0401 fitted a dominant model and DRB1*0901-DQB1*0303 fitted a recessive model. These data, which indicate that the contribution of HLA haplotypes to the genetic susceptibility to type 1 diabetes differs depending on the genotypic combination of HLA haplotypes, suggest the importance of extensive analysis of genotypes in studies on HLA and disease association in general.
Phasic Insulin Release and Metabolic Regulation in Type 2 Diabetes
Authors: Del Prato Stefano , Marchetti Piero , Bonadonna Riccardo C.
Published: March 2007

Type 2 diabetes is a heterogeneous disorder due to prevalent insulin resistance associated with deficient insulin secretion or to a prevalent defect of insulin secretion associated with impaired insulin action. The definition is supported by the high frequency at which insulin resistance can be demonstrated in type 2 diabetic patients. Nonetheless, insulin resistance is not a sufficient mechanism to cause diabetes. Impaired β-cell function is a necessary defect in all conditions of impaired glucose regulation; however, it manifests itself in a different manner in fasting and glucose-stimulated conditions. In the fasting state, the basal insulin secretory rate increases as a function of the progressive decline in insulin action. As such, the fasting plasma insulin concentration is often taken as a marker for insulin sensitivity. After glucose challenge, a specific alteration of acute insulin release is an early and progressive defect. The latter might represent an intrinsic defect, but its continuous decline is affected by glucotoxicity and lipotoxicity. To understand the impact of β-cell dysfunction in type 2 diabetes on metabolic homeostasis, it is useful to consider the different phases of insulin secretion separately. Insulin secretion can be divided into basal (postabsorptive) and stimulated (postprandial) states. The former prevails during the interprandial phases and plays a major role during the overnight fast; the latter regulates glucose metabolism when carbohydrate is abundant and must be disposed of. Data in animals and humans support a crucial physiological role of first-phase insulin secretion in postprandial glucose homeostasis. This effect is primarily achieved in the liver, allowing prompt inhibition of endogenous glucose production and limiting the postprandial rise in plasma glucose level. In type 2 diabetes, loss of the early surge of insulin release is an early and quite common defect that may have a pathogenetic role in the development of postprandial hyperglycemia, possibly requiring specific therapeutic intervention.
Effect of Acute Hyperglycemia on Insulin Secretion in Humans
Authors: Toschi Elena , Camastra Stefania , Sironi Anna Maria , Masoni Antonio , Gastaldelli Amalia , Mari Andrea , Ferrannini Ele , Natali Andrea
Published: March 2007

First-phase insulin response to intravenous glucose is impaired both in type 2 diabetic patients and in subjects at risk for the disease. Hyperglycemia can modify β-cell response by either inhibiting or potentiating both first- and second-phase insulin release. In normal subjects, the effect of acute hyperglycemia on insulin secretion is controversial. We measured (in 13 healthy volunteers) insulin secretion (by deconvolution of plasma C-peptide concentrations) during three consecutive 30-min hyperglycemic steps (2.8, 2.8, and 5.6 mmol/l), followed by an intravenous arginine bolus. First-phase insulin secretion in response to the first hyperglycemic step (456 ± 83 pmol · min−1 · m−2) was significantly larger than that in response to the second step (311 ± 37 pmol · min−1 · m−2, P < 0.01); the subsequent increase in glycemia failed to stimulate first-phase secretion any further (377 ± 60 pmol · min−1 · m−2, NS vs. the previous value). This inhibition was also evident when insulin release rates were corrected for the respective increments (absolute or percentage) in plasma glucose levels and was not due to β-cell exhaustion because the arginine bolus still elicited a large peak of insulin secretion (4,790 ± 2,330 pmol · min−1 · m−2). In contrast, second-phase insulin secretion was related to the prevailing glucose levels across the three hyperglycemic steps in a direct quasilinear manner. We conclude that first-phase insulin secretion is inhibited by short-term modest hyperglycemia, whereas the second-phase insulin secretion increases linearly with hyperglycemia.
Do Oscillations of Insulin Secretion Occur in the Absence of Cytoplasmic Ca2+ Oscillations in β-Cells?
Authors: Kjems Lise L. , Ravier Magalie A. , Jonas Jean-Christophe , Henquin Jean-Claude
Published: March 2007

That oscillations of the cytoplasmic free Ca2+ concentration ([Ca2+]i) in β-cells induce oscillations of insulin secretion is not disputed, but whether metabolism-driven oscillations of secretion can occur in the absence of [Ca2+]i oscillations is still debated. Because this possibility is based partly on the results of experiments using islets from aged, hyperglycemic, hyperinsulinemic ob/ob mice, we compared [Ca2+]i and insulin secretion patterns of single islets from 4- and 10-month-old, normal NMRI mice to those of islets from 7- and 10-month- old ob/ob mice (Swedish colony) and their lean littermates. The responses were subjected to cluster analysis to identify significant peaks. Control experiments without islets and with a constant insulin concentration were run to detect false peaks. Both ob/ob and NMRI islets displayed large synchronous oscillations of [Ca2+]i and insulin secretion in response to repetitive depolarizations with 30 mmol/l K+ in the presence of 0.1 mmol/l diazoxide and 12 mmol/l glucose. Continuous depolarization with high K+ steadily elevated [Ca2+]i in all types of islets, with no significant oscillation, and caused a biphasic insulin response. In islets from young (4-month-old) NMRI mice and 7-month-old lean mice, the insulin profile did not show significant peaks when [Ca2+]i was stable. In contrast, two or more peaks were detected over 20 min in the response of most ob/ob islets. Similar insulin peaks appeared in the insulin response of 10-month-old lean and NMRI mice. However, the size of the insulin peaks detected in the presence of stable [Ca2+]i was small, so that no more than 10–13% of total insulin secretion occurred in a pulsatile manner. In conclusion, insulin secretion does not oscillate when [Ca2+]i is stably elevated in β-cells from young normal mice. Some oscillations are observed in aged mice and are seen more often in ob/ob islets. These fluctuations of the insulin secretion rate at stably elevated [Ca2+]i, however, are small compared with the large oscillations induced by [Ca2+]i oscillations in β-cells.
Two-Photon Excitation Imaging of Pancreatic Islets With Various Fluorescent Probes
Authors: Takahashi Noriko , Nemoto Tomomi , Kimura Ryoichi , Tachikawa Akira , Miwa Akiko , Okado Haruo , Miyashita Yasushi , Iino Masamitsu , Kadowaki Takashi , Kasai Haruo
Published: March 2007

Various fluorescent probes were assessed for investigating intact islets of Langerhans using two-photon excitation imaging. Polar fluorescent tracers applied on the outside rapidly (within 3 min) penetrated deep into the islets via microvessels. Likewise, an adenovirus carrying a Ca2+-sensitive green fluorescent protein mutant gene, yellow cameleon 2.1, was successfully transfected and enabled ratiometric cytosolic Ca2+ measurement of cells in the deep layers of the islets. Interestingly, FM1-43, which is lipophilic and does not permeate the plasma membrane, also rapidly reached deep cell layers of the islets. In contrast, lipophilic fluorescent probes that permeate the plasma membrane (for example, fura-2-acetoxymethyl and BODIPY-forskolin) accumulated in the superficial cell layers of the islets, even 30 min after application. Thus, two-photon excitation imaging of pancreatic islets is a promising method for clarifying signaling mechanisms of islet cells, particularly when it is combined with membrane-impermeable probes. In addition, our data suggest that membrane-permeable antagonists may affect only the superficial cell layers of islets, and so their negative effects should be interpreted with caution.
Disturbances in β-Cell Function in Impaired Fasting Glycemia
Authors: van Haeften Timon W. , Pimenta Walkyria , Mitrakou Asimina , Korytkowski Mary , Jenssen Trond , Yki-Jarvinen Hannele , Gerich John E.
Published: March 2007

In a cross-sectional study, we assessed β-cell function and insulin sensitivity index (ISI) with hyperglycemic clamps (10 mmol/l) in 24 subjects with impaired fasting glycemia (IFG, fasting plasma glucose [FPG] between 6.1 and 7.0 mmol/l), 15 type 2 diabetic subjects (FPG >7.0 mmol/l), and 280 subjects with normal fasting glycemia (NFG, FPG <6.1 mmol/l). First-phase insulin release (0–10 min) was lower in IFG (geometric mean 541 pmol/l · 10 min; 95% confidence interval [CI] 416–702 pmol/l · 10 min) and in type 2 diabetes (geometric mean 376 pmol/l · 10 min; 95% CI 247–572 pmol/l · 10 min) than NFG (geometric mean 814 pmol/l · 10 min; 95% CI 759–873 pmol/l · 10 min) (P < 0.001). Second-phase insulin secretion (140–180 min) was also lower in IFG (geometric mean 251 pmol/l; 95% CI 198–318 pmol/l; P = 0.026) and type 2 diabetes (geometric mean 157 pmol/l; 95% CI 105–235 pmol/l; P < 0.001) than NFG (geometric mean 295 pmol/l; 95% CI 276–315 pmol/l). IFG and type 2 diabetic subjects had a lower ISI (0.15 ± 0.02 and 0.16 ± 0.02 μmol/kg fat-free mass [FFM]/min/pmol/l, respectively) than NFG (0.24 ± 0.01 μmol/kg FFM/min/pmol/l, P < 0.05). We found a stepwise decline in first-phase (and second-phase) secretion in NFG subjects with progressive decline in oral glucose tolerance (P < 0.05). IFG subjects with impaired glucose tolerance (IGT) had lower first-phase secretion than NFG subjects with IGT (P < 0.02), with comparable second-phase secretion and ISI. NFG and IFG subjects with a diabetic glucose tolerance (2-h glucose >11.1 mmol/l) had a lower ISI than their respective IGT counterparts (P < 0.05). We conclude that the early stages of glucose intolerance are associated with disturbances in β-cell function, while insulin resistance is seen more markedly in later stages.