bluuebunny/crossref_metadata_2025 huggingface dataset:
Induced Adiposity and Adipocyte Hypertrophy in Mice Lacking the AMP-Activated Protein Kinase-α2 Subunit
Authors: Villena Josep A. , Viollet Benoit , Andreelli Fabrizzio , Kahn Axel , Vaulont Sophie , Sul Hei Sook
Published: March 2007

AMP-activated protein kinase (AMPK) is considered as a cellular energy sensor that regulates glucose and lipid metabolism by phosphorylating key regulatory enzymes. Despite the major role of adipose tissue in regulating energy partitioning in the organism, the role of AMPK in this tissue has not been addressed. In the present study, we subjected AMPKα2 knockout (KO) mice to a high-fat diet to examine the effect of AMPK on adipose tissue formation. Compared with the wild type, AMPKα2 KO mice exhibited increased body weight and fat mass. The increase in adipose tissue mass was due to the enlargement of the preexisting adipocytes with increased lipid accumulation. However, we did not observe any changes in adipocyte marker expression, such as peroxisome proliferator–activated receptor-γ, CCAAT/enhancer-binding protein α (C/EBPα) and adipocyte fatty acid–binding protein (aFABP/aP2), or total cell number. Unlike impaired glucose homeostasis observed on normal diet feeding, when fed a high-fat diet AMPKα2 KO mice did not show differences in glucose tolerance and insulin sensitivity compared with wild-type mice. Our results suggest that the increase in lipid storage in adipose tissue in AMPKα2 KO mice may have protected these mice from further impairment of glucose homeostasis that normally accompanies high-fat feeding. Our study also demonstrates that lack of AMPKα2 subunit may be a factor contributing to the development of obesity.
Involvement of Hypothalamic Histamine H1 Receptor in the Regulation of Feeding Rhythm and Obesity
Authors: Masaki Takayuki , Chiba Seiichi , Yasuda Tohru , Noguchi Hitoshi , Kakuma Tetsuya , Watanabe Takeshi , Sakata Toshiie , Yoshimatsu Hironobu
Published: March 2007

Histamine H1 receptors (H1-Rs) are found in peripheral tissues and in regions of the hypothalamus that are concerned with regulating body composition. In the present study, we investigated the detailed mechanisms of histamine H1-Rs in the development of obesity. Histamine H1-R knockout (H1KO) mice gradually developed mature-onset obesity, which was accompanied by hyperphagia and decreased expression of uncoupling protein-1 (UCP-1) mRNA. Both younger nonobese (12-week-old) and older obese (48-week-old) H1KO mice exhibited impairment of the responsiveness to the leptin. In addition, disruption of the diurnal rhythm of feeding occurred before the onset of obesity in H1KO mice. Correction of these abnormal feeding rhythms by means of scheduled feeding caused a reduction in obesity and associated metabolic disorders in H1KO mice. Furthermore, central administration of a histamine H1-R agonist affected feeding behavior, body weight, and c-fos-like immunoreactivity in the hypothalamus. Taken together, these findings suggest that histamine H1-Rs are crucial for the regulation of feeding rhythm and in mediating the effects of leptin. Early disruption of H1-R-mediated functions in H1KO mice may lead to hyperphagia and decreased expression of UCP-1 mRNA, which may contribute to the development of obesity in these animals. In addition, centrally acting histamine H1-R may be a novel therapeutic target for the treatment of obesity and related metabolic disorders.
Mitochondrial Catalase Overexpression Protects Insulin-Producing Cells Against Toxicity of Reactive Oxygen Species and Proinflammatory Cytokines
Authors: Gurgul Ewa , Lortz Stephan , Tiedge Markus , Jörns Anne , Lenzen Sigurd
Published: March 2007

Insulin-producing cells are known for their extremely low antioxidant equipment with hydrogen peroxide (H2O2)-inactivating enzymes. Therefore, catalase was stably overexpressed in mitochondria and for comparison in the cytoplasmic compartment of insulin-producing RINm5F cells and analyzed for its protective effect against toxicity of reactive oxygen species (ROS) and proinflammatory cytokines. Only mitochondrial overexpression of catalase provided protection against menadione toxicity, a chemical agent that preferentially generates superoxide radicals intramitochondrially. On the other hand, the cytoplasmic catalase overexpression provided better protection against H2O2 toxicity. Mitochondrial catalase overexpression also preferentially protected against the toxicity of interleukin-1β (IL-1β) and a proinflammatory cytokine mixture (IL-1β, tumor necrosis factor-α [TNF-α], and γ-interferon [IFN-γ]) that is more toxic than IL-1β alone. Thus, it can be concluded that targeted overexpression of catalase in the mitochondria provides particularly effective protection against cell death in all situations in which ROS are generated intramitochondrially. The observed higher rate of cell death after exposure to a cytokine mixture in comparison with the weaker effect of IL-1β alone may be due to an additive toxicity of TNF-α through ROS formation in mitochondria. The results emphasize the central role of mitochondrially generated ROS in the cytokine-mediated cell destruction of insulin-producing cells.
Assessment of Cytotoxic Lymphocyte Gene Expression in the Peripheral Blood of Human Islet Allograft Recipients
Authors: Han Dongmei , Xu Xiumin , Baidal David , Leith Jenifer , Ricordi Camillo , Alejandro Rodolfo , Kenyon Norma S.
Published: March 2007

Studies in nonhuman primates have demonstrated that elevation of the cytotoxic lymphocyte (CL) genes granzyme B, perforin, and Fas ligand in peripheral blood precedes islet allograft rejection. The purpose of this study was to determine whether this approach has utility for prediction of human islet allograft loss. We studied 13 patients who had long-term type 1 diabetes and were treated with steroid-free immunosuppression and given sequential islet cell infusions. All recipients became insulin independent, and eight of them experienced deterioration in glycemic control, followed by reinitiation of insulin therapy. Frequent peripheral blood samples were collected to monitor CL gene mRNA levels with real-time PCR. For the eight back-to-insulin patients, there was a clear elevation of CL gene mRNA levels 25–203 days before the onset of frequent hyperglycemia. Granzyme B was the most reliable indicator of ongoing graft loss. Additional correlations with infection were noted; however, evidence of sensitization in antidonor mixed lymphocyte reaction was observed in seven of eight patients who experienced partial graft loss, whereas this was not seen when upregulated CL gene expression was associated with infection. The results suggest that, when taken into consideration with other clinical parameters, elevated CL gene levels may enable prediction of islet allograft loss.
Peptides From Common Viral and Bacterial Pathogens Can Efficiently Activate Diabetogenic T-Cells
Authors: Judkowski Valeria A. , Allicotti Gina M. , Sarvetnick Nora , Pinilla Clemencia
Published: March 2007

Cross-reactivity between an autoantigen and unknown microbial epitopes has been proposed as a molecular mechanism involved in the development of insulin-dependent diabetes (type 1 diabetes). Type 1 diabetes is an autoimmune disease that occurs in humans and the nonobese diabetic (NOD) mouse. BDC2.5 is an islet-specific CD4+ T-cell clone derived from the NOD mouse whose natural target antigen is unknown. A biometrical analysis of screening data from BDC2.5 T-cells and a positional scanning synthetic combinatorial library (PS-SCL) was used to analyze and rank all peptides in public viral and bacterial protein databases and identify potential molecular mimic sequences with predicted reactivity. Selected sequences were synthesized and tested for stimulatory activity with BDC2.5 T-cells. Active peptides were identified, and some of them were also able to stimulate spontaneously activated T-cells derived from young, pre-diabetic NOD mice, indicating that the reactivity of the BDC2.5 T-cell is directed at numerous mouse peptides. Our results provide evidence for their possible role as T-cell ligands involved in the activation of diabetogenic T-cells.
Progression to Islet Destruction in a Cyclophosphamide-Induced Transgenic Model
Authors: Matos Michael , Park Richard , Mathis Diane , Benoist Christophe
Published: March 2007

Type 1 diabetes appears to progress not as an uncontrolled autoimmune attack on the pancreatic islet β-cells, but rather in a highly regulated manner. Leukocytic infiltration of the pancreatic islets by autoimmune cells, or insulitis, can persist for long periods of time before the terminal destruction of β-cells. To gain insight on the final stage of diabetogenesis, we have studied progression to diabetes in a CD4+ T-cell receptor transgenic variant of the NOD mouse model, in which diabetes can be synchronously induced within days by a single injection of cyclophosphamide. A time-course analysis of the gene expression profiles of purified islets was performed using microarrays. Contrary to expectations, changes in transcripts subsequent to drug treatment did not reflect a perturbation of gene expression in CD4+ T-cells or a reduction in the expression of genes characteristic of regulatory T-cell populations. Instead, there was a marked decrease in transcripts of genes specific to B-cells, followed by an increase in transcripts of chemokine genes (cxcl1, cxcl5, and ccl7) and of other genes typical of the myelo-monocytic lineages. Interferon-γ dominated the changes in gene expression to a striking degree, because close to one-half of the induced transcripts issued from interferon-γ–regulated genes.
Long-Term Islet Graft Survival in NOD Mice by Abrogation of Recurrent Autoimmunity
Authors: Shi Qixin , Wang Donghua , Hadley Gregg A. , Bingaman Adam W. , Bartlett Stephen T. , Farber Donna L.
Published: March 2007

Islet transplantation has great potential for curing type 1 diabetes; however, long-term islet survival using conventional immunosuppression remains elusive. We present a novel strategy for inducing long-lasting islet graft survival in diabetic NOD mice in the absence of posttransplant immunosuppression by initial treatment with antilymphocyte serum (ALS) followed by coadministration of donor pancreatic lymph node cells (PLNCs). When treated with ALS/PLNC, diabetic NOD mice become normoglycemic and tolerated minor antigen-disparate islet grafts for >100 days and syngeneic islet grafts indefinitely. Donor T-cells are required for graft prolongation, and tolerant hosts have long-term donor T-cell chimerism. Strikingly, host autoreactive T-cells from mice with long-surviving islet grafts predominantly produce interleukin-4, whereas autoreactive T-cells from mice that rejected their islet grafts predominantly produce interferon-γ. We thus demonstrate a clinically relevant approach for ablation of recurrent autoimmunity in islet transplantation, involving donor lymphocyte-driven alteration of pathogenic autoreactive T-cells.
Glucokinase Is an Integral Component of the Insulin Granules in Glucose-Responsive Insulin Secretory Cells and Does Not Translocate During Glucose Stimulation
Authors: Arden Catherine , Harbottle Andrew , Baltrusch Simone , Tiedge Markus , Agius Loranne
Published: March 2007

The association of glucokinase with insulin secretory granules has been shown by cell microscopy techniques. We used MIN6 insulin-secretory cells and organelle fractionation to determine the effects of glucose on the subcellular distribution of glucokinase. After permeabilization with digitonin, 50% of total glucokinase remained bound intracellularly, while 30% was associated with the 13,000g particulate fraction. After density gradient fractionation of the organelles, immunoreactive glucokinase was distributed approximately equally between dense insulin granules and low-density organelles that cofractionate with mitochondria. Although MIN6 cells show glucose-responsive insulin secretion, glucokinase association with the granules and low-density organelles was not affected by glucose. Subfractionation of the insulin granule components by hypotonic lysis followed by sucrose gradient centrifugation showed that glucokinase colocalized with the granule membrane marker phogrin and not with insulin. PFK2 (6-phosphofructo-2-kinase-2/fructose-2,6-bisphosphatase)/FDPase-2, a glucokinase-binding protein, and glyceraldehyde phosphate dehydrogenase, which has been implicated in granule fusion, also colocalized with glucokinase after hypotonic lysis or detergent extaction of the granules. The results suggest that glucokinase is an integral component of the granule and does not translocate during glucose stimulation.
Glucose Intolerance in a Large Cohort of Mediterranean Women With Polycystic Ovary Syndrome
Authors: Gambineri Alessandra , Pelusi Carla , Manicardi Elisa , Vicennati Valentina , Cacciari Mauro , Morselli-Labate Antonio Maria , Pagotto Uberto , Pasquali Renato
Published: March 2007

The aim of this study was to investigate the phenotypic parameters and associated factors characterizing the development of glucose intolerance in polycystic ovary syndrome (PCOS). Among the 121 PCOS female subjects from the Mediterranean region, 15.7 and 2.5% displayed impaired glucose tolerance and type 2 diabetes, respectively. These subjects were included in a single group of overweight or obese subjects presenting with glucose intolerance (GI) states. PCOS women with normal glucose tolerance (81.8%) were subdivided into two groups: those who were overweight or obese and those of normal weight. Metabolic and hormonal characteristics of the GI group included significantly higher fasting and glucose-stimulated insulin levels, more severe insulin resistance, hyperandrogenemia, and significantly higher cortisol and androstenedione responses to 1–24 ACTH stimulation. One important finding was that lower birth weight and earlier age of menarche were associated with GI in PCOS women. Frequency of hirsutism, oligomenorrhea, acne, and acanthosis nigricans did not characterize women with GI. Our findings indicate that PCOS patients with GI represent a subgroup with specific clinical and hormonal characteristics. Our observations may have an important impact in preventative and therapeutic strategies.
Effect of a High-Protein, Low-Carbohydrate Diet on Blood Glucose Control in People With Type 2 Diabetes
Authors: Gannon Mary C. , Nuttall Frank Q.
Published: March 2007

There has been interest in the effect of various types and amounts of dietary carbohydrates and proteins on blood glucose. On the basis of our previous data, we designed a high-protein/low-carbohydrate, weight-maintaining, nonketogenic diet. Its effect on glucose control in people with untreated type 2 diabetes was determined. We refer to this as a low-biologically-available-glucose (LoBAG) diet. Eight men were studied using a randomized 5-week crossover design with a 5-week washout period. The carbohydrate:protein:fat ratio of the control diet was 55:15:30. The test diet ratio was 20:30:50. Plasma and urinary β-hydroxybutyrate were similar on both diets. The mean 24-h integrated serum glucose at the end of the control and LoBAG diets was 198 and 126 mg/dl, respectively. The percentage of glycohemoglobin was 9.8 ± 0.5 and 7.6 ± 0.3, respectively. It was still decreasing at the end of the LoBAG diet. Thus, the final calculated glycohemoglobin was estimated to be ∼6.3–5.4%. Serum insulin was decreased, and plasma glucagon was increased. Serum cholesterol was unchanged. Thus, a LoBAG diet ingested for 5 weeks dramatically reduced the circulating glucose concentration in people with untreated type 2 diabetes. Potentially, this could be a patient-empowering way to ameliorate hyperglycemia without pharmacological intervention. The long-term effects of such a diet remain to be determined.
Involvement of Brain-Derived Neurotrophic Factor in Early Retinal Neuropathy of Streptozotocin-Induced Diabetes in Rats
Authors: Seki Masaaki , Tanaka Takayuki , Nawa Hiroyuki , Usui Tomoaki , Fukuchi Takeo , Ikeda Kazuhito , Abe Haruki , Takei Nobuyuki
Published: March 2007

Although neurotrophins have been assessed as candidate therapeutic agents for neural complications of diabetes, their involvement in diabetic retinopathy has not been fully characterized. We found that the protein and mRNA levels of brain-derived neurotrophic factor (BDNF) in streptozotocin-induced diabetic rat retinas were reduced to 49% (P < 0.005) and 74% (P < 0.05), respectively, of those of normal control animals. In addition, dopaminergic amacrine cells appeared to be degenerating in the diabetic rat retinas, as revealed by tyrosine hydroxylase (TH) immunoreactivity. Overall TH protein levels in the retina were decreased to one-half that of controls (P < 0.01), reflecting reductions in the density of dopaminergic amacrine cells and the intensity of TH immunoreactivity within them. To confirm the neuropathological implications of BDNF reduction, we administered BDNF protein into the vitreous cavities of diabetic rats. Intraocular administration of BDNF rescued dopaminergic amacrine cells from neurodegeneration and counteracted the downregulation of TH expression, demonstrating its therapeutic potential. These findings suggest that the early retinal neuropathy of diabetes involves the reduced expression of BDNF and can be ameliorated by an exogenous supply of this neurotrophin.
Leptin Stimulates Ischemia-Induced Retinal Neovascularization
Authors: Suganami Eri , Takagi Hitoshi , Ohashi Hirokazu , Suzuma Kiyoshi , Suzuma Izumi , Oh Hideyasu , Watanabe Daisuke , Ojima Tomonari , Suganami Takayoshi , Fujio Yasushi , Nakao Kazuwa , Ogawa Yoshihiro , Yoshimura Nagahisa
Published: March 2007

Diabetic retinopathy is the leading cause of new blindness in adults in developed countries. Leptin, an adipocyte-derived hormone, stimulates endothelial proliferation and angiogenesis. This study was designed to elucidate the pathophysiologic role of leptin in the progression of retinal neovascularization. Using the retinopathy of prematurity model, a mouse model of ischemia-induced retinal neovascularization, we have demonstrated more pronounced retinal neovascularization in 17-day-old transgenic mice overexpressing leptin than in age-matched wild-type littermates. Ischemia-induced retinal neovascularization was markedly suppressed in 17-day-old leptin-deficient ob/ob mice. Western blot analysis revealed that a biologically active leptin receptor isoform is expressed in mouse retinal endothelial cells. Leptin receptor expression was also detected in primary cultures of porcine retinal endothelial cells, where it upregulated vascular endothelial growth factor (VEGF) mRNA expression. This effect was thought to be mediated at least partly through the activation of signal transducers and activators of transcription (STAT)3, because adenoviral transfection of the dominant-negative form of STAT3 abolished the leptin-induced upregulation of VEGF mRNA expression in retinal endothelial cells. This study provides evidence that leptin stimulates the ischemia-induced retinal neovasucularization possibly through the upregulation of endothelial VEGF, thereby suggesting that leptin antagonism may offer a novel therapeutic strategy to prevent or treat diabetic retinopathy.
Population-Based Assessment of Familial Clustering of Diabetic Nephropathy in Type 1 Diabetes
Authors: Harjutsalo Valma , Katoh Shuichi , Sarti Cinzia , Tajima Naoko , Tuomilehto Jaakko
Published: March 2007

We determined the magnitude of familial aggregation in the development of diabetic nephropathy (DN) among a population-based cohort of Finnish type 1 diabetic patients. Probands with type 1 diabetes were identified from the nationwide register of all Finnish cases diagnosed during 1965–1979. By 1998, there were 537 families with at least two siblings with type 1 diabetes. These 537 probands and their 616 diabetic siblings were followed for a diagnosis of DN until the end of 2001. We identified 323 cases of DN in these families. If the proband had DN, 38% of the siblings also had DN, whereas out of the diabetic siblings of the probands without DN, only 17% had DN (P = 0.001). Diabetic siblings of the nephropathic probands had a 2.3 times (95% CI 1.4–2.7) higher risk of DN compared with siblings of probands free of DN. The presence of a severe form of DN in the proband increases the risk threefold for diabetic siblings. Sex, the DN of the proband, the age at the onset of diabetes, and parental type 2 diabetes were significant predictors of DN among diabetic siblings. Although the majority of sibpairs with type 1 diabetes are discordant for DN, its presence in one sibling doubles the risk for the other diabetic siblings.
A Quantitative Trait Locus Influencing Fasting Plasma Glucose in Chromosome Region 18q22-23
Authors: Li Wei-Dong , Dong Chuanhui , Li Ding , Garrigan Cathleen , Price R. Arlen
Published: March 2007

Fasting plasma glucose is a multigenic trait related to both diabetes and obesity. We performed a genome scan for quantitative fasting plasma glucose levels in 320 families (1,514 subjects), segregating extreme obesity and normal weight using 382 autosomal microsatellite markers. We found significant linkages on 18q22-23 using family regression (logarithm of odds [LOD] 3.67, P = 0.00002, D18S1371 at 116 cM) and variance components (LOD 4.48, P < 0.00001) methods. Evidence for linkage remained strong when restricted to European Americans (260 families, 1,258 individuals). After an additional 60 families were added, the linkage signal strengthened (LOD 6.59). The result on 18q22-23 remained significant, even after controlling for both BMI and diabetes status. We also found suggestive linkages on chromosomes 2 (LOD 1.58, 216 cM) and 7 (LOD 1.78, 163 cM). Our results suggest that there is a quantitative trait locus in chromosome region 18q22-23 that influences fasting glucose levels and may play a role in the pathogenesis of type 2 diabetes. The strength of the serum glucose results after controlling for BMI suggests that this putative gene does not influence glucose levels merely through an effect on obesity.
A Recombinant Human Glucagon-Like Peptide (GLP)-1–Albumin Protein (Albugon) Mimics Peptidergic Activation of GLP-1 Receptor–Dependent Pathways Coupled With Satiety, Gastrointestinal Motility, and Glucose Homeostasis
Authors: Baggio Laurie L. , Huang Qingling , Brown Theodore J. , Drucker Daniel J.
Published: March 2007

Peptide hormones exert unique actions via specific G protein–coupled receptors; however, the therapeutic potential of regulatory peptides is frequently compromised by rapid enzymatic inactivation and clearance from the circulation. In contrast, recombinant or covalent coupling of smaller peptides to serum albumin represents an emerging strategy for extending the circulating t1/2 of the target peptide. However, whether larger peptide-albumin derivatives will exhibit the full spectrum of biological activities encompassed by the native peptide remains to be demonstrated. We report that Albugon, a human glucagon-like peptide (GLP)-1–albumin recombinant protein, activates GLP-1 receptor (GLP-1R)-dependent cAMP formation in BHK-GLP-1R cells, albeit with a reduced half-maximal concentration (EC50) (0.2 vs. 20 nmol/l) relative to the GLP-1R agonist exendin-4. Albugon decreased glycemic excursion and stimulated insulin secretion in wild-type but not GLP-1R−/− mice and reduced food intake after both intracerebroventricular and intraperitoneal administration. Moreover, intraperitoneal injection of Albugon inhibited gastric emptying and activated c-FOS expression in the area postrema, the nucleus of the solitary tract, the central nucleus of the amygdala, the parabrachial, and the paraventricular nuclei. These findings illustrate that peripheral administration of a larger peptide-albumin recombinant protein mimics GLP-1R–dependent activation of central and peripheral pathways regulating energy intake and glucose homeostasis in vivo.
Regeneration of the Heart in Diabetes by Selective Copper Chelation
Authors: Cooper Garth J.S. , Phillips Anthony R.J. , Choong Soon Y. , Leonard Bridget L. , Crossman David J. , Brunton Dianne H. , Saafi ’Etuate L. , Dissanayake Ajith M. , Cowan Brett R. , Young Alistair A. , Occleshaw Christopher J. , Chan Yih-Kai , Leahy Fiona E. , Keogh Geraldine F. , Gamble Gregory D. , Allen Grant R. , Pope Adèle J. , Boyd Peter D.W. , Poppitt Sally D. , Borg Thomas K. , Doughty Robert N. , Baker John R.
Published: March 2007

Heart disease is the major cause of death in diabetes, a disorder characterized by chronic hyperglycemia and cardiovascular complications. Although altered systemic regulation of transition metals in diabetes has been the subject of previous investigation, it is not known whether changed transition metal metabolism results in heart disease in common forms of diabetes and whether metal chelation can reverse the condition. We found that administration of the Cu-selective transition metal chelator trientine to rats with streptozotocin-induced diabetes caused increased urinary Cu excretion compared with matched controls. A CuII-trientine complex was demonstrated in the urine of treated rats. In diabetic animals with established heart failure, we show here for the first time that 7 weeks of oral trientine therapy significantly alleviated heart failure without lowering blood glucose, substantially improved cardiomyocyte structure, and reversed elevations in left ventricular collagen and β1 integrin. Oral trientine treatment also caused elevated Cu excretion in humans with type 2 diabetes, in whom 6 months of treatment caused elevated left ventricular mass to decline significantly toward normal. These data implicate accumulation of elevated loosely bound Cu in the mechanism of cardiac damage in diabetes and support the use of selective Cu chelation in the treatment of this condition.
Recombinant Human Erythropoietin Stimulates Angiogenesis and Wound Healing in the Genetically Diabetic Mouse
Authors: Galeano Mariarosaria , Altavilla Domenica , Cucinotta Domenico , Russo Giuseppina T. , Calò Margherita , Bitto Alessandra , Marini Herbert , Marini Rolando , Adamo Elena B. , Seminara Paolo , Minutoli Letteria , Torre Valerio , Squadrito Francesco
Published: March 2007

The effects of recombinant human erythropoietin (rHuEPO) in diabetes-related healing defects were investigated by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJ-m+/+Leptdb mice (db+/db+) and their normoglycemic littermates (db+/+m). Animals were treated with rHuEPO (400 units/kg in 100 μl s.c.) or its vehicle alone (100 μl). Mice were killed on different days (3, 6, and 12 days after skin injury) for measurement of vascular endothelial growth factor (VEGF) mRNA expression and protein synthesis, for monitoring angiogenesis by CD31 expression, and for evaluating histological changes. Furthermore, we evaluated wound-breaking strength at day 12. At day 6, rHuEPO injection in diabetic mice resulted in an increase in VEGF mRNA expression (vehicle = 0.33 ± 0.1 relative amount of mRNA; rHuEPO = 0.9 ± 0.09 relative amount of mRNA; P < 0.05) and protein wound content (vehicle = 23 ± 5 pg/wound; rHuEPO = 92 ± 12 pg/wound; P < 0.05) and caused a marked increase in CD31 gene expression (vehicle = 0.18 ± 0.05 relative amount of mRNA; rHuEPO = 0.98 ± 0.21 relative amount of mRNA; P < 0.05) and protein synthesis. Furthermore, rHuEPO injection improved the impaired wound healing and, at day 12, increased the wound-breaking strength in diabetic mice (vehicle = 12 ± 2 g/mm; rHuEPO 21 ± 5 g/mm; P < 0.05). Erythropoietin may have a potential application in diabetes-related wound disorders.
Effect of Two Amino Acids in TM17 of Sulfonylurea Receptor SUR1 on the Binding of ATP-Sensitive K+ Channel Modulators
Authors: Hambrock Annette , Kayar Tülay , Stumpp Demet , Osswald Hartmut
Published: March 2007

The sulfonylurea receptor (SUR) is the important regulatory subunit of ATP-sensitive K+ channels. It is an ATP-binding cassette protein comprising 17 transmembrane helices. SUR is endowed with binding sites for channel blockers like the antidiabetic sulfonylurea glibenclamide and for the chemically very heterogeneous channel openers. SUR1, the typical pancreatic SUR isoform, shows much higher affinity for glibenclamide but considerably lower affinity for most openers than SUR2. In radioligand binding assays, we investigated the role of two amino acids, T1285 and M1289, located in transmembrane helix (TM)-17, in opener binding to SUR1. These amino acids were exchanged for the corresponding amino acids of SUR2. In competition experiments using [3H]glibenclamide as radioligand, SUR1(T1285L, M1289T) showed much higher affinity toward the cyanoguanidine openers pinacidil and P1075 than SUR1 wild type. The affinity for the thioformamide aprikalim was also markedly increased. In contrast, the affinity for the benzopyrans rilmakalim and levcromakalim was unaffected; however, the amount of displaced [3H]glibenclamide binding was nearly doubled. The binding properties of the opener diazoxide and the blocker glibenclamide were unchanged. In conclusion, mutation of two amino acids in TM17 of SUR1, especially of M1289, leads to class-specific effects on opener binding by increasing opener affinity or by changing allosteric coupling between opener and glibenclamide binding.
Genetic Disruption of Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K+ Channel, Predisposes to Catecholamine-Induced Ventricular Dysrhythmia
Authors: Liu Xiao-Ke , Yamada Satsuki , Kane Garvan C. , Alekseev Alexey E. , Hodgson Denice M. , O’Cochlain Fearghas , Jahangir Arshad , Miki Takashi , Seino Susumu , Terzic Andre
Published: March 2007

Metabolic-sensing ATP-sensitive K+ channels (KATP channels) adjust membrane excitability to match cellular energetic demand. In the heart, KATP channel activity has been linked to homeostatic shortening of the action potential under stress, yet the requirement of channel function in securing cardiac electrical stability is only partially understood. Here, upon catecholamine challenge, disruption of KATP channels, by genetic deletion of the pore-forming Kir6.2 subunit, produced defective cardiac action potential shortening, predisposing the myocardium to early afterdepolarizations. This deficit in repolarization reserve, demonstrated in Kir6.2-knockout hearts, translated into a high risk for induction of triggered activity and ventricular dysrhythmia. Thus, intact KATP channel function is mandatory for adequate repolarization under sympathetic stress providing electrical tolerance against triggered arrhythmia.
ATP-Sensitive K+ Channel Knockout Compromises the Metabolic Benefit of Exercise Training, Resulting in Cardiac Deficits
Authors: Kane Garvan C. , Behfar Atta , Yamada Satsuki , Perez-Terzic Carmen , O’Cochlain Fearghas , Reyes Santiago , Dzeja Petras P. , Miki Takashi , Seino Susumu , Terzic Andre
Published: March 2007

Exercise training elicits a metabolic and cardiovascular response that underlies fitness. The molecular mechanisms that orchestrate this adaptive response and secure the wide-ranging gains of a regimented exercise program are poorly understood. Formed through association of the Kir6.2 pore and the sulfonylurea receptor, the stress-responsive ATP-sensitive K+ channels (KATP channels), with their metabolic-sensing capability and broad tissue expression, are potential candidates for integrating the systemic adaptive response to repetitive exercise. Here, the responses of mice lacking functional Kir6.2-containing KATP channels (Kir6.2-KO) were compared with wild-type controls following a 28-day endurance swimming protocol. While chronic aquatic training resulted in lighter, leaner, and fitter wild-type animals, the Kir6.2-KO manifested less augmentation in exercise capacity and lacked metabolic improvement in body fat composition and glycemic handling with myocellular defects. Moreover, the repetitive stress of swimming unmasked a survival disadvantage in the Kir6.2-KO, associated with pathologic calcium-dependent structural damage in the heart and impaired cardiac performance. Thus, Kir6.2-containing KATP channel activity is required for attainment of the physiologic benefits of exercise training without injury.
The Incretin Approach for Diabetes Treatment
Authors: Holst Jens Juul , Ørskov Cathrine
Published: March 2007

Glucagon-like peptide (GLP)-1 is a gut hormone that stimulates insulin secretion, gene expression, and β-cell growth. Together with the related hormone glucose-dependent insulinotropic polypeptide (GIP), it is responsible for the incretin effect, the augmentation of insulin secretion after oral as opposed to intravenous administration of glucose. Type 2 diabetic patients typically have little or no incretin-mediated augmentation of insulin secretion. This is due to decreased secretion of GLP-1 and loss of the insulinotropic effects of GIP. GLP-1, however, retains insulinotropic effects, and the hormone effectively improves metabolism in patients with type 2 diabetes. Continuous subcutaneous administration greatly improved glucose profiles and lowered body weight and HbA1c levels. Further, free fatty acid levels were lowered, insulin resistance was improved, and β-cell performance was greatly improved. The natural peptide is rapidly degraded by the enzyme dipeptidyl peptidase IV (DPP IV), but resistant analogs as well as inhibitors of DPP IV are now under development, and both approaches have shown remarkable efficacy in experimental and clinical studies.
The High-Fat Diet–Fed Mouse
Authors: Winzell Maria Sörhede , Ahrén Bo
Published: March 2007

This study characterizes the high-fat diet–fed mouse as a model for impaired glucose tolerance (IGT) and type 2 diabetes. Female C57BL/6J mice were fed a high-fat diet (58% energy by fat) or a normal diet (11% fat). Body weight was higher in mice fed the high-fat diet already after the first week, due to higher dietary intake in combination with lower metabolic efficiency. Circulating glucose increased after 1 week on high-fat diet and remained elevated at a level of ∼1 mmol/l throughout the 12-month study period. In contrast, circulating insulin increased progressively by time. Intravenous glucose challenge revealed a severely compromised insulin response in association with marked glucose intolerance already after 1 week. To illustrate the usefulness of this model for the development of new treatment, mice were fed an orally active inhibitor of dipeptidyl peptidase-IV (LAF237) in the drinking water (0.3 mg/ml) for 4 weeks. This normalized glucose tolerance, as judged by an oral glucose tolerance test, in association with augmented insulin secretion. We conclude that the high-fat diet–fed C57BL/6J mouse model is a robust model for IGT and early type 2 diabetes, which may be used for studies on pathophysiology and development of new treatment.
Signaling Elements Involved in the Metabolic Regulation of mTOR by Nutrients, Incretins, and Growth Factors in Islets
Authors: Kwon Guim , Marshall Connie A. , Pappan Kirk L. , Remedi Maria S. , McDaniel Michael L.
Published: March 2007

Mammalian target of rapamycin (mTOR) is a protein kinase that integrates signals from mitogens and the nutrients, glucose and amino acids, to regulate cellular growth and proliferation. Previous findings demonstrated that glucose robustly activates mTOR in an amino acid-dependent manner in rodent and human islets. Furthermore, activation of mTOR by glucose significantly increases rodent islet DNA synthesis that is abolished by rapamycin. Glucagon-like peptide-1 (GLP-1) agonists, through the production of cAMP, have been shown to enhance glucose-dependent proinsulin biosynthesis and secretion and to stimulate cellular growth and proliferation. The objective of this study was to determine if the glucose-dependent and cAMP-mediated mechanism by which GLP-1 agonists enhance β-cell growth and proliferation is mediated, in part, through mTOR. Our studies demonstrated that forskolin-generated cAMP resulted in activation of mTOR at basal glucose concentrations as assessed by phosphorylation of S6K1, a downstream effector of mTOR. Conversely, an adenylyl cyclase inhibitor partially blocked glucose-induced S6K1 phosphorylation. Furthermore, the GLP-1 receptor agonist, Exenatide, dose-dependently enhanced phosphorylation of S6K1 at an intermediate glucose concentration (8 mmol/l) in a rapamycin-sensitive manner. To determine the mechanism responsible for this potentiation of mTOR, the effects of intra- and extracellular Ca2+ were examined. Glyburide, an inhibitor of ATP-sensitive K+ channels (KATP channels), provided partial activation of mTOR at basal glucose concentrations due to the influx of extracellular Ca2+, and diazoxide, an activator of KATP channels, resulted in partial inhibition of S6K1 phosphorylation by 20 mmol/l glucose. Furthermore, Exenatide or forskolin reversed the inhibition by diazoxide, probably through mobilization of intracellular Ca2+ stores by cAMP. BAPTA, a chelator of intracellular Ca2+, resulted in inhibition of glucose-stimulated S6K1 phosphorylation due to a reduction in cytosolic Ca2+ concentrations. Selective blockade of glucose-stimulated Ca2+ influx unmasked a protein kinase A (PKA)-sensitive component involved in the mobilization of intracellular Ca2+ stores, as revealed with the PKA inhibitor H-89. Overall, these studies support our hypothesis that incretin-derived cAMP participates in the metabolic activation of mTOR by mobilizing intracellular Ca2+ stores that upregulate mitochondrial dehydrogenases and result in enhanced ATP production. ATP can then modulate KATP channels, serve as a substrate for adenylyl cyclase, and possibly directly regulate mTOR activation.
Prevention of Type 2 Diabetes
Authors: Chiasson J.-L. , Rabasa-Lhoret Rémi
Published: March 2007

Type 2 diabetes is increasing worldwide in epidemic proportions. Its associated morbidity and mortality is imposing a major burden on the health care system. Based on a better understanding of the pathophysiology of glucose intolerance, clinical trials on the prevention of diabetes have been performed. It has now been demonstrated that diet and exercise, metformin, acarbose, and troglitazone can prevent or at least delay the development of diabetes in subjects with impaired glucose tolerance (IGT). It is now generally accepted that insulin resistance and β-cell dysfunction are major factors involved in the development of diabetes. The relative contribution of insulin resistance versus β-cell dysfunction on the pathogenesis of diabetes has aroused much debate. These two processes should be studied in relation to one another: their relationship is best described as hyperbolic in nature. When this relationship is taken into consideration, it becomes evident that subjects at risk of developing type 2 diabetes have β-cell dysfunction before they develop glucose intolerance. Insulin resistance may be mostly explained by the presence of obesity and accelerate the progression to diabetes in subjects with the propensity to β-cell failure. By the time hyperglycemia occurs, impairment in both insulin sensitivity and insulin secretion are present. There are still few data on insulin sensitivity and insulin secretion from the trials on the prevention of diabetes. The few data that we do have suggest that most interventions mostly have an effect on insulin resistance. By reducing insulin resistance, they protect and preserve the β-cell function. No intervention has yet shown any direct effect on β-cell function.
Intensive Integrated Therapy of Type 2 Diabetes
Authors: Gæde Peter , Pedersen Oluf
Published: March 2007

The macro- and microvascular burden of type 2 diabetes is well established. A number of recent single risk factor intervention trials targeting hyperglycemia, dyslipidemia, hypertension, procoagulation, microalbumuria, and existing cardiovascular disorders have, however, shown major beneficial effects on long-term outcome. The results from these studies are anticipated to change the future management of type 2 diabetes, and most of the updated national guidelines for the treatment of type 2 diabetes recommend a multipronged approach driven by ambitious treatment targets. The outcome of this intensive integrated therapy has, however, only been investigated in a few studies of patients with type 2 diabetes. One of these trials, the Steno-2 Study, showed that intensive intervention for an average of 7.8 years cuts cardiovascular events as well as nephropathy, retinopathy, and autonomic neuropathy by about half when compared with a conventional multifactorial treatment. The challenge for now is to ensure that the trial experiences are widely adopted in daily clinical practice.
Integration of ATP, cAMP, and Ca2+ Signals in Insulin Granule Exocytosis
Authors: Shibasaki Tadao , Sunaga Yasuhiro , Seino Susumu
Published: March 2007

Intracellular ATP, cAMP, and Ca2+ are major signals involved in the regulation of insulin secretion in the pancreatic β-cell. We recently found that the ATP-sensitive K+ channel (KATP channel) as an ATP sensor, cAMP-GEFII as a cAMP sensor, Piccolo as a Ca2+ sensor, and l-type voltage-dependent Ca2+ channel (VDCC) can interact with each other. In the present study, we examined the effects of cAMP and ATP on the interaction of cAMP-GEFII and sulfonylurea receptor-1 (SUR1). Interaction of cAMP-GEFII with SUR1 was inhibited by the cAMP analog 8-bromo-cAMP but not by ATP, and the inhibition by 8-bromo-cAMP persisted in the presence of ATP. In addition, SUR1, cAMP-GEFII, and Piccolo could form a complex. Piccolo also interacted with the α11.2 subunit of VDCC in a Ca2+-independent manner. These data suggest that the interactions of the KATP channel, cAMP-GEFII, Piccolo, and l-type VDCC are regulated by intracellular signals such as cAMP and Ca2+ and that the ATP, cAMP, and Ca2+ signals are integrated at a specialized region of pancreatic β-cells.
Effect of PPAR-γ Activation and Inhibition on Glucose-Stimulated Insulin Release in INS-1e Cells
Authors: Santini Eleonora , Fallahi Poupak , Ferrari Silvia Martina , Masoni Antonio , Antonelli Alessandro , Ferrannini Ele
Published: March 2007

Peroxisome proliferator-activated receptor (PPAR)-γ is expressed in human β-cells and in the rat β-cell line INS-1. Previous studies have suggested that PPAR-γ agonism (e.g., thiazolidinediones) enhances glucose-stimulated insulin secretion (GSIS) from islets or INS-1 cells. We tested the direct effect on insulin release by INS-1e of a PPAR-γ agonist (Ro4389679-000-001 at 0.2 and 0.4 μmol/l) and a PPAR-γ antagonist (SR202 at 0.2 and 0.4 mmol/l). Cells were incubated in 11 mmol/l glucose for 96 h and then challenged with 3.3, 7.5, 11.0, and 20.0 mmol/l glucose for 1 h. Under these control conditions, insulin concentrations in the medium rose from 19 ± 4 ng/ml (mean ± SE) to 82 ± 5, 107 ± 11, and 103 ± 10 ng/ml (P < 0.0001 by ANOVA). Preincubation for 48 h with the PPAR-γ agonist potentiated GSIS (to 154 ± 14 and 156 ± 12 ng/ml at 20 mmol/l glucose, P < 0.01). Cell insulin content was not altered by either acute glucose challenge or PPAR-γ agonist coincubation. Preincubation for 48 h with SR202 at the higher dose caused a 30% inhibition of GSIS, with no change in cell insulin contents. When cells were preincubated with 11 mmol/l glucose plus 1 mmol/l oleate, GSIS was significantly potentiated (by 30%, P < 0.0001); adding Ro4389679-000-001 or SR202 to these preincubations reduced GSIS to the respective levels seen in the absence of oleate (P < 0.0001 for both effects). In conclusion, INS-1e cells display a PPAR-γ tone that is symmetrically modulated and competitively stimulated by oleate.
Beta-Cell-Targeted Expression of a Dominant-Negative Mutant of Hepatocyte Nuclear Factor-1α in Mice
Authors: Winzell Maria Sörhede , Pacini Giovanni , Wollheim Claes B. , Ahrén Bo
Published: March 2007

We studied islet function in mice with β-cell-targeted expression of a dominant-negative mutant of hepatocyte nuclear factor (HNF)-1α. At age 2–3 months, anesthetized transgenic and wild-type male mice underwent an intravenous glucose (1 g/kg) tolerance test (IVGTT). It was found that transgenic mice had an abolished insulin response in association with severe glucose intolerance. In other tests, the 5-min insulin response to intravenous arginine was impaired by 79% (P = 0.032) and the 15-min insulin response to gastric glucose was suppressed by 97% (P = 0.006). In islets incubated for 60 min, the insulin response to glucose (3.3–22.2 mmol/l) was impaired by >80% in transgenic mice. In contrast, insulin responses to nonglucose secretagogues were only partially suppressed (to GLP-1 [100 nmol/l] by 40%, to carbachol [1 μmol/l] by 20%, and to palmitate [0.5 mmol/l] by 15%), whereas the response to depolarization by KCl (50 mmol/l) was not reduced. Finally, the IVGTT data insulin sensitivity in transgenic mice was not significantly different from that of wild-type mice. Thus, mice with targeted suppression of β-cell HNF-1α represent a good diabetes model exhibiting severely impaired insulin secretion after glucose with marked glucose intolerance. In contrast, the insulin responses to nonglucose stimuli are not suppressed when the islet insulin content is taken into account.
Genetic Background Determines the Size and Structure of the Endocrine Pancreas
Authors: Bock Troels , Pakkenberg Bente , Buschard Karsten
Published: March 2007

Key parameters of the endocrine pancreas, such as islet number, islet mass, β-cell mass, and α-cell mass, were studied in different strains of inbred mice to investigate the impact of genetic background on the size and structure of the endocrine pancreas. Six mice from each of seven different strains of inbred mice were included in the study. For all parameters investigated, there was a pronounced interstrain variation. ANCOVA showed that only mouse strain was statistically significant as an explanatory parameter for the number of islets. Mouse strain, body weight, and pancreas mass reached statistical significance as explanatory parameters for the islet mass, with mouse strain as the most significant predictor. These data show that genetic background is the most important predictor of both the number of islets and total islet volume. We also conclude that inbred mice could be a valuable resource to identify the genes responsible for the size and structure of the endocrine pancreas.
Dynamic Changes in β-Cell Mass and Pancreatic Insulin During the Evolution of Nutrition-Dependent Diabetes in Psammomys obesus
Authors: Kaiser Nurit , Yuli Michal , Üçkaya Gökhan , Oprescu Andrei I. , Berthault Marie-France , Kargar Catherine , Donath Marc Y. , Cerasi Erol , Ktorza Alain
Published: March 2007

Recent studies ascribe a major role to pancreatic β-cell loss in type 2 diabetes. We investigated the dynamics of β-cell mass during diabetes evolution in Psammomys obesus, a model for nutrition-dependent type 2 diabetes, focusing on the very early and the advanced stages of the disease. P. obesus fed a high-calorie diet for 26 days developed severe hyperglycemia, β-cell degranulation, and markedly reduced pancreatic insulin content. Reducing calories for 7 days induced normoglycemia in 90% of the animals, restoring β-cell granulation and insulin content. To dissociate effects of diet from blood glucose reduction, diabetic animals received phlorizin for 2 days, which normalized glycemia and increased the pancreatic insulin reserve to 50% of control, despite a calorie-rich diet. During diabetes progression, β-cell mass decreased initially but recovered spontaneously to control levels, despite persistent hyperglycemia. Strikingly, however, β-cell mass did not correlate with degree of hyperglycemia or pancreatic insulin content. We conclude that reduced insulin reserve is the main cause of diabetes progression, whereas irreversible β-cell mass reduction is a late event in P. obesus. The rapid recovery of the pancreas by phlorizin-induced normoglycemia implies a causal relationship between hyperglycemia and islet dysfunction. Similar mechanisms could be operative during the evolution of type 2 diabetes in humans.