bluuebunny/crossref_metadata_2025 huggingface dataset:
Advanced Glycation End Product Precursors Impair Epidermal Growth Factor Receptor Signaling
Authors: Portero-Otín Manuel , Pamplona Reinald , Bellmunt Maria Josep , Ruiz Maria Cristina , Prat Joan , Salvayre Robert , Nègre-Salvayre Anne
Published: March 2007

Formation of advanced glycation end products (AGEs) is considered a potential link between hyperglycemia and chronic diabetic complications, including disturbances in cell signaling. It was hypothesized that AGEs alter cell signaling by interfering with growth factor receptors. Therefore, we studied the effects of two AGE precursors, glyoxal (GO) and methylglyoxal (MGO), on the epidermal growth factor receptor (EGFR) signaling pathway in cultured cells. Both compounds prevented tyrosine autophosphorylation induced by epidermal growth factor (EGF) in a time- and dose-dependent manner as well as phospholipase Cγ1 recruitment and subsequent activation of extracellular signal-regulated kinases. AGE precursors inhibit EGF-induced EGFR autophosphorylation and tyrosine kinase activity in cell membranes and in EGFR immunoprecipitates. In addition, AGE precursors strongly inhibited cellular phosphotyrosine phosphatase activities and residual EGFR dephosphorylation. AGE precursors induced the formation of EGFR cross-links, as shown by the cross-reactivity of modified EGFR with an anti-Nε(carboxymethyl)lysine antibody, suggesting that altered EGFR signaling was related to carbonyl-amine reactions on EGFR. Aminoguanidine, an inhibitor of AGE formation, partially prevented the EGFR dysfunction induced by GO and MGO. These data introduce a novel mechanism for impaired cellular homeostasis in situations that lead to increased production of these reactive aldehydes, such as diabetes.
The Early Natural History of Nephropathy in Type 1 Diabetes
Authors: Drummond Keith , Mauer Michael ,   for the International Diabetic Nephropathy Study Group
Published: March 2007

Renal structural abnormalities are known to precede the development of proteinuria, hypertension, and reduced renal function in patients with type 1 diabetes. The determinants of these early structural abnormalities are, however, largely unknown. The International Diabetic Nephropathy Study (IDNS) has recruited 243 children and adults (aged 10–40 years) in Montreal, Minneapolis, and Paris to identify and quantify these determinants. All study subjects were normotensive and had normal-to-high glomerular filtration rates (GFRs) and urinary albumin excretion rates (AERs) <100 μg/min at study entry. Only 8 of 243 had an AER ≥20 μg/min (microalbuminuria). Two renal biopsies are obtained at a 5-year intervals, with baseline and follow-up measures of renal function, blood pressure (BP), HbA1c, plasma lipids, and AER. Herein, we examine the baseline renal biopsy morphometric findings in these subjects and in 87 kidney donor control subjects and explore the associations between findings and clinical and demographic variables. The principal morphometric abnormalities were increased glomerular basement membrane (GBM) width and fractional volume of mesangium [Vv(Mes/glom)] and mesangial matrix [Vv(MM/glom)]. The frequency of these abnormalities increased with increasing duration of diabetes but was observed as early as 2–8 years after onset. Diastolic BP (DBP), but not HbA1c, was directly associated with these abnormalities. Elevated GFR was associated with only a small increase in peripheral glomerular capillary basement membrane filtration surface density. Center differences were detected in renal structural, renal functional, and BP parameters, especially between the Paris and North American centers. GBM width, Vv(Mes/glom), and Vv(MM/glom) are significantly increased even within a few years of onset of type 1 diabetes. These changes are detectable in normoalbuminuric patients and are related to duration, BP, and study site. Changes in these and other morphometric measures over 5-year follow-up should help clarify the roles of glycemia and other determinants of the rates of development of diabetic nephropathy lesions, as well as their relationships to early changes in BP, albumin excretion, and renal function.
Accumulation of 8-Hydroxy-2′-Deoxyguanosine and Mitochondrial DNA Deletion in Kidney of Diabetic Rats
Authors: Kakimoto Maiko , Inoguchi Toyoshi , Sonta Toshiyo , Yu Hai Yan , Imamura Minako , Etoh Takashi , Hashimoto Toshihiko , Nawata Hajime
Published: March 2007

Oxidative stress may contribute to the pathogenesis of diabetic nephropathy. However, the detailed molecular mechanism remains uncertain. Here, we report oxidative mitochondrial DNA (mtDNA) damage and accumulation of mtDNA with a 4,834-bp deletion in kidney of streptozotocin-induced diabetic rats. At 8 weeks after the onset of diabetes, levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), which is a marker of oxidative DNA damage, were significantly increased in mtDNA from kidney of diabetic rats but not in nuclear DNA, suggesting the predominant damage of mtDNA. Semiquantitative analysis using PCR showed that the frequency of 4,834-bp deleted mtDNA was markedly increased in kidney of diabetic rats at 8 weeks, but it did not change at 4 weeks. Intervention by insulin treatment starting at 8 weeks rapidly normalized an increase in renal 8-OHdG levels of diabetic rats, but it did not reverse an increase in the frequency of deleted mtDNA. Our study demonstrated for the first time that oxidative mtDNA damage and subsequent mtDNA deletion may be accumulated in kidney of diabetic rats. This may be involved in the pathogenesis of diabetic nephropathy.
Polymorphisms in the Oxygen-Regulated Protein 150 Gene (ORP150) Are Associated With Insulin Resistance in Pima Indians
Authors: Kovacs Peter , Yang Xiaolin , Permana Paska A. , Bogardus Clifton , Baier Leslie J.
Published: March 2007

The ORP150 gene that encodes the human oxygen-regulated protein (150 kDa) maps to chromosome 11q23, a region previously reported to be linked to type 2 diabetes and obesity in Pima Indians. This gene was also found to be differentially expressed in global gene expression studies comparing muscle mRNA from insulin-resistant versus insulin-sensitive subjects. Therefore, ORP150 was analyzed as a candidate gene for susceptibility to diabetes. Twelve variants were identified, and three unique representative polymorphisms were genotyped in 1,338 Pima Indians. None of these polymorphisms were associated with diabetes, but two polymorphisms were significantly associated with measures of insulin resistance. These data indicate that ORP150 has a role in insulin action but does not have a major role in determining susceptibility to type 2 diabetes in Pima Indians.
Mature Adipocytes Inhibit In Vitro Differentiation of Human Preadipocytes via Angiotensin Type 1 Receptors
Authors: Janke Jürgen , Engeli Stefan , Gorzelniak Kerstin , Luft Friedrich C. , Sharma Arya M.
Published: March 2007

Recent studies suggest that angiotensin II (Ang II) plays a role in the adipogenesis of murine preadipocytes. Here, we examined the role of Ang II for the differentiation of primary cultured human preadipocytes. Preadipocytes were isolated from human adipose tissue and stimulated to differentiate. Quantitation of gene expression during adipogenesis was performed for renin-angiotensin system (RAS) genes. The influence of the RAS on adipogenic differentiation was investigated by addition of either angiotensinogen (AGT), Ang II, or angiotensin receptor antagonists to the differentiation medium. We also examined the influence of adipocytes on adipogenesis by co-culture experiments. Expression of the RAS genes AGT, renin, angiotensin-converting enzyme, and Ang II type 1 receptor increased during adipogenesis. Stimulation of the Ang II type 1 receptor by Ang II reduced adipose conversion, whereas blockade of this receptor markedly enhanced adipogenesis. Adipocytes were able to inhibit preadipocyte differentiation in the co-culture, and this effect was abolished by blockade of the Ang II type 1 receptor. This finding points to a functional role of the RAS in the differentiation of human adipose tissue. Because AGT secretion and Ang II generation are characteristic features of adipogenesis, we postulate a paracrine negative-feedback loop that inhibits further recruitment of preadipocytes by maturing adipocytes.
A Nuclear Magnetic Resonance-Based Demonstration of Substantial Oxidative l-Alanine Metabolism and l-Alanine-Enhanced Glucose Metabolism in a Clonal Pancreatic β-Cell Line
Authors: Brennan Lorraine , Shine Aine , Hewage Chandralal , Malthouse J. Paul G. , Brindle Kevin M. , McClenaghan Neville , Flatt Peter R. , Newsholme Philip
Published: March 2007

Early experiments indicated that islet β-cells substantially metabolized l-alanine but that insulin secretion was largely unaffected by the amino acid. It was subsequently demonstrated using more intricate studies that l-alanine is a strong stimulus to insulin secretion in the presence of glucose in normal rodent islets and β-cell lines. Using 13C nuclear magnetic resonance (NMR), we have demonstrated substantial oxidative metabolism of l-alanine by the clonal β-cell line BRIN-BD11, with time-dependent increases in production of cellular glutamate and aspartate. Stimulatory effects of l-alanine on insulin secretion were attenuated by the inhibition of β-cell oxidative phosphorylation using oligomycin. Additionally, we detected substantial production of lactate, alanine, and glutamate from glucose (16.7 mmol/l) after 60 min. On addition of 10 mmol/l l-alanine to a stimulus of 16.7 mmol/l glucose, the utilization rate of glucose increased ∼2.4-fold. l-Alanine dramatically enhanced NMR-measurable aspects of glucose metabolism (both oxidative and nonoxidative). The enhanced rate of entry of glucose-derived pyruvate into the tricarboxylic acid (TCA) cycle in the presence of alanine may have stimulated rates of generation of key metabolites, including ATP, which affect the insulin secretory process. Thus l-alanine metabolism, in addition to the enhancing effect on glucose metabolism, contributes to the stimulatory effects of this amino acid on insulin secretion in vitro.
Inhibition of Thrombin Abrogates the Instant Blood-Mediated Inflammatory Reaction Triggered by Isolated Human Islets
Authors: Özmen Lisa , Ekdahl Kristina Nilsson , Elgue Graciela , Larsson Rolf , Korsgren Olle , Nilsson Bo
Published: March 2007

A thrombotic/inflammatory reaction is elicited when isolated islets of Langerhans come in contact with ABO-compatible blood. The detrimental effects of this instant blood-mediated inflammatory reaction (IBMIR) provide a reasonable explanation for the observation that an unexpectedly high number of islets, from several donors, are needed to produce normoglycemia in transplant patients with type 1 diabetes. In this study, the hypothesis that a specific thrombin inhibitor, Melagatran, could reduce IBMIR in an in vitro model in which human islets are exposed to ABO-compatible blood was tested. The administration of Melagatran abrogated IBMIR dose-dependently. Islets exposed to blood, in the absence or presence of 0.4 μmol/l Melagatran, exhibited a loss of integrity and were found to be trapped in macroscopic clots containing platelets and CD11b+ leukocytes. At concentrations from 1 to 10 μmol/l, Melagatran inhibited both coagulation and complement activation. Also, platelet and leukocyte activation and consumption were decreased. Islet morphology was maintained with almost no platelets adhering to the surface, and infiltration by CD11b+ leukocytes was considerably reduced. In conclusion, Melagatran significantly reduced IBMIR in this model system. This protective effect indicates that thrombin plays a pivotal role in IBMIR and suggests that thrombin inhibition can improve the outcome of clinical islet transplantation.
Hepatocyte Nuclear Factor-1α Modulates Pancreatic β-Cell Growth by Regulating the Expression of Insulin-Like Growth Factor-1 in INS-1 Cells
Authors: Yang Qin , Yamagata Kazuya , Fukui Kenji , Cao Yang , Nammo Takao , Iwahashi Hiromi , Wang Haiyan , Matsumura Itaru , Hanafusa Toshiaki , Bucala Richard , Wollheim Claes B. , Miyagawa Jun-ichiro , Matsuzawa Yuji
Published: March 2007

Maturity-onset diabetes of the young type 3 (MODY3) is characterized by impaired insulin secretion. Heterozygous mutations in the gene encoding hepatocyte nuclear factor (HNF)-1α are the cause of MODY3. Transgenic mice overexpressing dominant-negative HNF-1α mutant in pancreatic β-cells and HNF-1α knockout mice are animal models of MODY3. These mice exhibit defective glucose-stimulated insulin secretion and have reduced β-cell mass and β-cell proliferation rate. Here we examined the effect of HNF-1α on β-cell proliferation by overexpressing a human naturally occurring dominant- negative mutation P291fsinsC in INS-1 cells under the control of doxycycline-induction system. INS-1 cells overexpressing P291fsinsC showed apparent growth impairment. The proliferation rate estimated by [3H]thymidine incorporation was significantly reduced in P291fsinsC-expressing INS-1 cells compared with noninduced or wild-type HNF-1α-overexpressing INS-1 cells. Growth inhibition occurred at the transition from G1 to S cell cycle phase, with reduced expression of cyclin E and upregulation of p27. cDNA array analysis revealed that the expression levels of IGF-1, a major growth factor for β-cells, and macrophage migration inhibitory factor (MIF), a cytokine expressed in pancreatic β-cells, were reduced in P291fsinsC-HNF-1α–expressing INS-1 cells. Although MIF seemed to have proliferative function, blockade of MIF action by anti-MIF antibody stimulated INS-1 cell proliferation, excluding its direct role in the growth impairment. However, addition of IGF-1 to P291fsinsC–expressing INS-1 cells rescued the growth inhibition. Our data suggest that HNF-1α is critical for modulating pancreatic β-cell growth by regulating IGF-1 expression. IGF-1 might be a potential therapeutic target for the treatment of MODY3.
Overexpression of c-Myc in β-Cells of Transgenic Mice Causes Proliferation and Apoptosis, Downregulation of Insulin Gene Expression, and Diabetes
Authors: Laybutt D. Ross , Weir Gordon C. , Kaneto Hideaki , Lebet Judith , Palmiter Richard D. , Sharma Arun , Bonner-Weir Susan
Published: March 2007

To test the hypothesis that c-Myc plays an important role in β-cell growth and differentiation, we generated transgenic mice overexpressing c-Myc in β-cells under control of the rat insulin II promoter. F1 transgenic mice from two founders developed neonatal diabetes (associated with reduced plasma insulin levels) and died of hyperglycemia 3 days after birth. In pancreata of transgenic mice, marked hyperplasia of cells with an altered phenotype and amorphous islet organization was displayed: islet volume was increased threefold versus wild-type littermates. Apoptotic nuclei were increased fourfold in transgenic versus wild-type mice, suggesting an increased turnover of β-cells. Very few cells immunostained for insulin; pancreatic insulin mRNA and content were markedly reduced. GLUT2 mRNA was decreased, but other β-cell–associated genes (IAPP [islet amyloid pancreatic polypeptide], PDX-1 [pancreatic and duodenal homeobox-1], and BETA2/NeuroD) were expressed at near-normal levels. Immunostaining for both GLUT2 and Nkx6.1 was mainly cytoplasmic. The defect in β-cell phenotype in transgenic embryos (embryonic days 17–18) and neonates (days 1–2) was similar and, therefore, was not secondary to overt hyperglycemia. When pancreata were transplanted under the kidney capsules of athymic mice to analyze the long-term effects of c-Myc activation, β-cell depletion was found, suggesting that, ultimately, apoptosis predominates over proliferation. In conclusion, these studies demonstrate that activation of c-Myc in β-cells leads to 1) increased proliferation and apoptosis, 2) initial hyperplasia with amorphous islet organization, and 3) selective downregulation of insulin gene expression and the development of overt diabetes.
Na/Ca Exchanger Overexpression Induces Endoplasmic Reticulum–Related Apoptosis and Caspase-12 Activation in Insulin-Releasing BRIN-BD11 Cells
Authors: Diaz-Horta Oscar , Kamagate Adama , Herchuelz André , Van Eylen Françoise
Published: March 2007

Ca2+ may trigger programmed cell death (apoptosis) and regulate death-specific enzymes. Therefore, the development of strategies to control Ca2+ homeostasis may represent a potential approach to prevent or enhance cell apoptosis. To test this hypothesis, the plasma membrane Na/Ca exchanger (NCX1.7 isoform) was stably overexpressed in insulin-secreting tumoral cells. NCX1.7 overexpression increased apoptosis induced by endoplasmic reticulum (ER) Ca2+-ATPase inhibitors, but not by agents increasing intracellular calcium concentration ([Ca2+]i), through the opening of plasma membrane Ca2+-channels. NCX1.7 overexpression reduced the rise in [Ca2+]i induced by all agents, depleted ER Ca2+ stores, sensitized the cells to Ca2+-independent proapoptotic signaling pathways, and reduced cell proliferation by ∼40%. ER Ca2+ stores depletion was accompanied by the activation of the ER-specific caspase (caspase-12), and the activation was enhanced by ER Ca2+-ATPase inhibitors. Hence, Na/Ca exchanger overexpression, by depleting ER Ca2+ stores, triggers the activation of caspase-12 and increases apoptotic cell death. By increasing apoptosis and decreasing cell proliferation, overexpression of Na/Ca exchanger may represent a new potential approach in cancer gene therapy. On the other hand, our results open the way to the development of new strategies to control cellular Ca2+ homeostasis that could, on the contrary, prevent the process of apoptosis that mediates, in part, β-cell autoimmune destruction in type 1 diabetes.
Targeted Disruption of the Protein Tyrosine Phosphatase-Like Molecule IA-2 Results in Alterations in Glucose Tolerance Tests and Insulin Secretion
Authors: Saeki Keiichi , Zhu Min , Kubosaki Atsutaka , Xie Jingping , Lan Michael S. , Notkins Abner Louis
Published: March 2007

IA-2 is a major autoantigen in type 1 diabetes. Autoantibodies to IA-2 appear years before the development of clinical disease and are being widely used as predictive markers to identify individuals at risk for developing type 1 diabetes. IA-2 is an enzymatically inactive member of the transmembrane protein tyrosine phosphatase family and is an integral component of secretory granules in neuroendocrine cells. To study its function, we generated IA-2−deficient mice. Northern and Western blot analysis showed that neither IA-2 mRNA nor protein was expressed. Physical examination of the IA-2− /− animals and histological examination of tissues failed to reveal any abnormalities. Nonfasting blood glucose levels, measured over 6 months, were slightly elevated in male IA-2−/− as compared to IA-2+ /+ littermates, but remained within the nondiabetic range. Glucose tolerance tests, however, revealed statistically significant elevation of glucose in both male and female IA-2−/− mice and depressed insulin release. In vitro glucose stimulation of isolated islets showed that male and female mice carrying the disrupted gene released 48% (P < 0.001) and 42% (P < 0.01) less insulin, respectively, than mice carrying the wild-type gene. We concluded that IA-2 is involved in glucose-stimulated insulin secretion.
Tanis: A Link Between Type 2 Diabetes and Inflammation?
Authors: Walder Ken , Kantham Lakshmi , McMillan Janine S. , Trevaskis James , Kerr Lyndal , de Silva Andrea , Sunderland Terry , Godde Nathan , Gao Yuan , Bishara Natalie , Windmill Kelly , Tenne-Brown Janette , Augert Guy , Zimmet Paul Z. , Collier Greg R.
Published: March 2007

Here we describe a novel protein, which we have named Tanis, that is implicated in type 2 diabetes and inflammation. In Psammomys obesus, a unique polygenic animal model of type 2 diabetes and the metabolic syndrome, Tanis is expressed in the liver in inverse proportion to circulating glucose (P = 0.010) and insulin levels (P = 0.004) and in direct proportion with plasma triglyceride concentrations (P = 0.007). Hepatic Tanis gene expression was markedly increased (3.1-fold) after a 24-h fast in diabetic but not in nondiabetic P. obesus. In addition, glucose inhibited Tanis gene expression in cultured hepatocytes (P = 0.006) as well as in several other cell types (P = 0.001–0.011). Thus, Tanis seems to be regulated by glucose and is dysregulated in the diabetic state. Yeast-2 hybrid screening identified serum amyloid A (SAA), an acute-phase inflammatory response protein, as an interacting protein of Tanis, and this was confirmed by Biacore experiments. SAA and other acute-phase proteins have been the focus of recent attention as risk factors for cardiovascular disease, and we contend that Tanis and its interaction with SAA may provide a mechanistic link among type 2 diabetes, inflammation, and cardiovascular disease.
Altered Tumor Necrosis Factor-α (TNF-α) Processing in Adipocytes and Increased Expression of Transmembrane TNF-α in Obesity
Authors: Xu Haiyan , Uysal K. Teoman , Becherer J. David , Arner Peter , Hotamisligil Gökhan S.
Published: March 2007

Tumor necrosis factor-α (TNF-α) is synthesized as a 26-kDa transmembrane protein (mTNF-α), which may present on the cell surface or be processed to release the 17-kDa soluble form (sTNF-α). Because regulation of this ectodomain shedding might be critical in the generation of systemic versus local cytokine responses, we examined the rate of mTNF-α processing in adipocytes and its regulation in obesity. Here, we demonstrate that the 26-kDa mTNF-α is present in adipose tissue and that its production is significantly increased in different rodent obesity models as well as in obese humans. There was no apparent deficiency in the level of the major TNF-α converting enzyme in adipose tissue to account for the excess amount of mTNF-α produced in obesity. However, experiments in cultured fat cells stably expressing TNF-α demonstrated a significantly decreased rate of TNF-α cleavage in differentiated adipocytes compared with preadipocytes. Thus, a decreased processing rate of mTNF-α in mature adipocytes combined with an increase in TNF-α production may be a potential mechanism resulting in elevated membrane-associated TNF-α in adipose tissue in obesity.
High Alanine Aminotransferase Is Associated With Decreased Hepatic Insulin Sensitivity and Predicts the Development of Type 2 Diabetes
Authors: Vozarova Barbora , Stefan Norbert , Lindsay Robert S. , Saremi Aramesh , Pratley Richard E. , Bogardus Clifton , Tataranni P. Antonio
Published: March 2007

It has been proposed that liver dysfunction may contribute to the development of type 2 diabetes. The aim of the present study was to examine whether elevated hepatic enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], or γ -glutamyltranspeptidase [GGT]) are associated with prospective changes in liver or whole-body insulin sensitivity and/or insulin secretion and whether these elevated enzymes predict the development of type 2 diabetes in Pima Indians. We measured ALT, AST, and GGT in 451 nondiabetic (75-g oral glucose tolerance test) Pima Indians (aged 30 ± 6 years, body fat 33 ± 8%, ALT 45 ± 29 units/l, AST 34 ± 18 units/l, and GGT 56 ± 40 units/l [mean ± SD]) who were characterized for body composition (hydrodensitometry or dual-energy X-ray absorptiometry), whole-body insulin sensitivity (M), and hepatic insulin sensitivity (hepatic glucose output [HGO] during the low-dose insulin infusion of a hyperinsulinemic clamp) and acute insulin response (AIR) (25-g intravenous glucose challenge). Sixty-three subjects developed diabetes over an average follow-up of 6.9 ± 4.9 years. In 224 subjects, who remained nondiabetic, follow-up measurements of M and AIR were available. At baseline, ALT, AST, and GGT were related to percent body fat (r = 0.16, 0.17, and 0.11, respectively), M (r = −0.32, − 0.28, and −0.24), and HGO (r = 0.27, 0.12, and 0.14; all P < 0.01). In a proportional hazard analysis with adjustment for age, sex, body fat, M, and AIR, higher ALT [relative hazard 90th vs. 10th centiles (95% CI): 1.9 (1.1–3.3), P = 0.02], but not AST or GGT, predicted diabetes. Elevated ALT at baseline was associated prospectively with an increase in HGO (r = 0.21, P = 0.001) but not with changes in M or AIR (both P = 0.1). Higher ALT concentrations were cross-sectionally associated with obesity and whole-body and hepatic insulin resistance and prospectively associated with a decline in hepatic insulin sensitivity and the development of type 2 diabetes. Our findings indicate that high ALT is a marker of risk for type 2 diabetes and suggest a potential role of the liver in the pathogenesis of type 2 diabetes.
Differential Effects of Tumor Necrosis Factor-α on Protein Kinase C Isoforms α and δ Mediate Inhibition of Insulin Receptor Signaling
Authors: Rosenzweig Tovit , Braiman Liora , Bak Asia , Alt Addy , Kuroki Toshio , Sampson Sanford R.
Published: March 2007

Tumor necrosis factor-α (TNF-α) is a multifunctional cytokine that interferes with insulin signaling, but the molecular mechanisms of this effect are unclear. Because certain protein kinase C (PKC) isoforms are activated by insulin, we examined the role of PKC in TNF-α inhibition of insulin signaling in primary cultures of mouse skeletal muscle. TNF-α, given 5 min before insulin, inhibited insulin-induced tyrosine phosphorylation of insulin receptor (IR), IR substrate (IRS)-1, insulin-induced association of IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase (PI3-K), and insulin-induced glucose uptake. Insulin and TNF-α each caused tyrosine phosphorylation and activation of PKCs δ and α, but when TNF-α preceded insulin, the effects were less than that produced by each substance alone. Insulin induced PKCδ specifically to coprecipitate with IR, an effect blocked by TNF-α. Both PKCα and -δ are constitutively associated with IRS-1. Whereas insulin decreased coprecipitation of IRS-1 with PKCα, it increased coprecipitation of IRS-1 with PKCδ. TNF-α blocked the effects of insulin on association of both PKCs with IRS-1. To further investigate the involvement of PKCs in inhibitory actions of TNF-α on insulin signaling, we overexpressed specific PKC isoforms in mature myotubes. PKCα overexpression inhibited basal and insulin-induced IR autophosphorylation, whereas PKCδ overexpression increased IR autophosphorylation and abrogated the inhibitory effect of TNF-α on IR autophosphorylation and signaling to PI3-K. Blockade of PKCα antagonized the inhibitory effects of TNF-α on both insulin-induced IR tyrosine phosphorylation and IR signaling to PI3-K. We suggest that the effects of TNF-α on IR tyrosine phosphorylation are mediated via alteration of insulin-induced activation and association of PKCδ and -α with upstream signaling molecules.
Lipoprotein Subclasses and Particle Sizes and Their Relationship With Coronary Artery Calcification in Men and Women With and Without Type 1 Diabetes
Authors: Colhoun Helen M. , Otvos James D. , Rubens Mike B. , Taskinen M. R. , Underwood S. Richard , Fuller John H.
Published: March 2007

Type 1 diabetes is associated with increased coronary atherosclerosis, especially in women, even though such patients often have an apparently normal lipid profile. We examined whether lipoprotein particle sizes and subclasses differed between type 1 diabetic subjects (n = 194, age 30–55 years) and age- and sex-matched control subjects (n = 195). We examined whether any abnormalities were of similar magnitude in men and women. The relationship of particle size to electron beam computer tomography–defined coronary artery calcification, a measure of atherosclerosis, was also examined. Proton nuclear magnetic resonance (NMR) spectroscopy was used to quantify VLDL, LDL, and HDL subclass levels and average particle size on fasting samples. LDL size and subclass were similar in diabetic and nondiabetic men. In contrast, in women diabetes was associated with less large and more small LDL and a reduced LDL size (mean difference 0.2 nm; P = 0.0009). This greater 2effect of diabetes on LDL size in women compared with men was significant (P = 0.007). Diabetes was associated with more large and less small HDL and, to a similar degree in both sexes, a higher HDL size (difference of 0.4 nm in men and 0.3 nm in women; both P < 0.0001). There were no definitive abnormalities in VLDL size. In nondiabetic subjects, lower average HDL particle size, lower LDL size, and higher VLDL size were significantly associated with coronary calcification (P = 0.001, 0.02, and 0.04, respectively). Thus the HDL size differences with diabetes would be expected to be antiatherogenic and the LDL size differences pro-atherogenic. However, there was no clear relationship between particle size and calcification in diabetic subjects. We conclude that in the general population NMR spectroscopy–derived particle size reveals important information about the atherogenicity of lipoprotein profile. Type 1 diabetes is associated with differences in NMR-derived particle size, but their pathogenic significance is unclear.
Enterovirus RNA Is Found in Peripheral Blood Mononuclear Cells in a Majority of Type 1 Diabetic Children at Onset
Authors: Yin Hong , Berg Anna-Karin , Tuvemo Torsten , Frisk Gun
Published: March 2007

We have studied the occurrence of enterovirus (EV)-RNA at the onset of childhood type 1 diabetes in all 24 new cases of childhood type 1 diabetes during 1 year in Uppsala county, Sweden. We also studied 24 matched control subjects and 20 siblings of the patients. RNA was isolated from peripheral blood mononuclear cells and EV-RNA detected by RT-PCR. Primers (groups A and B) corresponding to conserved regions in the 5′ noncoding region (NCR) of EV were used in the PCRs, and the amplicons were sequenced. By the use of group A primers, EV-RNA was found in 12 (50%) of the 24 type 1 diabetic children, 5 (26%) of 19 siblings, and none of the control subjects. Both patients and siblings showed a higher frequency of EV-RNA compared with the control subjects. The group B primers detected EV-RNA in all three groups but did not show statistically significant differences between the groups. The EV-RNA positivity with the group B primers was 11 (46%) of 24 in the type 1 diabetic children, 11 (58%) of 19 in the siblings, and 7 (29%) of 24 in the control subjects. The significant difference between groups seen with the group A primers but not with the group B primers might indicate the existence of diabetogenic EV strains. The phylogenetic analysis of the PCR products revealed clustering of the sequences from patients and siblings into five major branches when the group A PCR primers were used. With the group B primers, the sequences from patients, siblings, and control subjects formed three major branches in the phylogenetic tree, where 6 of the 7 control subjects clustered together in a sub-branch of CBV-4/VD2921. Seven of the type 1 diabetic children clustered together in another sub-branch of CBV-4/VD2921. Five of the type 1 diabetic children formed a branch together with the CBV-4/E2 strain, four clustered together with CBV-5, and one formed a branch with echovirus serotype. The presence of EV-RNA in the blood cells of most newly diagnosed type 1 diabetic children supports the hypothesis that a viral infection acts as an exogenous factor. In addition, sequencing of the PCR amplicons from the type 1 diabetic children, their siblings, and matched control subjects might reveal differences related to diabetogenic properties of such a virus.
Functional Genomics of the Endocrine Pancreas
Authors: Scearce L. Marie , Brestelli John E. , McWeeney Shannon K. , Lee Catherine S. , Mazzarelli Joan , Pinney Deborah F. , Pizarro Angel , Stoeckert Christian J. , Clifton Sandra W. , Permutt M. Alan , Brown Juliana , Melton Douglas A. , Kaestner Klaus H.
Published: March 2007

Over the past 5 years, microarrays have greatly facilitated large-scale analysis of gene expression levels. Although these arrays were not specifically geared to represent tissues and pathways known to be affected by diabetes, they have been used in both type 1 and type 2 diabetes research. To prepare a tool that is particularly useful in the study of type 1 diabetes, we have assembled a nonredundant set of 3,400 clones representing genes expressed in the mouse pancreas or pathways known to be affected by diabetes. We have demonstrated the usefulness of this clone set by preparing a cDNA glass microarray, the PancChip, and using it to analyze pancreatic gene expression from embryonic day 14.5 through adulthood in mice. The clone set and corresponding array are useful resources for diabetes research.
Role of Peroxisome Proliferator-Activated Receptor-γ in Maintenance of the Characteristics of Mature 3T3-L1 Adipocytes
Authors: Tamori Yoshikazu , Masugi Jiro , Nishino Naonobu , Kasuga Masato
Published: March 2007

Peroxisome proliferator–activated receptor (PPAR)-γ plays an important role in adipogenesis. However, the functions of PPAR-γ in differentiated adipocytes have remained unclear. The role of PPAR-γ in mature 3T3-L1 adipocytes was therefore investigated by overexpression of a dominant negative mutant of this protein (PPAR-γ-ΔC) that lacks the 16 COOH-terminal amino acids and that has been shown to prevent the thiazolidinedione-induced differentiation of 3T3-L1 cells into adipocytes. Overexpression of PPAR-γ-ΔC in mature 3T3-L1 adipocytes by adenovirus gene transfer resulted in a decrease in both cell size and intracellular triglyceride content, an increase in the extent of lipolysis, and a reduction in the rate of free fatty acid uptake. Furthermore, overexpression of this mutant reduced the abundance of mRNAs for several key enzymes that contribute to triglyceride and free fatty acid metabolism as well as the amounts of GLUT4, insulin receptor, insulin receptor substrate (IRS), and C/EBPα mRNAs. It also reduced both the concentration of IRS2 and the insulin-stimulated glucose uptake. These results suggest that PPAR-γ plays an important role in mature 3T3-L1 adipocytes at least in part by maintaining the expression of genes that confer the characteristics of mature adipocytes.
Appetite Suppression and Weight Reduction by a Centrally Active Aminosterol
Authors: Ahima Rexford S. , Patel Hiralben R. , Takahashi Nobuhiko , Qi Yong , Hileman Stanley M. , Zasloff Michael A.
Published: March 2007

The rise in obesity and its complications has generated enormous interest in the regulation of feeding and body weight. We show that a spermine metabolite of cholesterol (MSI-1436) decreases body weight, specifically fat, by suppressing feeding and preventing the reduction in energy expenditure, hormonal changes, and patterns of neuropeptide expression normally associated with weight loss. MSI-1436 enters the brain after peripheral injection and is more potent when injected into the cerebral ventricle (intracerebroventricular [ICV]). Systemic or ICV MSI-1436 administration induced similar patterns of Fos immunoreactivity in the brain, especially the paraventricular hypothalamic nucleus (PVN). This brain region integrates neural signals from hypothalamic and brain stem nuclei and regulates feeding behavior, autonomic function, and neuroendocrine function. Microinjection of MSI-1436 into the PVN potently suppressed feeding and reduced body weight for several days. Unlike caloric restriction, MSI-1436 decreased mRNA levels of agouti-related peptide and neuropeptide Y in the hypothalamus. These findings indicate that MSI-1436 acts in the brain to regulate food intake and energy expenditure, likely through suppression of orexigenic hypothalamic pathways.
Estrogen Can Prevent or Reverse Obesity and Diabetes in Mice Expressing Human Islet Amyloid Polypeptide
Authors: Geisler John G. , Zawalich Walter , Zawalich Kathleen , Lakey Jonathan R.T. , Stukenbrok Hans , Milici Anthony J. , Soeller Walter C.
Published: March 2007

Type 2 diabetes is characterized by loss of β-cell mass and concomitant deposition of amyloid derived from islet amyloid polypeptide (IAPP). Previously we have shown that expression of human IAPP (huIAPP) in islets of transgenic mice results in either a rapid onset of hyperglycemia in mice homozygous for the huIAPP transgene on a lean background (FVB/N) or a gradual hyperglycemia in mice hemizygous for the huIAPP transgene on an obese background (Avy/A). In both strains, only the males routinely develop diabetes. To investigate this sexual dimorphism, we treated young prediabetic Avy/A mice transgenic for huIAPP (huIAPP-Avy) with 17β-estradiol (E2). The treatment completely blocked the progression to hyperglycemia but also prevented the associated weight gain in these mice. Immunohistochemistry of pancreatic sections demonstrated normal islet morphology with no apparent deposition of islet amyloid. E2 treatment of 1-year-old huIAPP-Avy diabetic males rapidly reverses obesity and hyperglycemia. To determine the effects of E2 in a nonobese model, we also treated prediabetic, ad libitum–fed and pair-fed Lean-huIAPP transgenic males. E2 completely blocked the progression to hyperglycemia with no significant effect on body weight. Pancreatic insulin content and plasma insulin concentration of Lean-huIAPP transgenic mice increased in a dose-dependent manner. We demonstrated the presence of estrogen receptor (ER)-α mRNA in mouse and human islets. By also confirming the presence of ER-α protein in islets, we discovered a novel 58-kDa ER-α isoform in mice and a 52-kDa isoform in humans, in the absence of the classic 67-kDa protein found in most tissues of both species. The demonstrated presence of ER-α in mouse and human islets is consistent with a direct effect on islet function. We conclude that exogenous E2 administered to male mice may block human IAPP-mediated β-cell loss both by direct action on β-cells and by decreasing insulin demand through inhibition of weight gain or increasing insulin action.
β-Cell Function Is a Major Contributor to Oral Glucose Tolerance in High-Risk Relatives of Four Ethnic Groups in the U.S.
Authors: Jensen Christine C. , Cnop Miriam , Hull Rebecca L. , Fujimoto Wilfred Y. , Kahn Steven E. ,   the American Diabetes Association GENNID Study Group
Published: March 2007

First-degree relatives of individuals with type 2 diabetes are at increased risk of developing hyperglycemia. To examine the prevalence and pathogenesis of abnormal glucose homeostasis in these subjects, 531 first-degree relatives with no known history of diabetes (aged 44.1 ± 0.7 years; BMI 29.0 ± 0.3 kg/m2) underwent an oral glucose tolerance test (OGTT). Newly identified diabetes was found in 19% (n = 100), and impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT) was found in 36% (n = 191). Thus, only 45% (n = 240) had normal glucose tolerance (NGT). The homeostasis model assessment of insulin resistance (HOMA-IR) was used to estimate insulin sensitivity; β-cell function was quantified as the ratio of the incremental insulin to glucose responses over the first 30 min during the OGTT (ΔI30/ΔG30). This latter measure was also adjusted for insulin sensitivity as it modulates β-cell function ([ΔI30/ΔG30]/HOMA-IR). Decreasing glucose tolerance was associated with increasing insulin resistance (HOMA: NGT 12.01 ± 0.54 pmol/mmol; IFG/IGT 16.14 ± 0.84; diabetes 26.99 ± 2.62; P < 0.001) and decreasing β-cell function (ΔI30/ΔG30: NGT 157.7 ± 9.7 pmol/mmol; IFG/IGT 100.4 ± 5.4; diabetes 57.5 ± 7.3; P < 0.001). Decreasing β-cell function was also identified when adjusting this measure for insulin sensitivity ([ΔI30/ΔG30]/HOMA-IR). In all four ethnic groups (African-American, n = 55; Asian-American, n = 66; Caucasian, n = 217; Hispanic-American, n = 193), IFG/IGT and diabetic subjects exhibited progressively increasing insulin resistance and decreasing β-cell function. The relationships of insulin sensitivity and β-cell function to glucose disposal, as measured by the incremental glucose area under the curve (AUCg), were examined in the whole cohort. Insulin sensitivity and AUCg were linearly related so that insulin resistance was associated with poorer glucose disposal (r2 = 0.084, P < 0.001). In contrast, there was a strong inverse curvilinear relationship between β-cell function and AUCg such that poorer insulin release was associated with poorer glucose disposal (log[ΔI30/ΔG30]: r2 = 0.29, P < 0.001; log[(ΔI30/ΔG30)/HOMA-IR]: r2 = 0.45, P < 0.001). Thus, abnormal glucose metabolism is common in first-degree relatives of subjects with type 2 diabetes. Both insulin resistance and impaired β-cell function are associated with impaired glucose metabolism in all ethnic groups, with β-cell function seeming to be more important in determining glucose disposal.
Inhibition of Glycogen Synthase Kinase 3 Improves Insulin Action and Glucose Metabolism in Human Skeletal Muscle
Authors: Nikoulina Svetlana E. , Ciaraldi Theodore P. , Mudaliar Sunder , Carter Leslie , Johnson Kirk , Henry Robert R.
Published: March 2007

Glycogen synthase kinase (GSK)-3 has been implicated in the regulation of multiple cellular physiological processes in skeletal muscle. Selective cell-permeable reversible inhibitors (INHs) of GSK-3 (CT98014 and CHIR98023 [Chiron, Emeryville, CA] and LiCl) were used to evaluate the role of GSK-3 in controlling glucose metabolism. Acute treatment (30 min) of cultured human skeletal muscle cells with either INH resulted in a dose-dependent activation of glycogen synthase (GS) with a maximally effective concentration of ∼2 μmol/l. The maximal acute effect of either INH on GS (103 ± 25% stimulation over basal) was greater than the maximal insulin response (48 ± 9%, P < 0.05 vs. INH); LiCl was as effective as insulin. The GSK-3 inhibitor effect, like that of insulin, was on the activation state (fractional velocity [FV]) of GS. Cotreatment of muscle cells with submaximal doses of INH and insulin resulted in an additive effect on GS FV (103 ± 10% stimulation, P < 0.05 vs. either agent alone). Glucose incorporation into glycogen was also acutely stimulated by INH. While prolonged (6–24 h) insulin exposure led to desensitization of GS, INH continued to activate GS FV for at least 24 h. Insulin and LiCl acutely activated glucose uptake, whereas INH stimulation of glucose uptake required more prolonged exposure, starting at 6 h and continuing to 24 h. Chronic (4-day) treatment with INH increased both basal (154 ± 32% of control) and insulin-stimulated (219 ± 74%) glucose uptake. Upregulation of uptake activity occurred without any change in total cellular GLUT1 or GLUT4 protein content. Yet the same chronic treatment resulted in a 65 ± 6% decrease in GSK-3 protein and a parallel decrease (61 ± 11%) in GSK-3 total activity. Together with the INH-induced increase in insulin-stimulated glucose uptake, there was an ∼3.5-fold increase (P < 0.05) in insulin receptor substrate (IRS)-1 protein abundance. Despite upregulation of IRS-1, maximal insulin stimulation of Akt phosphorylation was unaltered by INH treatment. The results suggest that selective inhibition of GSK-3 has an impact on both GS and glucose uptake, including effects on insulin action, using mechanisms that differ from and are additive to those of insulin.
Long-Term AICAR Administration Reduces Metabolic Disturbances and Lowers Blood Pressure in Rats Displaying Features of the Insulin Resistance Syndrome
Authors: Buhl Esben S. , Jessen Niels , Pold Rasmus , Ledet Thomas , Flyvbjerg Allan , Pedersen Steen B. , Pedersen Oluf , Schmitz Ole , Lund Sten
Published: March 2007

The insulin resistance syndrome is characterized by several risk factors for cardiovascular disease. Chronic chemical activation of AMP-activated protein kinase by the adenosine analog 5-aminoimidazole-4-carboxamide-1-β -d-ribofuranoside (AICAR) has been shown to augment insulin action, upregulate mitochondrial enzymes in skeletal muscles, and decrease the content of intra-abdominal fat. Furthermore, acute AICAR exposure has been found to reduce sterol and fatty acid synthesis in rat hepatocytes incubated in vitro as well as suppress endogenous glucose production in rats under euglycemic clamp conditions. To investigate whether chronic AICAR administration, in addition to the beneficial effects on insulin sensitivity, is capable of improving other phenotypes associated with the insulin resistance syndrome, obese Zucker (fa/fa) rats (n = 6) exhibiting insulin resistance, hyperlipidemia, and hypertension were subcutaneously injected with AICAR (0.5 mg/g body wt) daily for 7 weeks. Obese control rats were either pair-fed (PF) (n = 6) or ad libitum-fed (AL) (n = 6). Lean Zucker rats (fa/−) (n = 8) served as a reference group. AICAR administration significantly reduced plasma triglyceride levels (P < 0.01 for AICAR vs. AL, and P = 0.05 for AICAR vs. PF) and free fatty acids (P < 0.01 for AICAR vs. AL, and P < 0.05 for AICAR vs. PF) and increased HDL cholesterol levels (P < 0.01 for AICAR vs. AL and PF). AICAR treatment also lowered systolic blood pressure by 14.6 ± 4.3 mmHg (P < 0.05), and AICAR-treated animals exhibited a tendency toward decreased intra-abdominal fat content. Furthermore, AICAR administration normalized the oral glucose tolerance test and decreased fasting concentrations of glucose and insulin close to the level of the lean animals. Finally, in line with previous findings, AICAR treatment was also found to enhance GLUT4 protein expression and to increase maximally insulin-stimulated glucose transport in primarily white fast-twitch muscles. Our data provide strong evidence that long-term administration of AICAR improves glucose tolerance, improves the lipid profile, and reduces systolic blood pressure in an insulin-resistant animal model. The present study gives additional support to the hypothesis that AMPK activation might be a potential future pharmacological strategy for treating the insulin resistance syndrome.
The Effect of a 3-Month Low-Intensity Endurance Training Program on Fat Oxidation and Acetyl-CoA Carboxylase-2 Expression
Authors: Schrauwen Patrick , van Aggel-Leijssen Dorien P.C. , Hul Gabby , Wagenmakers Anton J.M. , Vidal Hubert , Saris Wim H.M. , van Baak Marleen A.
Published: March 2007

Endurance training has been shown to increase fat oxidation both at rest and during exercise. However, most exercise training studies have been performed at high exercise intensity in well-trained athletes, and not much is known about the effect of a low-intensity training program on fat oxidation capacity in lean sedentary humans. Here, we examine the effect of 3-month low-intensity training program on total and intramuscular triglyceride (IMTG)- and/or VLDL-derived fat oxidation capacity and skeletal muscle mRNA expression. Six healthy untrained subjects (aged 43 ± 2 years, BMI 22.7 ± 1.1 kg/ m2, Vo2max 3.2 ± 0.2 l/min) participated in a supervised 12-week training program at 40% Vo2max three times weekly. Total and plasma-derived fatty acid oxidation at rest and during 1 h exercise was measured using [13C]palmitate, and in a separate test, [13C]acetate recovery was determined. Muscle biopsies were taken after an overnight fast. Total fat oxidation during exercise increased from 1,241 ± 93 to 1,591 ± 130 μmol/min (P = 0.06), and IMTG- and/or VLDL-derived fatty acid oxidation increased from 236 ± 84 to 639 ± 172 μmol/min (P = 0.09). Acetyl-CoA carboxylase-2 mRNA expression was significantly decreased after training (P = 0.005), whereas lipoprotein lipase mRNA expression tended to increase (P = 0.07). In conclusion, a minimal amount of physical activity tends to increase fat oxidation and leads to marked changes in the expression of genes encoding for key enzymes in fat metabolism.