bluuebunny/crossref_metadata_2025 huggingface dataset:
Modular Kinetic Analysis of the Adenine Nucleotide Translocator–Mediated Effects of Palmitoyl-CoA on the Oxidative Phosphorylation in Isolated Rat Liver Mitochondria
Authors: Ciapaite Jolita , Van Eikenhorst Gerco , Bakker Stephan J.L. , Diamant Michaela , Heine Robert J. , Wagner Marijke J. , Westerhoff Hans V. , Krab Klaas
Published: March 2007

To test whether long-chain fatty acyl-CoA esters link obesity with type 2 diabetes through inhibition of the mitochondrial adenine nucleotide translocator, we applied a system-biology approach, dual modular kinetic analysis, with mitochondrial membrane potential (Δψ) and the fraction of matrix ATP as intermediates. We found that 5 μmol/l palmitoyl-CoA inhibited adenine nucleotide translocator, without direct effect on other components of oxidative phosphorylation. Indirect effects depended on how oxidative phosphorylation was regulated. When the electron donor and phosphate acceptor were in excess, and the mitochondrial “work” flux was allowed to vary, palmitoyl-CoA decreased phosphorylation flux by 38% and the fraction of ATP in the medium by 39%. Δψ increased by 15 mV, and the fraction of matrix ATP increased by 46%. Palmitoyl-CoA had a stronger effect when the flux through the mitochondrial electron transfer chain was maintained constant: Δψ increased by 27 mV, and the fraction of matrix ATP increased 2.6 times. When oxidative phosphorylation flux was kept constant by adjusting the rate using hexokinase, Δψ and the fraction of ATP were not affected. Palmitoyl-CoA increased the extramitochondrial AMP concentration significantly. The effects of palmitoyl-CoA in our model system support the proposed mechanism linking obesity and type 2 diabetes through an effect on adenine nucleotide translocator.
S-Nitrosation of the Insulin Receptor, Insulin Receptor Substrate 1, and Protein Kinase B/Akt
Authors: Carvalho-Filho Marco A. , Ueno Mirian , Hirabara Sandro M. , Seabra Amedea B. , Carvalheira José B.C. , de Oliveira Marcelo G. , Velloso Lício A. , Curi Rui , Saad Mario J.A.
Published: March 2007

Evidence demonstrates that exogenous nitric oxide (NO) and the NO produced by inducible nitric oxide synthase (iNOS) can induce insulin resistance in muscle. Here, we investigated whether this insulin resistance could be mediated by S-nitrosation of proteins involved in early steps of the insulin signal transduction pathway. Exogenous NO donated by S-nitrosoglutathione (GSNO) induced in vitro and in vivo S-nitrosation of the insulin receptor β subunit (IRβ) and protein kinase B/Akt (Akt) and reduced their kinase activity in muscle. Insulin receptor substrate (IRS)-1 was also rapidly S-nitrosated, and its expression was reduced after chronic GSNO treatment. In two distinct models of insulin resistance associated with enhanced iNOS expression—diet-induced obesity and the ob/ob diabetic mice—we observed enhanced S-nitrosation of IRβ/IRS-1 and Akt in muscle. Reversal of S-nitrosation of these proteins by reducing iNOS expression yielded an improvement in insulin action in both animal models. Thus, S-nitrosation of proteins involved in insulin signal transduction is a novel molecular mechanism of iNOS-induced insulin resistance.
Impact of Genetic Versus Environmental Factors on the Control of Muscle Glycogen Synthase Activation in Twins
Authors: Poulsen Pernille , Wojtaszewski Jørgen F.P. , Petersen Inge , Christensen Kaare , Richter Erik A. , Beck-Nielsen Henning , Vaag Allan
Published: March 2007

Storage of glucose as glycogen accounts for the largest proportion of muscle glucose metabolism during insulin infusion in normal and insulin-resistant subjects. Studies in first-degree relatives have indicated a genetic origin of the defective insulin activation of muscle glycogen synthase (GS) in type 2 diabetes. The aim of this study was to evaluate the relative impact of genetic versus nongenetic factors on muscle GS activation and regulation in young and elderly twins examined with a 2-h euglycemic-hyperinsulinemic (40 mU · m−2 · min−1) clamp combined with indirect calorimetry and excision of muscle biopsies. The etiological components were determined using structural equation modeling. Fractional GS activity; GS phosphorylation at sites 2, 2 + 2a, and 3a + 3b corrected for total GS protein; and GS kinase 3 (GSK3) activity were similar in both age groups, whereas total GS activity and protein were lower in elderly compared with younger twins. GS fractional activity increased significantly during insulin stimulation in both young and elderly twins. Conversely, there was a significant decrease in GS phosphorylation at site 3a + 3b and GSK3 activity during insulin stimulation in both age groups, whereas GS phosphorylation at site 2 and 2 + 2a only decreased on insulin stimulation in the younger twins. The increment in whole-body glucose disposal (Rd) and nonoxidative glucose metabolism (insulin − basal) correlated significantly with the increment in GS fractional activity. Fractional GS activity had a major environmental component in both age groups. GSK3 activity exhibited a genetic component in young (basal: a2 = 0.42; insulin: a2 = 0.58) and elderly (insulin: a2 = 0.56) twins. Furthermore, GS phosphorylation at site 2 (insulin: a2 = 0.69) in the elderly and at site 3a + 3b (insulin: a2 = 0.50) in the young twins had a genetic component. In conclusion, GSK3 activity and GS phosphorylation, particularly at sites 2 and 3a + 3b, had major genetic components. Total and fractional GS activities per se were, on the other hand, predominantly controlled by environmental factors. Moreover, GS activity was intact with increasing age, despite a significant reduction in nonoxidative glucose metabolism.
Oscillations of Fatty Acid and Glycerol Release From Human Subcutaneous Adipose Tissue In Vivo
Authors: Karpe Fredrik , Fielding Barbara A. , Coppack Simon W. , Lawrence Victor J. , Macdonald Ian A. , Frayn Keith N.
Published: March 2007

We sought evidence for pulsatility of lipolysis in human subcutaneous adipose tissue in vivo. Arterialized and adipose tissue venous blood samples were drawn at 2-min intervals from nine healthy subjects. This procedure was repeated during hyperinsulinemic-euglycemic clamp to remove insulin pulsatility. We found evidence for pulsatile release of both nonesterified fatty acids (NEFAs) (seven of nine subjects) and glycerol (five of six subjects) with a period of ∼12–14 min. This pulsatility was maintained even during the hyperinsuline-mic clamp. Checks were made for spurious pulse detection, including the creation of “mock” venoarterialized differences by subtracting one subject’s arterialized concentrations from another’s venous; the peaks detected were less consistent in character than with real data (peak width, P = 0.006; peak interval, P < 0.004). Significant cross-correlations between NEFA and glycerol release also provided evidence of a real effect. Arterialized norepinephrine concentrations were also pulsatile, but the period did not match that of NEFA and glycerol release. Insulin concentrations were pulsatile with a typical period of 12 min, but this was not significantly cross-correlated with lipolysis. We conclude that release from adipose tissue of the products of lipolysis is pulsatile in humans.
Effects of Diet and Genetic Background on Sterol Regulatory Element-Binding Protein-1c, Stearoyl-CoA Desaturase 1, and the Development of the Metabolic Syndrome
Authors: Biddinger Sudha B. , Almind Katrine , Miyazaki Makoto , Kokkotou Efi , Ntambi James M. , Kahn C. Ronald
Published: March 2007

Both environmental and genetic factors play important roles in the development of the metabolic syndrome. To elucidate how these factors interact under normal conditions, C57Bl/6 (B6) and 129S6/SvEvTac (129) mice were placed on a low-fat or high-fat diet. Over 18 weeks, the 129 strain developed features of the metabolic syndrome, notably obesity, hyperinsulinemia, and glucose intolerance only on the high-fat diet; the B6 strain on the other hand developed these features on both diets. High-fat feeding of both strains led to decreased serum triglycerides, hepatic steatosis, and hypercholesterolemia; however, B6 mice developed worse steatosis and a larger increase in LDL cholesterol. Both B6 background and high-fat feeding increased sterol regulatory element-binding protein-1c (SREBP-1c), a key regulator of lipogenic gene transcription, and its downstream targets. Stearoyl-CoA desaturase 1 (SCD1), an enzyme that regulates monounsaturated fatty acid (MUFA) synthesis, was also increased at the mRNA and enzyme activity levels by both high-fat feeding and B6 background. Furthermore, lipid analysis revealed increased hepatic triglycerides and MUFAs in B6 and high-fat-fed mice. Thus, dietary fat and genetic background act through SREBP-1c and SCD1 to affect hepatic lipid metabolism contributing to the development of the metabolic syndrome.
Short-Term Overexpression of a Constitutively Active Form of AMP-Activated Protein Kinase in the Liver Leads to Mild Hypoglycemia and Fatty Liver
Authors: Foretz Marc , Ancellin Nicolas , Andreelli Fabrizio , Saintillan Yannick , Grondin Pascal , Kahn Axel , Thorens Bernard , Vaulont Sophie , Viollet Benoît
Published: March 2007

AMP-activated protein kinase (AMPK) is a major therapeutic target for the treatment of diabetes. We investigated the effect of a short-term overexpression of AMPK specifically in the liver by adenovirus-mediated transfer of a gene encoding a constitutively active form of AMPKα2 (AMPKα2-CA). Hepatic AMPKα2-CA expression significantly decreased blood glucose levels and gluconeogenic gene expression. Hepatic expression of AMPKα2-CA in streptozotocin-induced and ob/ob diabetic mice abolished hyperglycemia and decreased gluconeogenic gene expression. In normal mouse liver, AMPKα2-CA considerably decreased the refeeding-induced transcriptional activation of genes encoding proteins involved in glycolysis and lipogenesis and their upstream regulators, SREBP-1 (sterol regulatory element–binding protein-1) and ChREBP (carbohydrate response element–binding protein). This resulted in decreases in hepatic glycogen synthesis and circulating lipid levels. Surprisingly, despite the inhibition of hepatic lipogenesis, expression of AMPKα2-CA led to fatty liver due to the accumulation of lipids released from adipose tissue. The relative scarcity of glucose due to AMPKα2-CA expression led to an increase in hepatic fatty acid oxidation and ketone bodies production as an alternative source of energy for peripheral tissues. Thus, short-term AMPK activation in the liver reduces blood glucose levels and results in a switch from glucose to fatty acid utilization to supply energy needs.
Essential Role of Chicken Ovalbumin Upstream Promoter-Transcription Factor II in Insulin Secretion and Insulin Sensitivity Revealed by Conditional Gene Knockout
Authors: Bardoux Pascale , Zhang Pili , Flamez Daisy , Perilhou Anaïs , Lavin Tiphaine Aguirre , Tanti Jean-François , Hellemans Karine , Gomas Emmanuel , Godard Cécile , Andreelli Fabrizio , Buccheri Maria Antonietta , Kahn Axel , Le Marchand-Brustel Yannick , Burcelin Rémy , Schuit Frans , Vasseur-Cognet Mireille
Published: March 2007

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) has been implicated in the control of blood glucose by its potent effect on expression and signaling of various nuclear receptors. To understand the role of COUP-TFII in glucose homeostasis, conditional COUP-TFII-deficient mice were generated and crossed with mice expressing Cre under the control of rat insulin II gene promoter, resulting in deletion of COUP-TFII in pancreatic β-cells. Homozygous mutants died before birth for yet undetermined reasons. Heterozygous mice appeared healthy at birth and showed normal growth and fertility. When challenged intraperitoneally, the animals had glucose intolerance associated with reduced glucose-stimulated insulin secretion. Moreover, these heterozygous mice presented a mild increase in fasting and random-fed circulating insulin levels. In accordance, islets isolated from these animals exhibited higher insulin secretion in low glucose conditions and markedly decreased glucose-stimulated insulin secretion. Their pancreata presented normal microscopic architecture and insulin content up to 16 weeks of study. Altered insulin secretion was associated with peripheral insulin resistance in whole animals. It can be concluded that COUP-TFII is a new, important regulator of glucose homeostasis and insulin sensitivity.
Uncoupling Protein 1 Is Necessary for Norepinephrine-Induced Glucose Utilization in Brown Adipose Tissue
Authors: Inokuma Ken-ichi , Ogura-Okamatsu Yuko , Toda Chitoku , Kimura Kazuhiro , Yamashita Hitoshi , Saito Masayuki
Published: March 2007

Sympathetic stimulation activates glucose utilization in parallel with fatty acid oxidation and thermogenesis in brown adipose tissue (BAT) through the β-adrenergic receptors. To clarify the roles of the principal thermogenic molecule mitochondrial uncoupling protein 1 (UCP1) in the sympathetically stimulated glucose utilization, we investigated the uptake of 2-deoxyglucose (2-DG) into BAT and some other tissues of UCP1-knockout (KO) mice in vivo. In wild-type (WT) mice, administration of norepinephrine (NE) accelerated the disappearance of plasma 2-DG and increased 2-DG uptake into BAT and heart without any rise of plasma insulin level. In UCP1-KO mice, the stimulatory effect of NE on 2-DG uptake into BAT, but not into heart, disappeared completely. Insulin administration increased 2-DG uptake into BAT and also heart similarly in WT and UCP1-KO mice. NE also increased the activity of AMP-activated protein kinase (AMP kinase) in BAT of WT but not UCP1-KO mice. Our results, together with reports that the activation of AMP kinase increases glucose transport in myocytes, suggest that the sympathetically stimulated glucose utilization in BAT is due to the serial activation of UCP1 and AMP kinase.
Functional Defects and the Influence of Age on the Frequency of CD4+CD25+ T-Cells in Type 1 Diabetes
Authors: Brusko Todd M. , Wasserfall Clive H. , Clare-Salzler Michael J. , Schatz Desmond A. , Atkinson Mark A.
Published: March 2007

CD4+CD25+ T-cells appear to play a crucial role in regulating the immune response. Therefore, we evaluated the peripheral blood frequency and function of CD4+CD25+ T-cells in 70 type 1 diabetic patients and 37 healthy individuals. Interestingly, a positive correlation was observed between increasing age and CD4+CD25+ T-cell frequency in both subject groups. In contrast to previous studies of nonobese diabetic mice and type 1 diabetic patients, similar frequencies of CD4+CD25+ and CD4+CD25+Bright T-cells were observed in healthy control subjects and type 1 diabetic patients of similar age. There was no difference between type 1 diabetic subjects of recent-onset versus those with established disease in terms of their CD4+CD25+ or CD4+CD25+BrightT-cell frequency. However, type 1 diabetic patients were markedly defective in their ability to suppress the proliferation of autologous effector T-cells in vitro. This type 1 diabetes-associated defect in suppression was associated with reduced production of interleukin (IL)-2, γ-interferon, and transforming growth factor-β, whereas other cytokines including those of adaptive and innate immunity (IL-10, IL-1β, IL-6, IL-8, IL-12p70, and tumor necrosis factor-α) were similar in control subjects and type 1 diabetic patients. These data suggest that age strongly influences the frequency of CD4+CD25+ T-cells and that function, rather than frequency, may represent the means by which these cells associate with type 1 diabetes in humans.
Autoimmune Diabetes Onset Results From Qualitative Rather Than Quantitative Age-Dependent Changes in Pathogenic T-Cells
Authors: You Sylvaine , Belghith Mériam , Cobbold Stephen , Alyanakian Marie-Alexandra , Gouarin Christine , Barriot Samia , Garcia Corinne , Waldmann Herman , Bach Jean-François , Chatenoud Lucienne
Published: March 2007

Diabetogenic T-cells can be detected in pre-diabetic nonobese diabetic (NOD) mice after transfer in NOD-SCID recipients. Here we demonstrate that 6-week-old pre-diabetic NOD mice, >2 months before disease onset, already harbor pathogenic T-cells in equal numbers to overtly diabetic animals. The delay in diabetes appearance is explained by the presence of regulatory CD4+CD25+ T-cells that control diabetogenic effectors and that are, in our hands, transforming growth factor (TGF)-β–dependent. Our present results suggest, however, that diabetes onset is only partly explained by a decline in this regulatory T-cell activity. Another major factor appears to be the progressive resistance of diabetogenic cells to TGF-β–dependent mediated inhibition. We propose that progression to overt disease correlates with the pathogenic T-cell’s escape from TGF-β–dependent T-cell–mediated regulation.
The Effect of C-Peptide on Cognitive Dysfunction and Hippocampal Apoptosis in Type 1 Diabetic Rats
Authors: Sima Anders A.F. , Li Zhen-guo
Published: March 2007

Primary diabetic encephalopathy is a recently recognized late complication of diabetes resulting in a progressive decline in cognitive faculties. In the spontaneously type 1 diabetic BB/Wor rat, we recently demonstrated that cognitive impairment was associated with hippocampal apoptotic neuronal loss. Here, we demonstrate that replacement of proinsulin C-peptide in this insulinopenic model significantly prevented spatial learning and memory deficits and hippocampal neuronal loss. C-peptide replacement prevented oxidative stress–, endoplasmic reticulum–, nerve growth factor receptor p75–, and poly(ADP-ribose) polymerase–related apoptotic activities. It partially ameliorated apoptotic stresses mediated via impaired insulin and IGF activities. These findings were associated with the prevention of increased expression of Bax and active caspase 3 and the frequency of caspase 3–positive neurons. The results show that several partially interrelated apoptotic mechanisms are involved in primary encephalopathy and suggest that impaired insulinomimetic action by C-peptide plays a prominent role in cognitive dysfunction and hippocampal apoptosis in type 1 diabetes. Although these abnormalities were not fully prevented by C-peptide replacement, the findings suggest that this regime will substantially prevent cognitive decline in the type 1 diabetic population.
Wound Inflammation in Diabeticob/obMice
Authors: Kämpfer Heiko , Schmidt Ronald , Geisslinger Gerd , Pfeilschifter Josef , Frank Stefan
Published: March 2007

This study focused on the regulation of prostaglandin (PG) production in diabetes-impaired wound tissue. Cyclooxygenase (COX)- 1 and -2 expression and activity were severely dysregulated in chronic wounds of diabetic ob/ob mice. Those wounds were characterized by a reduced expression of COX-1 and the presence of strongly elevated levels of COX-2 when compared with conditions observed in healthy animals. Resolution of the diabetic and impaired wound-healing phenotype by systemic administration of leptin into ob/ob mice increased COX-1 expression in wound margin keratinocytes and decreased COX-2 expression in inner wound areas to levels found in wild-type animals. Notably, improved wound healing was characterized by a marked increase in PGE2/PGD2 biosynthesis that colocalized with induced COX-1 in new tissue at the margin of the wound. COX-2 expression did not significantly contribute to PGE2/PGD2 production in impaired wound tissue. Accordingly, only late wound tissue from SC-560–treated (selective COX-1 inhibitor) but not celecoxib-treated (selective COX-2 inhibitor) ob/ob mice exhibited a severe loss in PGE2, PGD2, and prostacyclin at the wound site, and this change was associated with reduced keratinocyte numbers in the neo-epithelia. These data constitute strong evidence that a dysregulation of COX-1–coupled prostaglandin contributes to diabetes-impaired wound healing.
Single Nucleotide Polymorphisms in KATP Channels
Authors: Li Li , Shi Yun , Wang Xueren , Shi Weiwei , Jiang Chun
Published: March 2007

ATP-sensitive K+ channels (KATP channels) play an important role in glucose homeostasis. A single nucleotide polymorphism (SNP) in the Kir6.2 subunit causes a point mutation of Glu23 to lysine and reduces the ATP sensitivity of pancreatic KATP channels. The SNP found in 58% of Caucasians accounts for 15% of type 2 diabetes. Here we show evidence for dysregulations of muscular KATP channels with the E23K variation. We were particularly interested in the channel modulation by intracellular protons, as pH changes widely and frequently in skeletal muscles. Surprisingly, we found that the defect of the E23K variant was more related to pH than ATP. A level of intracellular acidification seen during exercise not only activated the E23K channel more readily than the wild type, but also relieved the channel inhibition by ATP, leading to a vast increase in the channel open-state probability by approximately sevenfold at pH 6.8 over the wild-type channel at pH 7.4. Considering the reduction in sarcolemmal excitability, muscle fatigue, and impairment of muscular glucose uptake found previously by genetically disrupting KATP channels, it is likely that the E23K variant in muscular KATP channels affects systemic glucose homeostasis and poses an important risk factor for type 2 diabetes and obesity.
Variations in Peptide YY and Y2 Receptor Genes Are Associated With Severe Obesity in Pima Indian Men
Authors: Ma Lijun , Tataranni P. Antonio , Hanson Robert L. , Infante Aniello M. , Kobes Sayuko , Bogardus Clifton , Baier Leslie J.
Published: March 2007

Peptide YY (PYY) and Y2 receptor (Y2R) may be important in the central regulation of body weight and food intake. To determine whether genetic variation in PYY and/or Y2R may contribute to morbid obesity in humans, these genes were sequenced in 83 extremely obese Pima Indians (BMI ≥50 kg/m2). Sequencing of PYY identified three single nucleotide polymorphsms (SNPs) in the untranslated region. Sequencing of the Y2R coding region identified one missense (Ala172Thr) substitution and two silent substitutions. Eight additional SNPs in the 5′ untranslated region of Y2R were identified from public databases. These SNPs were genotyped in 489 full-heritage adult Pimas (362 severely obese and 127 nondiabetic, nonobese subjects), who are not first-degree relatives, for association analysis. The PYY variants were not associated with obesity, whereas four variants from two haplotype blocks in Y2R were marginally associated (P = 0.054–0.067) with obesity. However, if the analysis was restricted to men (n = 167, 100 obese and 67 lean), the PYY variants and two SNPs in Y2R that were in complete linkage disequilibrium were significantly associated with severe obesity (P = 0.001 and P = 0.002, respectively). Our data suggest that the PYY-Y2R pathway may influence body weight through a sex-specific mechanism, but this finding requires confirmation in other populations.
Major Histocompatibility Complex–Linked Diabetes Susceptibility in NOD/Lt Mice
Authors: Pomerleau Darcy P. , Bagley Rebecca J. , Serreze David V. , Mathews Clayton E. , Leiter Edward H.
Published: March 2007

The diabetogenic major histocompatibility complex (MHC) (H2g7) of NOD mice comprises contributions from several class II loci collectively designated as Idd1. Introduction of the H2gx haplotype from the related but diabetes-resistant cataract Shionogi (CTS) strain demonstrated an additional MHC-linked locus designated Idd16. The NOD-related alloxan resistant (ALR)/Lt strain is also characterized by the H2gx haplotype, which does not differ from H2g7 from the class I H2-Kd gene distally through the class II and into the class III region. Polymorphisms distal to the heat shock protein 70 locus (Hspa1b) include a rare H2-Ddx rather than the H2g7 encoded Db allele. Two differential-length NOD.ALR-H2gx congenic stocks (D.R1 and D.R2), both containing H2-Ddx, significantly suppressed diabetogenesis. This protection was lost when ALR alleles between the class III region and H2-D were removed in a shorter interval congenic (D.R3). Because no differences were observed in the ALR-derived interval extending 0.41 mB proximal to H2-K in any of these congenic stocks, a component of what was originally designated “Idd16” was sited to an interval shorter than 7.33 mB, distinguishing D.R2 from D.R3. Evidence supporting the candidacy of the ALR/CTS-shared H2-Ddx MHC class I variant present in both diabetes-resistant stocks, but not the susceptible stock, is discussed.
Phosphatidylinositol 4,5-Bisphosphate Reverses Endothelin-1–Induced Insulin Resistance via an Actin-Dependent Mechanism
Authors: Strawbridge Andrew B. , Elmendorf Jeffrey S.
Published: March 2007

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) plays a pivotal role in insulin-stimulated glucose transport as an important precursor to PI 3,4,5-trisphosphate (PIP3) and a key regulator of actin polymerization. Since endothelin (ET)-1 impairs insulin sensitivity and PIP2 is a target of ET-1–induced signaling, we tested whether a change in insulin-stimulated PIP3 generation and signaling, PIP2-regulated actin polymerization, or a combination of both accounted for ET-1–induced insulin resistance. Concomitant with a time-dependent loss of insulin sensitivity, ET-1 caused a parallel reduction in plasma membrane PIP2. Despite decreased insulin-stimulated PI 3-kinase activity and PIP3 generation, ET-1 did not diminish downstream signaling to Akt-2. Furthermore, addition of exogenous PIP2, but not PIP3, restored insulin-regulated GLUT4 translocation and glucose transport impaired by ET-1. Microscopic and biochemical analyses revealed a PIP2-dependent loss of cortical filamentous actin (F-actin) in ET-1–treated cells. Restoration of insulin sensitivity by PIP2 add-back occurred concomitant with a reestablishment of cortical F-actin. The corrective effect of exogenous PIP2 in ET-1–induced insulin-resistant cells was not present in cells where cortical F-actin remained experimentally depolymerized. These data suggest that ET-1–induced insulin resistance results from reversible changes in PIP2-regulated actin polymerization and not PIP2-dependent signaling.
A Single Course of Anti-CD3 Monoclonal Antibody hOKT3γ1(Ala-Ala) Results in Improvement in C-Peptide Responses and Clinical Parameters for at Least 2 Years after Onset of Type 1 Diabetes
Authors: Herold Kevan C. , Gitelman Stephen E. , Masharani Umesh , Hagopian William , Bisikirska Brygida , Donaldson David , Rother Kristina , Diamond Beverly , Harlan David M. , Bluestone Jeffrey A.
Published: March 2007

Despite advances in understanding autoimmune diabetes in animal models, there has been little progress in altering the natural course of the human disease, which involves progression to insulin deficiency. Studies with immunosuppressive agents have shown short-term effectiveness, but they have not induced tolerance, and continuous treatment is needed. We studied the effects of hOKT3γ1(Ala-Ala), a humanized Fc mutated anti-CD3 monoclonal antibody, on the progression of type 1 diabetes in patients with recent-onset disease in a randomized controlled trial. In general, the drug was well tolerated. A single course of treatment, within the first 6 weeks after diagnosis, preserved C-peptide responses to a mixed meal for 1 year after diagnosis (97 ± 9.6% of response at study entry in drug-treated patients vs. 53 ± 7.6% in control subjects, P < 0.01), with significant improvement in C-peptide responses to a mixed meal even 2 years after treatment (P < 0.02). The improved C-peptide responses were accompanied by reduced HbA1c and insulin requirements. Clinical responses to drug treatment were predicted by an increase in the relative number of CD8+ T-cells in the peripheral blood after the lymphocyte count recovered 2 weeks after the last dose of drug. We conclude that treatment with the anti-CD3 monoclonal antibody hOKT3γ1(Ala-Ala) results in improved C-peptide responses and clinical parameters in type 1 diabetes for at least 2 years in the absence of continued immunosuppressive medications.
Imaging β-Cell Death With a Near-Infrared Probe
Authors: Medarova Zdravka , Bonner-Weir Susan , Lipes Myra , Moore Anna
Published: March 2007

Evidence exists for an essential role of β-cell apoptosis in the pathology of type 1 and type 2 diabetes. Current methods for diabetes-associated apoptosis detection, however, suffer the drawbacks of relying on in situ–based strategies. In this study, we attempted to measure, both in vitro and ex vivo, levels of β-cell apoptosis in diabetic mice using Cy5.5-labeled annexin V. We used streptozotocin-treated BALB/c mice and NOD mice of different ages as models of type 1 diabetes and db/db mice as a model of type 2 diabetes. With annexin V Cy5.5, we established differences in levels of apoptosis between diabetic and control animals. Intravenously administered annexin V Cy5.5 accumulated in pancreata of diabetic mice but not in nondiabetic controls. Furthermore, its localization was specific to apoptotic events within diabetic islets; its selectivity was supported by transferase-mediated dUTP nick-end labeling staining. Because annexin V defines an early marker of apoptosis and the developed probe is suitable for in vivo administration, it may provide a promising tool for real-time identification in intact animals of the earliest stages of diabetes-associated β-cell death and for tracing the events that characterize the pathology of the disease.
β-Cell Secretory Products Activate α-Cell ATP-Dependent Potassium Channels to Inhibit Glucagon Release
Authors: Franklin Isobel , Gromada Jesper , Gjinovci Asllan , Theander Sten , Wollheim Claes B.
Published: March 2007

Glucagon, secreted from islet α-cells, mobilizes liver glucose. During hyperglycemia, glucagon secretion is inhibited by paracrine factors from other islet cells, but in type 1 and type 2 diabetic patients, this suppression is lost. We investigated the effects of β-cell secretory products zinc and insulin on isolated rat α-cells, intact islets, and perfused pancreata. Islet glucagon secretion was markedly zinc sensitive (IC50 = 2.7 μmol/l) more than insulin release (IC50 = 10.7 μmol/l). Glucose, the mitochondrial substrate pyruvate, and the ATP-sensitive K+ channel (KATP channel) inhibitor tolbutamide stimulated isolated α-cell electrical activity and glucagon secretion. Zinc opened KATP channels and inhibited both electrical activity and pyruvate (but not arginine)-stimulated glucagon secretion in α-cells. Insulin tran-siently increased KATP channel activity, inhibited electrical activity and glucagon secretion in α-cells, and inhibited pancreatic glucagon output. Insulin receptor and KATP channel subunit transcripts were more abundant in α- than β-cells. Transcript for the glucagon-like peptide 1 (GLP-1) receptor was not detected in α-cells nor did GLP-1 stimulate α-cell glucagon release. β-Cell secretory products zinc and insulin therefore inhibit glucagon secretion most probably by direct activation of KATP channels, thereby masking an α-cell metabolism secretion coupling pathway similar to β-cells.
Reduction of Hepatic and Adipose Tissue Glucocorticoid Receptor Expression With Antisense Oligonucleotides Improves Hyperglycemia and Hyperlipidemia in Diabetic Rodents Without Causing Systemic Glucocorticoid Antagonism
Authors: Watts Lynnetta M. , Manchem Vara Prasad , Leedom Thomas A. , Rivard Amber L. , McKay Robert A. , Bao Dingjiu , Neroladakis Teri , Monia Brett P. , Bodenmiller Diane M. , Cao Julia Xiao-Chun , Zhang Hong Yan , Cox Amy L. , Jacobs Steven J. , Michael M. Dodson , Sloop Kyle W. , Bhanot Sanjay
Published: March 2007

Glucocorticoids (GCs) increase hepatic gluconeogenesis and play an important role in the regulation of hepatic glucose output. Whereas systemic GC inhibition can alleviate hyperglycemia in rodents and humans, it results in adrenal insufficiency and stimulation of the hypothalamic-pituitary-adrenal axis. In the present study, we used optimized antisense oligonucleotides (ASOs) to cause selective reduction of the glucocorticoid receptor (GCCR) in liver and white adipose tissue (WAT) and evaluated the resultant changes in glucose and lipid metabolism in several rodent models of diabetes. Treatment of ob/ob mice with GCCR ASOs for 4 weeks resulted in ∼75 and ∼40% reduction in GCCR mRNA expression in liver and WAT, respectively. This was accompanied by ∼65% decrease in fed and ∼30% decrease in fasted glucose levels, a 60% decrease in plasma insulin concentration, and ∼20 and 35% decrease in plasma resistin and tumor necrosis factor-α levels, respectively. Furthermore, GCCR ASO reduced hepatic glucose production and inhibited hepatic gluconeogenesis in liver slices from basal and dexamethasone-treated animals. In db/db mice, a similar reduction in GCCR expression caused ∼40% decrease in fed and fasted glucose levels and ∼50% reduction in plasma triglycerides. In ZDF and high-fat diet–fed streptozotocin-treated (HFD-STZ) rats, GCCR ASO treatment caused ∼60% reduction in GCCR expression in the liver and WAT, which was accompanied by a 40–70% decrease in fasted glucose levels and a robust reduction in plasma triglyceride, cholesterol, and free fatty acids. No change in circulating corticosterone levels was seen in any model after GCCR ASO treatment. To further demonstrate that GCCR ASO does not cause systemic GC antagonism, normal Sprague-Dawley rats were challenged with dexamethasone after treating with GCCR ASO. Dexamethasone increased the expression of GC-responsive genes such as PEPCK in the liver and decreased circulating lymphocytes. GCCR ASO treatment completely inhibited the increase in dexamethasone-induced PEPCK expression in the liver without causing any change in the dexamethasone-induced lymphopenia. These studies demonstrate that tissue-selective GCCR antagonism with ASOs may be a viable therapeutic strategy for the treatment of the metabolic syndrome.
Type A Insulin Resistance Syndrome Revealing a Novel Lamin A Mutation
Authors: Young Jacques , Morbois-Trabut Louise , Couzinet Béatrice , Lascols Olivier , Dion Elisabeth , Béréziat Véronique , Fève Bruno , Richard Isabelle , Capeau Jacqueline , Chanson Philippe , Vigouroux Corinne
Published: March 2007

Particular forms of polycystic ovary syndrome with severe hyperandrogenism, acanthosis nigricans, and marked insulin resistance, defining the type A insulin resistance syndrome, are due to insulin receptor gene mutations. However, the majority of affected individuals do not have such mutation, arguing for the genetic heterogeneity of this syndrome. The familial partial lipodystrophy of the Dunnigan type, one of the diseases due to mutations in the lamin A/C (LMNA) gene, is characterized by a lipodystrophic phenotype and shares some clinical and metabolic features with the type A syndrome. We describe here the case of a nonobese 24-year-old woman affected with type A syndrome without clinical lipodystrophy. We linked this phenotype to a novel heterozygous missense mutation in the LMNA, predicting a G602S amino acid substitution in lamin A. This mutation cosegregated with impaired glucose tolerance, insulin resistance, and acanthosis nigricans in the absence of clinical lipodystrophy in the family. The skin fibroblasts from the proband exhibited nuclear alterations similar to those described in other laminopathies, and showed several defects in the insulin transduction pathway. This study further extends the vast range of diseases linked to LMNA mutations and identifies another genetic cause for the type A insulin resistance syndrome.
Assessing the Predictive Accuracy of QUICKI as a Surrogate Index for Insulin Sensitivity Using a Calibration Model
Authors: Chen Hui , Sullivan Gail , Quon Michael J.
Published: March 2007

The quantitative insulin-sensitivity check index (QUICKI) has an excellent linear correlation with the glucose clamp index of insulin sensitivity (SIClamp) that is better than that of many other surrogate indexes. However, correlation between a surrogate and reference standard may improve as variability between subjects in a cohort increases (i.e., with an increased range of values). Correlation may be excellent even when prediction of reference values by the surrogate is poor. Thus, it is important to evaluate the ability of QUICKI to accurately predict insulin sensitivity as determined by the reference glucose clamp method. In the present study, we used a calibration model to compare the ability of QUICKI and other simple surrogates to predict SIClamp. Predictive accuracy was evaluated by both root mean squared error of prediction as well as a more robust leave-one-out cross-validation–type root mean squared error of prediction (CVPE). Based on data from 116 glucose clamps obtained from nonobese, obese, type 2 diabetic, and hypertensive subjects, we found that QUICKI and log (homeostasis model assessment [HOMA]) were both excellent at predicting SIClamp (CVPE = 1.45 and 1.51, respectively) and significantly better than HOMA, 1/HOMA, and fasting insulin (CVPE = 3.17, P < 0.001; 1.67, P < 0.02; and 2.85, P < 0.001, respectively). QUICKI and log(HOMA) also had the narrowest distribution of residuals (measured SIClamp − predicted SIClamp). In a subset of subjects (n = 78) who also underwent a frequently sampled intravenous glucose tolerance test with minimal model analysis, QUICKI was significantly better than the minimal model index of insulin sensitivity (SIMM) at predicting SIClamp (CVPE = 1.54 vs. 1.98, P = 0.001). We conclude that QUICKI and log(HOMA) are among the most accurate surrogate indexes for determining insulin sensitivity in humans.
Role of Upstream Stimulatory Factors in Regulation of Renal Transforming Growth Factor-β1
Authors: Zhu Yanqing , Casado Marta , Vaulont Sophie , Sharma Kumar
Published: March 2007

We previously identified an E-box to be implicated in high-glucose–induced transforming growth factor-β1 (TGF-β1) gene stimulation in murine mesangial cells. In the present study, we evaluated the role of upstream stimulatory factors (USFs) in mediating glucose-induced stimulation of TGF-β1. Mesangial cells cultured in glucose concentrations exceeding 2.7 mmol/l d-glucose exhibited increased levels of USF1 and USF2 protein by Western analysis and electrophoretic mobility shift assay (EMSA). An E-box element from the murine TGF-β1 promoter revealed USF1 and USF2 binding by EMSA. Chromatin immunoprecipitation assay revealed in vivo binding of USF1 to a glucose-responsive region of the TGF-β1 promoter. Transient cotransfection studies of 293 cells with USF1 led to a twofold increase in TGF-β1 promoter activity and a 46% increase in secreted TGF-β1 protein levels. Wild-type and USF2 knockout mice exhibited a 2.5-fold stimulation of renal TGF-β1 expression upon fasting and refeeding with a carbohydrate-rich diet, whereas USF1 knockout mice exhibited only a minimal increase of renal TGF-β1 upon refeeding. USF1 mRNA levels were increased in mouse kidneys with carbohydrate refeeding, and USF1 protein was increased in diabetic rat kidneys compared with controls. We conclude that USF1 is stimulated by modest increases in glucose concentration in murine mesangial cells, bind to the murine TGF-β1 promoter, contribute to carbohydrate-induced renal TGF-β1 expression, and may play a role in diabetes-related gene regulation in the kidney.
Long-Term Efficacy of Leptin Replacement in Patients With Generalized Lipodystrophy
Authors: Javor Edward D. , Cochran Elaine K. , Musso Carla , Young Janice Ryan , DePaoli Alex M. , Gorden Phillip
Published: March 2007

Ectopic fat accumulation has been implicated as a contributing factor in the abnormal metabolic state of obesity. One human model of ectopic fat deposition is generalized lipodystrophy. Generalized lipodystrophy is a rare disorder characterized by a profound deficiency of adipose tissue with resultant loss of triglyceride storage capacity and reduced adipokines, including leptin. Subjects with generalized lipodystrophy and reduced leptin levels often have an increased appetite leading to hyperphagia. Excess fuel consumption, coupled with a lack of adipose tissue, contributes to the significant ectopic triglyceride accumulation in the muscle and liver seen in these subjects. This ectopic fat, along with the deficiency in leptin signaling and perhaps other adipokines, likely contributes to insulin resistance, diabetes, and hepatic steatosis. We report here the long-term effects of leptin replacement in a cohort of these subjects. Fifteen patients with generalized lipodystrophy were treated with twice-daily recombinant methionyl human leptin (r-metHuLeptin) for 12 months. We evaluated metabolic parameters at baseline and every 4 months. Antidiabetes medications were decreased or discontinued as necessary. Reductions were seen in serum fasting glucose (from 205 ± 19 to 126 ± 11 mg/dl; P < 0.001), HbA1c (from 9 ± 0.4 to 7.1 ± 0.5%; P < 0.001), triglycerides (from 1,380 ± 500 to 516 ± 236 mg/dl; P < 0.001), LDL (from 139 ± 16 to 85 ± 7 mg/dl; P < 0.01), and total cholesterol (from 284 ± 40 to 167 ± 21 mg/dl; P < 0.01). HDL was unchanged (from 31 ± 3 to 29 ± 2 mg/dl; P = 0.9). Liver volumes were significantly reduced (from 3,663 ± 326 to 2,190 ± 159 cm3; P < 0.001), representing loss of steatosis. Decreases were seen in total body weight (from 61.8 ± 3.6 to 57.4 ± 3.4 kg; P = 0.02) and resting energy expenditure (from 1,929 ± 86 to 1,611 ± 101 kcal/24 h; P < 0.001). R-metHuLeptin led to significant and sustained improvements in glycemia, dyslipidemia, and hepatic steatosis. Leptin represents the first novel, effective, long-term treatment for severe forms of lipodystrophy.
Five-Year Follow-Up After Clinical Islet Transplantation
Authors: Ryan Edmond A. , Paty Breay W. , Senior Peter A. , Bigam David , Alfadhli Eman , Kneteman Norman M. , Lakey Jonathan R.T. , Shapiro A.M. James
Published: March 2007

Islet transplantation can restore endogenous β-cell function to subjects with type 1 diabetes. Sixty-five patients received an islet transplant in Edmonton as of 1 November 2004. Their mean age was 42.9 ± 1.2 years, their mean duration of diabetes was 27.1 ± 1.3 years, and 57% were women. The main indication was problematic hypoglycemia. Forty-four patients completed the islet transplant as defined by insulin independence, and three further patients received >16,000 islet equivalents (IE)/kg but remained on insulin and are deemed complete. Those who became insulin independent received a total of 799,912 ± 30,220 IE (11,910 ± 469 IE/kg). Five subjects became insulin independent after one transplant. Fifty-two patients had two transplants, and 11 subjects had three transplants. In the completed patients, 5-year follow-up reveals that the majority (∼80%) have C-peptide present post–islet transplant, but only a minority (∼10%) maintain insulin independence. The median duration of insulin independence was 15 months (interquartile range 6.2–25.5). The HbA1c (A1C) level was well controlled in those off insulin (6.4% [6.1–6.7]) and in those back on insulin but C-peptide positive (6.7% [5.9–7.5]) and higher in those who lost all graft function (9.0% [6.7–9.3]) (P < 0.05). Those who resumed insulin therapy did not appear more insulin resistant compared with those off insulin and required half their pretransplant daily dose of insulin but had a lower increment of C-peptide to a standard meal challenge (0.44 ± 0.06 vs. 0.76 ± 0.06 nmol/l, P < 0.001). The Hypoglycemic score and lability index both improved significantly posttransplant. In the 128 procedures performed, bleeding occurred in 15 and branch portal vein thrombosis in 5 subjects. Complications of immunosuppressive therapy included mouth ulcers, diarrhea, anemia, and ovarian cysts. Of the 47 completed patients, 4 required retinal laser photocoagulation or vitrectomy and 5 patients with microalbuminuria developed macroproteinuria. The need for multiple antihypertensive medications increased from 6% pretransplant to 42% posttransplant, while the use of statin therapy increased from 23 to 83% posttransplant. There was no change in the neurothesiometer scores pre- versus posttransplant. In conclusion, islet transplantation can relieve glucose instability and problems with hypoglycemia. C-peptide secretion was maintained in the majority of subjects for up to 5 years, although most reverted to using some insulin. The results, though promising, still point to the need for further progress in the availability of transplantable islets, improving islet engraftment, preserving islet function, and reducing toxic immunosuppression.
Role for β1 Integrin and Its Associated α3, α5, and α6 Subunits in Development of the Human Fetal Pancreas
Authors: Wang Rennian , Li Jinming , Lyte Kristina , Yashpal Nina K. , Fellows Fraser , Goodyer Cynthia G.
Published: March 2007

The integrin receptors play a major role in tissue morphogenesis and homeostasis by regulating cell interactions with extracellular matrix proteins. We have examined the expression pattern of integrin subunits in the human fetal pancreas (8–20 weeks fetal age) and the relevance of β1 integrin function for insulin gene expression and islet cell survival. Its subunits α3, α5, and α6 β1 integrins are expressed in ductal cells at 8 weeks, before glucagon- and insulin-immunoreactive cells bud off; their levels gradually increase in both ductal cells and islet clusters up to 20 weeks. Colocalization of α3, α5 and α6 β1 integrins with endocrine cell markers was frequently observed in 8- to 20-week fetal pancreatic cells. When the β1 integrin receptor was functionally blocked in cultured islet-epithelial clusters with a β1 immunoneutralizing antibody or following transient β1 integrin small interfering RNA treatment, there was inhibition of cell adhesion to extracellular matrices, decreased expression of insulin, and increased cell apoptosis. These data offer evidence for dynamic and cell-specific changes in integrin expression during human pancreatic islet neogenesis. They also provide an initial insight into a molecular basis for cell-matrix interactions during islet development and suggest that β1 integrin plays a vital role in regulating islet cell adhesion, gene expression, and survival.
Aberrant Processing of Human Proislet Amyloid Polypeptide Results in Increased Amyloid Formation
Authors: Paulsson Johan F. , Westermark Gunilla T.
Published: March 2007

The amyloid present in the islets of Langerhans in type 2 diabetes is polymerized islet amyloid polypeptide (IAPP). The precursor protein proIAPP is posttranslationally modified, a process involving the removal of NH2- and COOH-terminal flanking peptides. This step is performed by the prohormone convertases PC2 and PC1/3. PC2 processes proIAPP preferably at the NH2-terminal processing site, and PC1/3 processes proIAPP exclusively at the COOH-terminal site. Little is known regarding the exact circumstances leading to islet amyloid formation. In this study, we have examined the possible significance of aberrant processing of proIAPP on amyloid formation in several in vitro cellular systems. In our studies, human (h)-proIAPP was transfected into β-TC-6 cells expressing both prohormone convertases and in which proIAPP is processed into IAPP. Additionally, h-proIAPP was transfected into three different pituitary-derived cell lines with different prohormone convertase profiles: AtT-20 cells (deficient in PC2), GH3 cells (deficient in PC1/3), and GH4C1 cells (deficient in both convertases). We followed the processing of h-proIAPP with antibodies specific for the respective cleavage sites and stained the cells with Congo red to verify the accumulation of amyloid. Incomplete processing of h-proIAPP that occurs in AtT-20 and GH4C1 cells resulted in the formation of intracellular amyloid. No amyloid developed in β-TC-6 and GH3 cells lines with full processing of proIAPP. An intracellular increase in proIAPP and/or its metabolic products may thus promote intracellular amyloid formation, thereby causing cell death. When extracellularly exposed, this amyloid might act as template for continuing amyloid formation from processed IAPP released from the surrounding β-cells.