bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Fatty Acid Re-esterification by PEPCK Overexpression in Adipose Tissue Leads to Obesity Without Insulin Resistance
Authors: Franckhauser Sylvie , Muñoz Sergio , Pujol Anna , Casellas Alba , Riu Efren , Otaegui Pedro , Su Benli , Bosch Fatima
Published: March 2007

Adipose tissue glyceroneogenesis generates glycerol 3-phosphate, which could be used for fatty acid esterification during starvation. To determine whether increased glyceroneogenesis leads to increased fat mass and to explore the role of obesity in the development of insulin resistance, we overexpressed PEPCK, a regulatory enzyme of glyceroneogenesis in adipose tissue. Transgenic mice showed a chronic increase in PEPCK activity, which led to increased glyceroneogenesis, re-esterification of free fatty acids (FFAs), increased adipocyte size and fat mass, and higher body weight. In spite of increased fat mass, transgenic mice showed decreased circulating FFAs and normal leptin levels. Moreover, glucose tolerance and whole-body insulin sensitivity were preserved. Skeletal muscle basal and insulin-stimulated glucose uptake and glycogen content were not affected, suggesting that skeletal muscle insulin sensitivity is normal in transgenic obese mice. Our results indicate the key role of PEPCK in the control of FFA re-esterification in adipose tissue and, thus, the contribution of glyceroneogenesis to fat accumulation. Moreover, they suggest that higher fat mass without increased circulating FFAs does not lead to insulin resistance or type 2 diabetes in these mice.
Genetic Risk Determines the Emergence of Diabetes-Associated Autoantibodies in Young Children
Authors: Kupila Antti , Keskinen Päivi , Simell Tuula , Erkkilä Satu , Arvilommi Paula , Korhonen Sari , Kimpimäki Teija , Sjöroos Minna , Ronkainen Matti , Ilonen Jorma , Knip Mikael , Simell Olli
Published: March 2007

Timing of onset of autoimmunity is a prerequisite for unmasking triggers and pathogenesis of type 1 diabetes. We followed 4,590 consecutive newborns with 8 or 3% HLA-DQB1 conferred risk for type 1 diabetes at 3-, 6-, or 12-month intervals up to 5.5 years of age. Islet cell autoantibodies (ICAs) and, in the 137 children with ICAs, insulin autoantibodies (IAAs), GAD65 autoantibodies (GADAs), and IA-2 protein autoantibodies (IA-2As) were measured. Children with high genetic risk developed ICAs more often than those with moderate risk (log-rank P = 0.0015); 85 and 91% remained ICA negative by 5 years of age, respectively. The time of appearance of biochemical autoantibodies was then compared with the appearance of ICAs. IAAs and GADAs emerged usually before ICAs (means −1.8 and −1.5 months, respectively) and IA-2As after ICAs (mean 2.0 months). Ninety-five percent of all IAAs, GADAs, and IA-2As seroconversions occurred in a cluster (−12 to 8 months) around the ICA seroconversion. We conclude that diabetes-associated autoantibodies emerged in children with predisposing HLA-DQB1 alleles after 3 months of age at a constant tempo, determined by the genetic risk level, usually in the order of IAA, GADA, ICA, and IA-2A. Seroconversion to multiple autoantibody positivity usually occurred tightly clustered in time.
Differential Effects of Hyperlipidemia on Insulin Secretion in Islets of Langerhans From Hyperglycemic Versus Normoglycemic Rats
Authors: Briaud Isabelle , Kelpe Cynthia L. , Johnson Lisa M. , Tran Phuong Oanh T. , Poitout Vincent
Published: March 2007

Chronic elevations in plasma levels of fatty acids (FAs) adversely affect pancreatic β-cell function in type 2 diabetes. In vitro, we have previously shown that deleterious effects of prolonged exposure of isolated islets to FAs were dependent on the presence of elevated glucose concentration. This led us to hypothesize that both hyperlipidemia and hyperglycemia must be present simultaneously for FAs to affect β-cell function. To test this hypothesis in vivo, we administered a high-fat diet for 6 weeks to Goto-Kakizaki (GK) rats. High-fat feeding had no effect on insulin secretion, insulin content, or insulin mRNA levels in islets from normoglycemic Wistar rats. In contrast, high-fat feeding markedly impaired glucose-induced insulin secretion in islets from GK rats. High-fat feeding did not affect triglyceride (TG) content or the rate of glucose oxidation in islets. It was, however, accompanied by a twofold increase in uncoupling protein (UCP)-2 levels in GK rat islets. Insulin treatment completely normalized glucose-induced insulin secretion and prevented the increase in UCP-2 expression in islets from high-fat-fed GK rats. We conclude that hyperlipidemia induced by high-fat feeding affects insulin secretion in islets from hyperglycemic GK rats only, by a mechanism which may involve, at least in part, modulation of UCP-2 expression.
Association of Fasting Plasma Glucose With Heart Rate Recovery in Healthy Adults
Authors: Panzer Claudia , Lauer Michael S. , Brieke Andreas , Blackstone Eugene , Hoogwerf Byron
Published: March 2007

Diabetes is associated with abnormal autonomic function and increased mortality. Abnormal heart rate recovery after exercise, a measure of autonomic dysfunction, is also associated with increased mortality. The objective of this study was to determine the association of fasting plasma glucose with abnormal heart rate recovery and its prognostic importance in healthy adults. We studied 5,190 healthy adults who did not have medically treated diabetes (mean age 45 years, 39% women), were enrolled in the Lipid Research Clinics’ Prevalence Study, and underwent exercise testing. Heart rate recovery was defined as the change from peak heart rate to that after 2 min of recovery; an abnormal value was ≤42 bpm. All-cause mortality was assessed over 12 years. A total of 504 participants (10%) had impaired fasting glucose, and 131 (3%) had untreated diabetes. An abnormal heart rate recovery was found in 1,699 (33%). Compared with participants who had normal fasting plasma glucose, abnormal heart rate recovery was more common among those with impaired fasting glucose (42 vs. 31%; relative risk, 1.34; 95% confidence interval [CI], 1.20–1.50; P < 0.0001) and those with diabetes (50 vs. 31%; relative risk, 1.61; 95% CI, 1.35–1.92; P < 0.0001). Fasting plasma glucose remained an independent predictor of abnormal heart rate recovery even after adjustment for age, sex, and other confounders (P = 0.0003). An abnormal heart rate recovery added to impaired fasting plasma glucose for the prediction of death. Fasting plasma glucose is strongly and independently associated with abnormal heart rate recovery, even at nondiabetic levels.
Morphometry of Dorsal Root Ganglion in Chronic Experimental Diabetic Neuropathy
Authors: Kishi Motoko , Tanabe James , Schmelzer James D. , Low Phillip A.
Published: March 2007

Chronic hyperglycemia results in a predominantly sensory neuropathy. Recent studies suggest that dorsal root ganglion (DRG) neurons comprise a specific target and may be responsible for the important complication of diabetic sensory neuropathy, since hyperglycemia for longer than 6 months results in a vacuolar ganglionopathy with associated radiculopathy and distal sensory neuropathy. We undertook morphometric analysis of L5 DRG neurons in seven diabetic rats and six age- and sex-matched littermates. Nerve conduction studies were also performed, and neuropathy was confirmed. Diabetes was induced with streptozotocin; duration of diabetes was 12 months. The DRG count for control rats was 15,304 ± 991 neurons. Two of seven diabetic DRG counts were reduced, but the group mean count at 14,847 ± 1,524 was not significantly reduced. The number of small neurons (type B) considerably exceeded that of large neurons (type A), at a ratio of 71:29. The percentage of large cells was significantly reduced in diabetic compared with control rats (P = 0.01). The large-diameter population can be subdivided into two groups; with this subdivision, the number of neurons <50 μm was not reduced in samples from diabetic rats, but the neurons of largest size (≥50 μm) were significantly reduced (by 41%).
Factors of Insulin Resistance Syndrome–Related Phenotypes Are Linked to Genetic Locations on Chromosomes 6 and 7 in Nondiabetic Mexican-Americans
Authors: Arya Rector , Blangero John , Williams Ken , Almasy Laura , Dyer Thomas D. , Leach Robin J. , O’Connell Peter , Stern Michael P. , Duggirala Ravindranath
Published: March 2007

Insulin resistance syndrome (IRS)−related phenotypes, such as hyperinsulinemia, obesity-related traits, impaired glucose tolerance, dyslipidemia, and hypertension, tend to cluster into factors. We attempted to identify loci influencing the factors of IRS-related phenotypes using phenotypic data from 261 nondiabetic subjects distributed across 27 low-income Mexican-American extended families. Principal component factor analyses were performed using eight IRS-related phenotypes: fasting glucose (FG), fasting specific insulin (FSI), BMI, systolic blood pressure (SBP), diastolic blood pressure (DBP), HDL cholesterol, ln triglycerides (ln TGs), and leptin (LEP). The factor analysis yielded three factors: factor 1 (BMI, LEP, and FSI), factor 2 (DBP and SBP), and factor 3 (HDL and ln TG). We conducted multipoint variance components linkage analyses on these factors with the program SOLAR using a 10–15 cM map. We found significant evidence for linkage of factor 1 to two regions on chromosome 6 near markers D6S403 (logarithm of odds [LOD] = 4.2) and D6S264 (LOD = 4.9). We also found strong evidence for linkage of factor 3 to a genetic location on chromosome 7 between markers D7S479 and D7S471 (LOD = 3.2). In conclusion, we found substantial evidence for susceptibility loci on chromosomes 6 and 7 that appear to influence the factors representing the IRS-related phenotypes in Mexican-Americans.
A Genome-Wide Search for Linkage-Disequilibrium With Type 1 Diabetes in a Recent Genetically Isolated Population From the Netherlands
Authors: Vaessen Norbert , Heutink Peter , Houwing-Duistermaat Jeanine J. , Snijders Pieter J.L.M. , Rademaker Tessa , Testers Leon , Batstra Manou R. , Sandkuijl Lodewijk A. , van Duijn Cornelia M. , Oostra Ben A.
Published: March 2007

Type 1 diabetes has a substantial genetic component, with consistent evidence for a susceptibility locus in the HLA-DR/DQ region (chromosome 6p) and the insulin gene region (chromosome 11p). Genome scans have identified >18 other genomic regions that may harbor putative type 1 diabetes genes. However, evidence for most regions varies in different data sets. Given the genetic heterogeneity of type 1 diabetes, studies in homogeneous genetically isolated populations may be more successful in mapping susceptibility loci than in complex outbred populations. We describe a genome-wide search in a recently Dutch isolated population. We identified 43 patients that could be traced back to a common ancestor within 15 generations and performed a genome-wide scan using a combined linkage- and association-based approach. In addition to the HLA locus, evidence for type 1 diabetes loci was observed on chromosome 8q24 (marker D8S1128) and on chromosome 17q24 (marker D17S2059). Both the 8q and 17q localization are supported by allele-sharing at adjacent markers in affected individuals. Statistical evidence for a conserved ancestral haplotype was found for chromosome 8q24.
Human Resistin Gene, Obesity, and Type 2 Diabetes
Authors: Sentinelli Federica , Romeo Stefano , Arca Marcello , Filippi Emanuela , Leonetti Frida , Banchieri Michela , Di Mario Umberto , Baroni Marco Giorgio
Published: March 2007

The hormone resistin has been suggested to link obesity to type 2 diabetes by modulating steps in the insulin-signaling pathway and inducing insulin resistance. Thus, the resistin gene represents a potential candidate for the etiology of insulin resistance and type 2 diabetes. In this study, we analyzed the coding sequence of the three exons of the resistin gene, together with its 5′ regulatory region and 3′ untranslated region (UTR), by single-strand conformation polymorphism (SSCP) in 58 type 2 diabetic subjects, 59 obese subjects, and 60 normal subjects. Only one sequence variant was detected in the resistin gene. Sequencing of this variant revealed the presence of a single nucleotide substitution (SNP) in the 3′-UTR of exon 3 (G1326C). Because 3′-UTR SNPs have been shown to affect gene expression, we examined the frequency of this SNP in 591 subjects (198 obese subjects, 207 diabetic subjects, and 186 control subjects) by PCR amplification and BseRI digestion. No significant association was found between the G1326C variant and diabetes and obesity. Comparison of clinical and metabolic parameters between G1326C carriers and noncarriers again showed no significant difference. In conclusion, our data suggest that genetic defects of the resistin gene are unlikely to play a role in the etiology of these common disorders in our population.
Systematic Search for Single Nucleotide Polymorphisms in the Resistin Gene
Authors: Osawa Haruhiko , Onuma Hiroshi , Murakami Akiko , Ochi Masaaki , Nishimiya Tatsuya , Kato Kenichi , Shimizu Ikki , Fujii Yasuhisa , Ohashi Jun , Makino Hideichi
Published: March 2007

Resistin is a novel polypeptide specifically secreted from adipocytes, and its serum levels are increased in obese diabetic mice. Resistin antagonizes insulin and could account for insulin resistance. To determine whether there are single nucleotide polymorphisms (SNPs) in the resistin gene associated with type 2 diabetes, sequences for 24 Japanese type 2 diabetic patients were initially analyzed using PCR direct sequencing. Three SNPs were found in the introns, but none were present in the coding regions. The allele frequencies of genomic −167C>T, +157C>T, and +299G>A in 99 Japanese control subjects were determined to be 3.5, 6.6, and 39.4%, respectively. In each pair of these SNPs, linkage disequilibria were found between either −167C>T and +299G>A or +157C>T and +299G>A. A linkage disequilibrium was also detected among −167C>T, +157C>T, and +299G>A, and only four of the eight possible haplotypes defined by these SNPs were found. A comparison of the frequencies of these SNPs and haplotypes between 99 type 2 diabetes and 99 control subjects revealed no evidence for any association. These identified SNPs, which were in linkage disequilibrium, represent potentially useful tools for searching for their association with specific phenotypes of diabetes.
Assessment of Skeletal Muscle Triglyceride Content by 1H Nuclear Magnetic Resonance Spectroscopy in Lean and Obese Adolescents
Authors: Sinha Ranjana , Dufour Sylvie , Petersen Kitt Falk , LeBon Vincent , Enoksson Staffan , Ma Yong-Zhan , Savoye Mary , Rothman Douglas L. , Shulman Gerald I. , Caprio Sonia
Published: March 2007

The metabolism and composition of skeletal muscle tissue is of special interest because it is a primary site of insulin action and plays a key role in the pathogenesis of insulin resistance. Intramyocellular (IMCL) triglyceride stores are an accessible form of energy that may decrease skeletal muscle glucose utilization, thereby contributing to impaired glucose metabolism. Because of the invasive nature of muscle biopsies, there is limited, if any, information about intramuscular lipid stores in children. The development of 1H nuclear magnetic resonance (NMR) spectroscopy provides a unique noninvasive alternative method that differentiates intracellular fat from intercellular fat in muscle tissue. The present study was performed to determine whether IMCL and extramyocellular (EMCL) lipid contents are increased early in the development of juvenile obesity and to explore the relationships between IMCL and EMCL to in vivo insulin sensitivity, independently of total body fat and central adiposity in obese and nonobese adolescents. Eight nonobese (BMI 21 kg/m2, age 11–16 years) and 14 obese (BMI 35 ± 1.5 kg/m2, age 11–15 years) adolescents underwent 1) 1H-NMR spectroscopy to noninvasively quantify IMCL and EMCL triglyceride content of the soleus muscle, 2) a 2-h euglycemic-hyperinsulinemic clamp (40 mU · m−2 · min−1) to assess insulin sensitivity, 3) a dual-energy X-ray absorptiometry scan to measure total percent body fat, and 4) magnetic resonance imaging to measure abdominal fat distribution. Both the IMCL and EMCL content of the soleus muscle were significantly greater in the obese adolescents than in the lean control subjects. A strong inverse correlation was found between IMCL and insulin sensitivity, which persisted and became even stronger after controlling for percent total body fat and abdominal subcutaneous fat mass (partial correlation r = −0.73, P < 0.01) but not when adjusting for visceral fat (r = − 0.54, P < 0.08). In obese adolescents, increase in total body fat and central adiposity were accompanied by higher IMCL and EMCL lipid stores. The striking relationships between both IMCL and EMCL with insulin sensitivity in childhood suggest that these findings are not a consequence of aging but occur early in the natural course of obesity.
Insulin Resistance, Defective Insulin Receptor Substrate 2—Associated Phosphatidylinositol-3′ Kinase Activation, and Impaired Atypical Protein Kinase C (ζ/λ) Activation in Myotubes From Obese Patients With Impaired Glucose Tolerance
Authors: Vollenweider Peter , Ménard Barbara , Nicod Pascal
Published: March 2007

Impaired glucose tolerance (IGT) is characterized by insulin resistance. Recently, defects in the insulin-signaling cascade have been implicated in the pathogenesis of insulin resistance. To study insulin signaling in IGT, we used human skeletal muscle cells in primary culture from patients with IGT and control subjects. In these cultured myotubes, we assessed insulin-induced 2-deoxyglucose uptake and early steps of the metabolic insulin-signaling cascade. Myotubes in culture from patients with IGT had insulin-induced glucose uptake that was roughly 30–50% less than that from control subjects. This insulin resistance was associated with impaired insulin receptor substrate (IRS)-2–associated phosphatidylinositol 3′ (PI3) kinase activation and IRS-2 tyrosine phosphorylation as well as significantly decreased protein kinase C (PKC)-ζ/λ activation in response to insulin. IRS-1– associated PI3 kinase activation and insulin receptor autophosphorylation were comparable in the two groups. Protein expression levels for the insulin receptor, IRS-1, IRS-2, the p85 regulatory subunit of PI3 kinase, Akt, PKC-ζ/λ, GLUT1, and GLUT4 were also similar in the two groups. In conclusion, myotubes from patients with IGT have impaired insulin-induced glucose uptake. This is associated with impaired IRS-2–associated PI3 kinase activation and PKC-ζ/λ activation. Our results suggest that these defects may contribute to insulin resistance in IGT patients.
Modulation of Adipose Tissue Expression of Murine Matrix Metalloproteinases and Their Tissue Inhibitors With Obesity
Authors: Maquoi Erik , Munaut Carine , Colige Alain , Collen Désiré , Lijnen H. Roger
Published: March 2007

The potential role of the matrix metalloproteinase (MMP) system in the pathophysiology of the adipose tissue was investigated in a mouse model of nutritionally induced obesity. mRNA levels of 16 MMPs and 4 tissue inhibitors of MMPs (TIMPs) were measured by semiquantitative RT-PCR in adipose tissue isolated from mice maintained for 15 weeks on a standard or high-fat diet. In mice on standard diet, with the exception of MMP-8, all MMP and TIMP transcripts were detected in both gonadal and subcutaneous depots. In obese mice, the expression of MMP-3, -11, -12, -13, and -14 and TIMP-1 mRNAs was upregulated, whereas that of MMP-7, -9, -16, and -24 and TIMP-4 was downregulated. Most MMP and TIMP mRNAs were expressed at higher levels in stromal-vascular cells than in mature adipocytes. Analysis of adipose tissue by in situ fluorescent zymography revealed MMP-dependent proteolytic activities, demonstrating the presence of active MMPs in the intact tissue. In vitro conversion of adipogenic 3T3-F442A cells into mature adipocytes was associated with substantial modulations of MMP and TIMP expression. Moreover, this in vitro adipogenesis was reduced in the presence of a synthetic MMP inhibitor. Thus, the adipose tissue expresses a large array of MMPs and TIMPs, which modulate adipocyte differentiation.
Xanthine Oxidase Is Involved in Free Radical Production in Type 1 Diabetes
Authors: Desco Marí-Carmen , Asensi Miguel , Márquez Rafael , Martínez-Valls José , Vento Máximo , Pallardó Federico V. , Sastre Juan , Viña José
Published: March 2007

The aim of this work was to study the mechanism of free radical formation in type 1 diabetes and its possible prevention. We have found oxidation of blood glutathione and an increase in plasma lipoperoxide levels in both human type 1 diabetes and experimental diabetes. Peroxide production by mitochondria does not increase in diabetes. On the contrary, the activity of xanthine oxidase, a superoxide-generating enzyme, increases in liver and plasma of diabetic animals. The increase in plasma xanthine oxidase activity may be explained by the increase in the hepatic release of this enzyme, which is not due to nonspecific membrane damage: release of other hepatic enzymes, such as the amino transferases, does not increase in diabetes. Superoxide formation by aortic rings of rabbits increases significantly in diabetes. This is completely inhibited by allopurinol, an inhibitor of xanthine oxidase. Heparin, which releases xanthine oxidase from the vessel wall, also decreases superoxide formation by aortic rings of diabetic animals. Treatment with allopurinol decreases oxidative stress in type 1 diabetic patients: hemoglobin glycation, glutathione oxidation, and the increase in lipid peroxidation are prevented. These results may have clinical significance in the prevention of late-onset vascular complications of diabetes.
Flux Through the Hexosamine Pathway Is a Determinant of Nuclear Factor κB– Dependent Promoter Activation
Authors: James Leighton R. , Tang Damu , Ingram Alistair , Ly Hao , Thai Kerri , Cai Lu , Scholey James W.
Published: March 2007

The hexosamine pathway may mediate some of the toxic effects of glucose. We hypothesized that flux through this pathway might regulate the activity of nuclear factor κB (NF-κB)-dependent genes in mesangial cells (MCs). In MCs, RT-PCR revealed that high glucose (30 mmol/l) and glucosamine (1 mmol/l) increased mRNA levels for vascular cell adhesion molecule 1 (VCAM-1) and increased the activity of an NF-κB enhancer by 1.5- and 2-fold, respectively. Overexpression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme for flux through the hexosamine pathway, led to a 2.2-fold increase in NF-κB enhancer activity; the combination of GFAT overexpression and high glucose increased activity 2.8-fold, and these increases were prevented by 40 μmol/l O-diazoacetyl-l-serine (azaserine) or 6-diazo-5-oxonorleucine. High glucose, glucosamine, and GFAT overexpression increased binding of MC nuclear proteins to NF-κB consensus sequences. Immunoblotting revealed that the p65 subunit of NF-κB was O-glycosylated in MC cultured in physiologic glucose and that significant enhancement occurred with high glucose and glucosamine. Both glucose and glucosamine dose-dependently increased human VCAM-1 promoter activity. In addition, GFAT overexpression activated the VCAM-1 promoter (2.25-fold), with further augmentation by high glucose and abrogation by inhibitors of GFAT, NF-κB, and O-glycosylation. Inactivation of the two NF-κB sites in the VCAM-1 promoter abolished its response to high glucose, glucosamine, and GFAT overexpression. These results suggest that increased flux through the hexosamine pathway leads to NF-κB–dependent promoter activation in MCs.
Homocysteine Induces Protein Kinase C Activation and Stimulates c-Fos and Lipoprotein Lipase Expression in Macrophages
Authors: Beauchamp Marie-Claude , Renier Geneviève
Published: March 2007

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease in human diabetes. Among the multiple factors that may account for the atherogenicity of homocysteine (Hcys) in patients with diabetes, macrophage (Mo) lipoprotein lipase (LPL) has unique features in that it is increased in human diabetes and acts as a proatherogenic factor in the arterial wall. In the present study, we determined the direct regulatory effect of Hcys on Mo LPL gene expression and secretion. Incubation of J774 Mo with Hcys increased, in a time- and dose-dependent manner, LPL mRNA expression and secretion. Induction of LPL gene expression was biphasic, peaking at 1 and 6 h. Whereas Hcys treatment increased protein kinase C (PKC) activity in Mo, pretreatment of Mo with PKC inhibitors totally suppressed Hcys-induced LPL mRNA expression. Hcys also increases the levels of c-fos mRNA in Mo and enhanced nuclear protein binding to the AP-1 sequence of the LPL gene promoter. Overall, these results demonstrate that Hcys stimulates Mo LPL at both the gene and protein levels and that Hcys-induced LPL mRNA expression requires PKC activation. They also suggest a possible role of c-fos in the stimulatory effect of Hcys on Mo LPL mRNA expression. These observations suggest a new mechanism by which Hcys may exert its proatherogenic effects in human diabetes.
Altered Phosphodiesterase 3-Mediated cAMP Hydrolysis Contributes to a Hypermotile Phenotype in Obese JCR:LA-cp Rat Aortic Vascular Smooth Muscle Cells
Authors: Netherton Stuart J. , Jimmo Sandra L. , Palmer Daniel , Tilley Douglas G. , Dunkerley Heather A. , Raymond Daniel R. , Russell James C. , Absher P. Marlene , Sage E. Helene , Vernon Robert B. , Maurice Donald H.
Published: March 2007

Cardiovascular diseases represent a significant cause of morbidity and mortality in diabetes. Of the many animal models used in the study of non-insulin-dependent (type 2) diabetes, the JCR:LA-cp rat is unique in that it develops insulin resistance in the presence of obesity and manifests both peripheral and coronary vasculopathies. In this animal model, arterial vascular smooth muscle cells (VSMCs) from homozygous obese (cp/cp) rats, but not from age-matched healthy (+/+ or + /cp, collectively defined +/?) littermates, display an “ activated” phenotype in vitro and in vivo and have an elevated level of cAMP phosphodiesterase (PDE) activity. In this report, we confirm that cp/cp rat aortic VSMCs have an elevated level of PDE3 activity and show that only particulate PDE3 (PDE3B) activity is elevated. In marked contrast to results obtained in + /? VSMCs, simultaneous activation of adenylyl cyclase and inhibition of PDE3 activity in cp/cp VSMCs synergistically increased cAMP. Although PDE3 inhibition did not potentiate the antimigratory effects of forskolin on +/? VSMCs, PDE3 inhibition did markedly potentiate the forskolin-induced inhibition of migration of cp/cp-derived VSMCs. Although PDE3 activity was elevated in cp/cp rat aortic VSMCs, levels of expression of cytosolic PDE3 (PDE3A) and PDE3B in +/? and cp/cp VSMCs, as well as activation of these enzymes following activation of the cAMP-protein kinase A signaling cascade, were not different. Our data are consistent with an increased role for PDE3 in regulating cAMP-dependent signaling in cp/cp VSMCs and identify PDE3 as a cellular activity potentially responsible for the phenotype of cp/cp VSMCs.
ATP-Dependent K+ Channels Contribute to Local Metabolic Coronary Vasodilation in Experimental Diabetes
Authors: Tune Johnathan D. , Yeh Clement , Setty Srinath , Downey H. Fred
Published: March 2007

This study tested whether ATP-dependent K+ channels (KATP channels) are an important mechanism of functional coronary hyperemia in conscious, instrument-implanted diabetic dogs. Data were collected at rest and during exercise before and after induction of diabetes with alloxan monohydrate (40–60 mg/kg intravenously). KATP channels were inhibited with glibenclamide (1 mg/kg intravenously). In nondiabetic dogs, arterial plasma glucose concentration increased from 4.8 ± 0.3 to 21.5 ± 2.2 mmol/l 1 week after alloxan injection. In nondiabetic dogs, exercise increased myocardial oxygen consumption (MVo2) 3.4-fold, myocardial O2 delivery 3.0-fold, and heart rate 2.4-fold. Coronary venous Po2 decreased from 19.9 ± 0.8 mmHg at rest to 14.8 ± 0.8 mmHg during exercise. Diabetes significantly reduced myocardial O2 delivery and lowered coronary venous Po2 from 16.3 ± 0.6 mmHg at rest to 13.1 ± 0.9 mmHg during exercise. Glibenclamide did not alter the slope of the coronary venous Po2 versus MVo2 relationship in nondiabetic dogs. In diabetic dogs, however, glibenclamide further reduced myocardial O2 delivery; coronary venous Po2 fell to 9.0 ± 1.0 mmHg during exercise, and the slope of the coronary venous PO2 versus MVo2 relationship steepened. These findings indicate that KATP channels contribute to local metabolic coronary vasodilation in alloxan-induced diabetic dogs.
Early Differential Defects of Insulin Secretion and Action in 19-Year-Old Caucasian Men Who Had Low Birth Weight
Authors: Jensen Christine B. , Storgaard Heidi , Dela Flemming , Holst Jens Juul , Madsbad Sten , Vaag Allan A.
Published: March 2007

Several studies have linked low birth weight (LBW) and type 2 diabetes. We investigated hepatic and peripheral insulin action including intracellular glucose metabolism in 40 19-year-old men (20 LBW, 20 matched control subjects), using the hyperinsulinemic-euglycemic clamp technique at two physiological insulin levels (10 and 40 mU/m2 per min), indirect calorimetry, and [3-3H]glucose. Insulin secretion was examined during an oral and intravenous glucose tolerance test. Fasting p-glucose was higher in the LBW group (5.6 ± 0.1 vs. 5.4 ± 0.1; P < 0.05). Basal plasma glycerol concentrations were significantly lower in the LBW group. Insulin-stimulated glycolytic flux was significantly reduced, and suppression of endogenous glucose production was enhanced in the LBW group. Nevertheless, basal and insulin-stimulated rates of whole-body peripheral glucose disposal, glucose oxidation, lipid oxidation, exogenous glucose storage, and nonoxidative glucose metabolism were similar in the two groups. Insulin secretion was reduced by 30% in the LBW group, when expressed relative to insulin sensitivity (disposition index = insulin secretion × insulin action). We propose that reduced insulin-stimulated glycolysis precedes overt insulin resistance in LBW men. A lower insulin secretion may contribute to impaired glucose tolerance and ultimately lead to diabetes.
Thyroid Hormone Receptor Interacting Protein 3 (Trip3) Is a Novel Coactivator of Hepatocyte Nuclear Factor-4α
Authors: Iwahashi Hiromi , Yamagata Kazuya , Yoshiuchi Issei , Terasaki Jungo , Yang Qin , Fukui Kenji , Ihara Arisa , Zhu Qian , Asakura Takeshi , Cao Yang , Imagawa Akihisa , Namba Mitsuyoshi , Hanafusa Toshiaki , Miyagawa Jun-ichiro , Matsuzawa Yuji
Published: March 2007

Mutations of the hepatocyte nuclear factor-4α (HNF-4α) gene are associated with a subtype of maturity-onset diabetes of the young (MODY1) that is characterized by impaired insulin secretion in response to a glucose load. HNF-4α, which is a transcription factor expressed in pancreatic β-cells, plays an important role in regulating the expression of genes involved in glucose metabolism. Thus, cofactors that interact with HNF-4α and modify its transcriptional activity might also play an important role in regulating the metabolic pathways in pancreatic β-cells, and the genes of such cofactors are plausible candidate genes for MODY. In the present study, we showed, using a yeast two-hybrid screening assay, that thyroid hormone receptor interacting protein 3 (Trip3) interacted with HNF-4α, and their interaction was confirmed by the glutathione S-transferase pull-down assay. Human Trip3 cDNA contained an open reading frame for a protein of 155 amino acids, and the gene was expressed in both pancreatic islets and MIN6 cells. Cotransfection experiments indicated that Trip3 could enhance (two- to threefold) the transcription activity of HNF-4α in COS-7 cells and MIN6 cells. These results suggest that Trip3 is a coactivator of HNF-4α. Mutation screening revealed that variation of the Trip3 gene is not a common cause of MODY/early-onset type 2 diabetes in Japanese individuals. Trip3 may play an important role in glucose metabolism by regulating the transcription activity of HNF-4α.
The Diabetic Phenotype Is Conserved in Myotubes Established From Diabetic Subjects
Authors: Gaster Michael , Petersen Ingrid , Højlund Kurt , Poulsen Pernille , Beck-Nielsen Henning
Published: March 2007

The most well-described defect in the pathophysiology of type 2 diabetes is reduced insulin-mediated glycogen synthesis in skeletal muscles. It is unclear whether this defect is primary or acquired secondary to dyslipidemia, hyperinsulinemia, or hyperglycemia. We determined the glycogen synthase (GS) activity; the content of glucose-6-phosphate, glucose, and glycogen; and the glucose transport in satellite cell cultures established from diabetic and control subjects. Myotubes were precultured in increasing insulin concentrations for 4 days and subsequently stimulated acutely by insulin. The present study shows that the basal glucose uptake as well as insulin-stimulated GS activity is reduced in satellite cell cultures established from patients with type 2 diabetes. Moreover, increasing insulin concentrations could compensate for the reduced GS activity to a certain extent, whereas chronic supraphysiological insulin concentrations induced insulin resistance in GS and glucose transport activity. Our data suggest that insulin resistance in patients with type 2 diabetes comprises at least two important defects under physiological insulin concentrations: a reduced glucose transport under basal conditions and a reduced GS activity under acute insulin stimulation, implicating a reduced glucose uptake in the fasting state and a diminished insulin-mediated storage of glucose as glycogen after a meal.
Orally Administered Leucine Enhances Protein Synthesis in Skeletal Muscle of Diabetic Rats in the Absence of Increases in 4E-BP1 or S6K1 Phosphorylation
Authors: Anthony Joshua C. , Reiter Ali K. , Anthony Tracy G. , Crozier Stephen J. , Lang Charles H. , MacLean David A. , Kimball Scot R. , Jefferson Leonard S.
Published: March 2007

In this study, food-deprived (18 h) control rats and rats with alloxan-induced diabetes were orally administered saline or the amino acid leucine to assess whether it regulates protein synthesis independently of a change in serum insulin concentrations. Immediately after leucine administration, diabetic rats were infused with insulin (0.0, 4.0, or 20 pmol · min−1 · kg−1) for 1 h to examine the role of the hormone in the protein synthetic response to leucine. In control rats, leucine stimulated protein synthesis by 58% and increased phosphorylation of the translational repressor, eukaryotic initiation factor (eIF) 4E-binding protein (BP)-1, 4E-BP1, fivefold. Consequently, association of the mRNA cap-binding protein eukaryotic initiation factor (eIF)4E with 4E-BP1 was reduced to 50% of control values, and eIF4G•eIF4E complex assembly was increased 80%. Furthermore, leucine increased the phosphorylation of the 70-kDa ribosomal protein S6 (rp S6) and the ribosomal protein S6 kinase (S6K1). Diabetes attenuated protein synthesis compared with control rats. Nonetheless, in diabetic rats, leucine increased protein synthesis by 53% without concomitant changes in the phosphorylation of 4E-BP1 or S6K1. Skeletal muscle protein synthesis was stimulated in diabetic rats infused with insulin, but rates of synthesis remained less than values in nondiabetic controls that were administered leucine. Phosphorylation of 4E-BP1 and S6K1 was increased in diabetic rats infused with insulin in a dose-dependent manner, and the response was enhanced by leucine. The results suggest that leucine enhances protein synthesis in skeletal muscle through both insulin-dependent and -independent mechanisms. The insulin-dependent mechanism is associated with increased phosphorylation of 4E-BP1 and S6K1. In contrast, the insulin-independent effect on protein synthesis is mediated by an unknown mechanism.
Intraislet Hyperinsulinemia Prevents the Glucagon Response to Hypoglycemia Despite an Intact Autonomic Response
Authors: Banarer Salomon , McGregor Veronica P. , Cryer Philip E.
Published: March 2007

Because absence of the glucagon response to falling plasma glucose concentrations plays a key role in the pathogenesis of iatrogenic hypoglycemia in patients with insulin-deficient diabetes and the mechanism of this defect is unknown, and given evidence in experimental animals that a decrease in intraislet insulin is a signal to increased glucagon secretion, we examined the role of endogenous insulin in the physiological glucagon response to hypoglycemia. We tested the hypothesis that intraislet hyperinsulinemia prevents the glucagon response to hypoglycemia despite an intact autonomic—adrenomedullary, sympathetic neural, and parasympathetic neural—response and a low α-cell glucose concentration. Twelve healthy young adults were studied on three separate occasions. Insulin was infused in hourly steps in relatively low doses (1.5, 3.0, 4.5, and 6.0 pmol · kg−1 · min−1) from 60 through 300 min on all three occasions. Plasma glucose levels were clamped at euglycemia (∼5.0 mmol/l, ∼90 mg/dl) on one occasion and at hourly steps of ∼4.7, 4.2, 3.6, and 3.0 mmol/l (∼85, 75, 65, and 55 mg/dl) from 60 through 300 min on the other two occasions. On one of the latter occasions, the β-cell secretagogue tolbutamide was infused in a dose of 1.0 g/h from 60 through 300 min. Hypoglycemia with tolbutamide infusion, compared with similar hypoglycemia alone, was associated with higher (P < 0.0001) C-peptide levels (final values of 1.0 ± 0.2 vs. 0.1 ± 0.0 nmol/l), higher (P < 0.0001) rates of insulin secretion (final values of 198 ± 60 vs. 15 ± 4 pmol/min), and higher (P < 0.0001) insulin levels (final values of 325 ± 30 vs. 245 ± 20 pmol/l) as expected. The glucagon response to hypoglycemia was prevented during tolbutamide infusion (P < 0.0001). Glucagon levels were 17 ± 1 pmol/l at baseline on both occasions, 14 ± 1 vs. 15 ± 1 pmol/l, respectively, during the initial hyperinsulinemic euglycemia, and 15 ± 1 vs. 22 ± 2 pmol/l, respectively, during hypoglycemia with and without tolbutamide infusion. Autonomic—adrenomedullary (plasma epinephrine), sympathetic neural (plasma norepinephrine), and parasympathetic neural (plasma pancreatic polypeptide)—responses to hypoglycemia were not reduced during tolbutamide infusion. We conclude that intraislet hyperinsulinemia prevents the glucagon response to hypoglycemia despite an intact autonomic response and a low α-cell glucose concentration.
Activation of IRS-2—Mediated Signal Transduction by IGF-1, but not TGF-α or EGF, Augments Pancreatic β-Cell Proliferation
Authors: Lingohr Melissa K. , Dickson Lorna M. , McCuaig Jill F. , Hugl Sigrun R. , Twardzik Daniel R. , Rhodes Christopher J.
Published: March 2007

Transforming growth factor (TGF)-α- and epidermal growth factor (EGF)-induced signal transduction was directly compared with that of glucose and insulin-like growth factor-1 (IGF-1) in INS-1 cells. TGF-α/EGF transiently (<20 min) induced phosphorylation of extracellular-regulated kinase (Erk)-1/2 (>20-fold), glycogen synthase kinase (GSK)-3 (>10-fold), and protein kinase B (PKB) (Ser473 and Thr308), but did not increase [3H]thymidine incorporation. In contrast, phosphorylation of Erk1/2, GSK-3, and PKB in response to glucose and IGF-1 was more prolonged (>24 h) and, though not as robust as TGF-α/EGF, did increase β-cell proliferation. Phosphorylation of p70S6K was also increased by IGF-1/glucose, but not by TGF-α/EGF, despite upstream PKB activation. It was found that IGF-1 induced phosphatidylinositol 3-kinase (PI3K) association with insulin receptor substrate (IRS)-1 and -2 in a glucose-dependent manner, whereas TGF-α/EGF did not. The importance of specific IRS-2-mediated signaling events was emphasized in that adenoviral-mediated overexpression of IRS-2 further increased glucose/IGF-1-induced β-cell proliferation (more than twofold; P < 0.05) compared with control or adenoviral-mediated IRS-1 overexpressing INS-1 cells. Neither IRS-1 nor IRS-2 overexpression induced a β-cell proliferative response to TGF-α/EGF. Thus, a prolonged activation of Erk1/2 and PI3K signaling pathways is important in committing a β-cell to a mitogenic event, and it is likely that this sustained activation is instigated by signal transduction occurring specifically through IRS-2.
Leptin Accelerates Autoimmune Diabetes in Female NOD Mice
Authors: Matarese Giuseppe , Sanna Veronica , Lechler Robert I. , Sarvetnick Nora , Fontana Silvia , Zappacosta Serafino , La Cava Antonio
Published: March 2007

We have recently shown that leptin, the product of the obese gene, can directly influence T-cell function. In the work presented here, we explored the role of leptin in the development of spontaneous autoimmunity in the nonobese diabetic (NOD) mouse, an animal model for the study of human insulin-dependent diabetes mellitus (type 1 diabetes). We found that expression of serum leptin increased soon before the onset of hyperglycemia and diabetes in susceptible females. A pathogenetic role of leptin was assessed by administering recombinant leptin to young female and male NOD mice. Intraperitoneal injections of leptin accelerated autoimmune destruction of insulin-producing β-cells and significantly increased interferon-γ production in peripheral T-cells. These findings indicate that leptin can favor proinflammatory cell responses and directly influence development of autoimmune disease mediated by Th1 responses.
Combined Expression of Pancreatic Duodenal Homeobox 1 and Islet Factor 1 Induces Immature Enterocytes to Produce Insulin
Authors: Kojima Hideto , Nakamura Takaaki , Fujita Yukihiro , Kishi Akio , Fujimiya Mineko , Yamada Syu , Kudo Motoi , Nishio Yoshihiko , Maegawa Hiroshi , Haneda Masakazu , Yasuda Hitoshi , Kojima Itaru , Seno Masaharu , Wong Norman C.W. , Kikkawa Ryuichi , Kashiwagi Atsunori
Published: March 2007

Immature rat intestinal stem cells (IEC-6) given the ability to express the transcription factor, pancreatic duodenal homeobox 1 (Pdx-1), yielded YK cells. Although these cells produced multiple enteroendocrine hormones, they did not produce insulin. Exposure of YK cells to 2 nmol/l betacellulin yielded BYK cells that showed the presence of insulin expression in cytoplasm and that secreted insulin into culture media. By examining the mechanism of differentiation in BYK cells, we found that another transcription factor, islet factor 1 (Isl-1) was newly expressed with the disappearance of Pax-6 expression in those cells after exposure to betacellulin. These results indicated that combined expression of Pdx-1 and Isl-1 in IEC-6 cells was required for the production of insulin. In fact, overexpression of both Pdx-1 and Isl-1 in IEC-6 cells (Isl-YK-12, -14, and -15 cells) gave them the ability to express insulin without exposure to betacellulin. Furthermore, implantation of the Isl-YK-14 cells into diabetic rats reduced the animals’ plasma glucose levels; glucose levels dropped from 19.4 to 16.9 mmol/l 1 day after the injection of cells. As expected, the plasma insulin concentrations were 2.7 times higher in the diabetic rats injected with Isl-YK-14 cells compared to in controls. In summary, our results indicated that immature intestinal stem cells can differentiate into insulin-producing cells given the ability to express the transcription factors Pdx-1 and Isl-1.
Prolonged Exposure to Free Fatty Acids Has Cytostatic and Pro-Apoptotic Effects on Human Pancreatic Islets
Authors: Lupi Roberto , Dotta Francesco , Marselli Lorella , Del Guerra Silvia , Masini Matilde , Santangelo Carmela , Patané Giovanni , Boggi Ugo , Piro Salvatore , Anello Marcello , Bergamini Ettore , Mosca Franco , Di Mario Umberto , Del Prato Stefano , Marchetti Piero
Published: March 2007

In an effort to better understand the phenomenon of lipotoxicity in human β-cells, we evaluated the effects of 48-h preculture with 1.0 or 2.0 mmol/l free fatty acid (FFA) (2:1 oleate to palmitate) on the function and survival of isolated human islets and investigated some of the possible mechanisms. Compared with control islets, triglyceride content was significantly increased and insulin content and glucose-stimulated insulin release were significantly reduced in islets precultured with increased FFA concentrations. These changes were accompanied by a significant reduction of glucose utilization and oxidation. By cell death detection techniques, it was observed that exposure to FFAs induced a significant increase of the amount of dead cells. Electron microscopy showed the involvement of β-cells, with morphological appearance compatible with the presence of apoptotic phenomena. FFA-induced islet cell death was blocked by inhibition of upstream caspases and partially prevented by inhibiton of ceramide synthesis or serine protease activity, whereas inhibition of nitric oxide synthesis had no effect. RT-PCR studies revealed no major change of iNOS and Bax mRNA expression and a marked decrease of Bcl-2 mRNA expression in the islets cultured with FFA. Thus, prolonged exposure to FFAs has cytostatic and pro-apoptotic effects on human pancreatic β-cells. The cytostatic action is likely to be due to the FFA-induced reduction of intraislet glucose metabolism, and the proapoptotic effects are mostly caspase mediated, partially dependent on ceramide pathway, and possibly Bcl-2 regulated.
Chronic Inhibition of Circulating Dipeptidyl Peptidase IV by FE 999011 Delays the Occurrence of Diabetes in Male Zucker Diabetic Fatty Rats
Authors: Sudre Béatrice , Broqua Pierre , White Richard B. , Ashworth Doreen , Evans D. Michael , Haigh Robert , Junien Jean-Louis , Aubert Michel L.
Published: March 2007

Acute suppression of dipeptidyl peptidase IV (DPP-IV) activity improves glucose tolerance in the Zucker fatty rat, a rodent model of impaired glucose tolerance, through stabilization of glucagon-like peptide (GLP)-1. This study describes the effects of a new and potent DPP-IV inhibitor, FE 999011, which is able to suppress plasma DPP-IV activity for 12 h after a single oral administration. In the Zucker fatty rat, FE 999011 dose-dependently attenuated glucose excursion during an oral glucose tolerance test and increased GLP-1 (7-36) release in response to intraduodenal glucose. Chronic treatment with FE 999011 (10 mg/kg, twice a day for 7 days) improved glucose tolerance, as suggested by a decrease in the insulin-to-glucose ratio. In the Zucker diabetic fatty (ZDF) rat, a rodent model of type 2 diabetes, chronic treatment with FE 999011 (10 mg/kg per os, once or twice a day) postponed the development of diabetes, with the twice-a-day treatment delaying the onset of hyperglycemia by 21 days. In addition, treatment with FE 999011 stabilized food and water intake to prediabetic levels and reduced hypertriglyceridemia while preventing the rise in circulating free fatty acids. At the end of treatment, basal plasma GLP-1 levels were increased, and pancreatic gene expression for GLP-1 receptor was significantly upregulated. This study demonstrates that DPP-IV inhibitors such as FE 999011 could be of clinical value to delay the progression from impaired glucose tolerance to type 2 diabetes.