bluuebunny/crossref_metadata_2025 huggingface dataset:
Herpes Simplex-Mediated Gene Transfer of Nerve Growth Factor Protects Against Peripheral Neuropathy in Streptozotocin-Induced Diabetes in the Mouse
Authors: Goss James R. , Goins William F. , Lacomis David , Mata Marina , Glorioso Joseph C. , Fink David J.
Published: March 2007

Peripheral neuropathy is a common and debilitating complication of diabetes. In animal models, neurotrophic factors can prevent progression of the neuropathy, but adverse effects prevent systemic administration in adequate doses to treat human disease. We examined whether gene transfer with replication-defective genomic herpes simplex virus (HSV) vectors modified to express nerve growth factor (NGF) could be used to prevent progression of neuropathy in mice. Diabetes induced by streptozotocin (STZ) resulted in a sensory neuropathy manifest by a decrease in the foot sensory nerve amplitude (FSA; control = 20 ± 0.1 μV, treated = 14 ± 0.1 μV). Transduction of dorsal root ganglia in vivo with an HSV-based vector expressing NGF under the control of the human cytomegalovirus immediate early promoter (vector SHN) or the HSV latency active promoter 2 (vector SLN) by footpad inoculation 2 weeks after STZ administration protected against the decrease in FSA (22 ± 1.4 μV and 21 ± 1.7 μV, respectively) measured 4 weeks later. Injection of SHN into inguinal adipose tissue 2 weeks after onset of diabetes also prevented the decrease in FSA (20 ± 3.3 μV). These results suggest that gene transfer with an NGF-producing herpes-based vector may prove useful in the treatment of diabetic neuropathy.
Negative Regulation of Rho Signaling by Insulin and Its Impact on Actin Cytoskeleton Organization in Vascular Smooth Muscle Cells
Authors: Begum Najma , Sandu Oana A. , Duddy Noreen
Published: March 2007

Recent studies from our laboratory have shown that insulin induces relaxation of vascular smooth muscle cells (VSMCs) via stimulation of myosin phosphatase and inhibition of Rho kinase activity. In this study, we examined the mechanism whereby insulin inhibits Rho signaling and its impact on actin cytoskeleton organization. Incubation of confluent serum-starved VSMCs with thrombin or phenylephrine (PE) caused a rapid increase in glutathione S-transferase-Rhotekin-Rho binding domain-associated RhoA, Rho kinase activation, and actin cytoskeleton organization, which was blocked by preincubation with insulin. Preexposure to NG-monomethyl l-arginine acetate (l-NMMA), a nitric oxide synthase inhibitor, and Rp-8 CPT-cyclic guanosine monophosphate (RpcGMP), a cyclic guanosine monophosphate (cGMP) antagonist, attenuated the inhibitory effect of insulin on RhoA activation and restored thrombin-induced Rho kinase activation, and site-specific phosphorylation of the myosin-bound regulatory subunit (MBSThr695) of myosin-bound phosphatase (MBP), and caused actin fiber reorganization. In contrast, 8-bromo-cGMP, a cGMP agonist, mimicked the inhibitory effects of insulin and abolished thrombin-mediated Rho activation. Insulin inactivation of RhoA was accompanied by inhibition of isoprenylation via reductions in geranylgeranyl transferase-1 activity as well as increased RhoA phosphorylation, which was reversed by pretreatment with RpcGMP and l-NMMA. We conclude that insulin may inhibit Rho signaling by affecting posttranslational modification of RhoA via nitric oxide/cGMP signaling pathway to cause MBP activation, actin cytoskeletal disorganization, and vasodilation.
Loss of Kinase Activity in a Patient With Wolcott-Rallison Syndrome Caused By a Novel Mutation in the EIF2AK3 Gene
Authors: Biason-Lauber Anna , Lang-Muritano Mariarosaria , Vaccaro Tindara , Schoenle Eugen J.
Published: March 2007

Wolcott-Rallison syndrome (WRS) is an autosomal recessive disorder characterized by neonatal or early infancy type 1 diabetes, epiphyseal dysplasia, and growth retardation. Mutations in the EIF2AK3 gene, encoding the eukaryotic initiation factor 2α-kinase 3 (EIF2AK3), have been found in WRS patients. Here we describe a girl who came to our attention at 2 months of age with severe hypertonic dehydration and diabetic ketoacidosis. A diagnosis of type 1 diabetes was made and insulin treatment initiated. Growth retardation and microcephaly were also present. Anti-islet cell autoantibodies were negative, and mitochondrial diabetes was excluded. Imaging revealed a hypoplastic pancreas and typical signs of spondylo-epiphyseal dysplasia. The diagnosis of WRS was therefore made at age 5 years. Sequencing analysis of her EIF2AK3 gene revealed the presence of a homozygous T to C exchange in exon 13 leading to the missense serine 877 proline mutation. The mutated kinase, although it partly retains the ability of autophosphorylation, is unable to phosphorylate its natural substrate, eukaryotic initiation factor 2α (eIF2α). This is the first case in which the pathophysiological role of EIF2AK3 deficiency in WRS is confirmed at the molecular level. Our data demonstrate that EIF2AK3 kinase activity is essential for pancreas islet function and bone development in humans, and we suggest EIF2AK3 as a possible target for therapeutic intervention in diabetes.
The Effects of the Pro12Ala Polymorphism of the Peroxisome Proliferator-Activated Receptor-γ2 Gene on Insulin Sensitivity and Insulin Metabolism Interact With Size at Birth
Authors: Eriksson Johan G. , Lindi Virpi , Uusitupa Matti , Forsén Tom J. , Laakso Markku , Osmond Clive , Barker David J.P.
Published: March 2007

Type 2 diabetes is known to be associated with a small body size at birth. Body size at birth is an indicator of the intrauterine environment. There is also a well-established association between the peroxisome proliferator-activated receptor (PPAR)-γ2 gene and type 2 diabetes. We therefore assessed whether the effects of the Pro12Ala polymorphism of the PPAR-γ2 gene on insulin sensitivity and insulin concentrations in adult life are modified by size at birth. We found that the effects of the Pro12Pro and Pro12Ala polymorphisms of the PPAR-γ2 gene in elderly people depended on their body size at birth. The well-known association between small body size at birth and insulin resistance was seen only in individuals with the high-risk Pro12Pro allele. In those who had low birth weight, the Pro12Pro polymorphism of the PPAR-γ2 gene was associated with increased insulin resistance (P < 0.002) and elevated insulin concentrations (P < 0.003). These interactions between the effects of the Pro12Ala polymorphisms of the PPAR-γ2 gene on adult traits and the effects of birth weight link two previously unknown associations together within the context of type 2 diabetes. We suggest that these findings reflect gene-environment interaction.
The Peroxisome Proliferator-Activated Receptor-γ2 Pro12Ala Polymorphism
Authors: Stumvoll Michael , Häring Hans
Published: March 2007

Peroxisome proliferator-activated receptor (PPAR)-γ is a transcription factor with a key role in adipocyte differentiation. The Ala allele of the common Pro12Ala polymorphism in the isoform PPAR-γ2 is associated with reduced risk for type 2 diabetes. The effect on the individual is weak, but because of a prevalence of >75% of the high-risk Pro allele, the population-attributable risk is enormous. The in vivo effects of the polymorphism are secondary to alterations in adipose tissue, where PPAR-γ2 is predominantly expressed. Moderate reduction in transcriptional activity of PPAR-γ as a result of the polymorphism modulates production and release of adipose-derived factors. Both decreased release of insulin-desensitizing free fatty acids, tumor necrosis factor-α, and resistin and increased release of the insulin-sensitizing hormone adiponectin result in secondary improvement of insulin sensitivity of glucose uptake and suppression of glucose production. The population effect of this polymorphism may be modulated by environmental or genetic factors such as obesity, ethnicity, ratio of unsaturated to saturated fatty acids, and genetic background. Once diabetes has developed, the protective effect of the Ala allele may be lost, since increased vascular complications and more pronounced β-cell dysfunction have been reported. These observations, however, are currently unexplained. In conclusion, the Pro12Ala polymorphism in PPAR-γ2 represents the first genetic variant with a broad impact on the risk of common type 2 diabetes. The precise understanding of its mechanism may lead to novel diagnostic, preventive, and therapeutic approaches for improving the management of type 2 diabetes.
Effects of Benfluorex on Fatty Acid and Glucose Metabolism in Isolated Rat Hepatocytes
Authors: Kohl Claude , Ravel Denis , Girard Jean , Pégorier Jean-Paul
Published: March 2007

The effects of benfluorex and two of its metabolites (S 422-1 and S 1475-1) on fatty acid and glucose metabolic fluxes and specific gene expression were studied in hepatocytes isolated from 24-h fasted rats. Both benfluorex and S 422-1 (0.1 or 1 mmol/l) reduced β-oxidation rates and ketogenesis, whereas S 1475-1 had no effect. At the same concentration, benfluorex and S 422-1 were more efficient in reducing gluconeogenesis from lactate/pyruvate than S 1475-1. Benfluorex inhibited gluconeogenesis at the level of pyruvate carboxylase (45% fall in acetyl-CoA concentration) and of glyceraldehyde-3-phosphate dehydrogenase (decrease in ATP/ADP and NAD+/NADH ratios). Accordingly, neither benfluorex nor S 422-1 inhibited gluconeogenesis from dihydroxyacetone, but both stimulated gluconeogenesis from glycerol. In hepatocytes cultured in the presence of benfluorex or S 422-1 (10 or 100 μmol/l), the expression of genes encoding enzymes of fatty acid oxidation (carnitine palmitoyltransferase [CPT] I), ketogenesis (hydroxymethylglutaryl-CoA synthase), and gluconeogenesis (glucose-6-phosphatase, PEPCK) was decreased, whereas mRNAs encoding glucokinase and pyruvate kinase were increased. By contrast, Glut-2, acyl-CoA synthetase, and CPT II gene expression was not affected by benfluorex or S 422-1. In conclusion, this work suggests that benfluorex mainly via S 422-1 reduces gluconeogenesis by affecting gene expression and metabolic status of hepatocytes.
Impairment of Insulin Signaling in Human Skeletal Muscle Cells by Co-Culture With Human Adipocytes
Authors: Dietze Daniela , Koenen Marlis , Röhrig Karin , Horikoshi Hiroyoshi , Hauner Hans , Eckel Jürgen
Published: March 2007

Adipocyte factors play a major role in the induction of insulin resistance in skeletal muscle. To analyze this cross-talk, we established a system of co-culture of human fat and skeletal muscle cells. Cells of three muscle donors were kept in co-culture with cells of various fat cell donors, and insulin signaling was subsequently analyzed in myocytes. Insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 was completely blocked, with unaltered expression of IRS-1. Troglitazone increased insulin action on IRS-1 phosphorylation, in both the absence and presence of co-culture. Insulin-regulated activation of Akt kinase in the myocytes was significantly reduced after co-culture, with troglitazone restoring insulin action. Addition of tumor necrosis factor (TNF)-α (2.5 nmol/l) to myocytes for 48 h reduced IRS-1 expression and inhibited IRS-1 and Akt phosphorylation comparable to the effect of co-culture. Lower doses of TNF-α were ineffective. After co-culture, TNF-α in the culture medium was below the detection limit of 0.3 pmol/l. A very low level of resistin was detected in the supernatant of myocytes, but not of adipocytes. In conclusion, the release of fat cell factors induces insulin resistance in human skeletal muscle cells; however, TNF-α and resistin appear not to be involved in this process.
Regulatory Effects of HMG CoA Reductase Inhibitor and Fish Oils on Apolipoprotein B-100 Kinetics in Insulin-Resistant Obese Male Subjects With Dyslipidemia
Authors: Chan Dick C. , Watts Gerald F. , Barrett P. Hugh R. , Beilin Lawrence J. , Redgrave Trevor G. , Mori Trevor A.
Published: March 2007

Hepatic accumulation of lipid substrates perturbs apolipoproteinB-100 (apoB) metabolism in insulin-resistant, obese subjects and may account for increased risk of cardiovascular disease. In a placebo-controlled trial, we examined the independent and combined effects of decreasing cholesterol synthesis with atorvastatin (40 mg/day) and triglyceride synthesis with fish oils (4 g/day) on apoB kinetics. The subjects were 48 viscerally obese, insulin-resistant men with dyslipidemia who were studied in a fasted state. We found that atorvastatin significantly decreased plasma apoB-containing lipoproteins (P < 0.001, main effect) through increases in the fractional catabolic rate (FCR) of VLDL-, IDL-, and LDL-apoB (P < 0.01). Fish oils significantly decreased plasma levels of triglycerides and VLDL-apoB (P < 0.001), decreased the VLDL-apoB secretion rate (P < 0.01), but increased the conversion of VLDL to LDL (P < 0.001). Compared with placebo, combined treatment with atorvastatin and fish oils decreased VLDL-apoB secretion (P < 0.03) and increased the FCR of apoB in each lipoprotein fraction (P < 0.03) and the percent conversion of VLDL to LDL (P < 0.05). None of the treatments altered insulin resistance. In conclusion, in visceral obesity, atorvastatin increased hepatic clearance of all apoB-containing lipoproteins, whereas fish oils decreased hepatic secretion of VLDL-apoB. The differential effects of atorvastatin and fish oils on apoB kinetics support their combined use in correcting defective apoB metabolism in obese, insulin-resistant subjects.
The Antidiabetic Drug Metformin Activates the AMP-Activated Protein Kinase Cascade via an Adenine Nucleotide-Independent Mechanism
Authors: Hawley Simon A. , Gadalla Anne E. , Olsen Grith Skytte , Hardie D. Grahame
Published: March 2007

Metformin, a drug widely used to treat type 2 diabetes, was recently shown to activate the AMP-activated protein kinase (AMPK) in intact cells and in vivo. In this study we addressed the mechanism for this effect. In intact cells, metformin stimulated phosphorylation of the key regulatory site (Thr-172) on the catalytic (α) subunit of AMPK. It did not affect phosphorylation of this site by either of two upstream kinases in cell-free assays, although we were able to detect an increase in upstream kinase activity in extracts of metformin-treated cells. Metformin has been reported to be an inhibitor of complex 1 of the respiratory chain, but we present evidence that activation of AMPK in two different cell types is not a consequence of depletion of cellular energy charge via this mechanism. Whereas we have not established the definitive mechanism by which metformin activates AMPK, our results show that the mechanism is different from that of the existing AMPK-activating agent, 5-aminoimidazole-4-carboxamide (AICA) riboside. Metformin therefore represents a useful new tool to study the consequences of AMPK activation in intact cells and in vivo. Our results also show that AMPK can be activated by mechanisms other than changes in the cellular AMP-to-ATP ratio.
Impaired Postprandial Adipose Tissue Blood Flow Response Is Related to Aspects of Insulin Sensitivity
Authors: Karpe Fredrik , Fielding Barbara A. , Ilic Vera , Macdonald Ian A. , Summers Lucinda K.M. , Frayn Keith N.
Published: March 2007

Obesity has been associated with dysfunctional postprandial adipose tissue blood flow (ATBF), but it has also been recognized that the interindividual response is highly variable. The present work aimed at characterizing this variability. Fifteen subjects were given 75 g oral glucose, and abdominal subcutaneous ATBF was monitored by the 133Xe washout method. Determinants of insulin sensitivity based on nonesterified fatty acid (NEFA) suppression after oral glucose administration [ISI(NEFA)] were higher in the top tertile ATBF response group (1.29 ± 0.09 vs. 0.90 ± 0.08 in the lower tertiles, P = 0.01). ISI(NEFA) was related to ATBF response (rs = 0.73, P < 0.002) as well as insulin sensitivity based on postprandial glycemia [ISI(gly), rs = 0.58, P < 0.05], whereas the homeostasis model assessment (HOMA) index (rs = −0.39, P = 0.16) was not. The relationship between increase in ATBF and ISI(NEFA) was independent of BMI (P = 0.015) in multivariate analysis. Subjects with a high ATBF response had significantly higher increase of plasma norepinephrine (P < 0.05), indicating a link between postprandial insulinemia, sympathetic activation, and ATBF response. There is a close relationship between insulin sensitivity and the regulation of postprandial ATBF, independent of adiposity. Impaired regulation of ATBF seems to be another facet of the insulin resistance syndrome.
Stimulation of Acetyl-CoA Carboxylase Gene Expression by Glucose Requires Insulin Release and Sterol Regulatory Element Binding Protein 1c in Pancreatic MIN6 β-Cells
Authors: Andreolas Chrysovalantis , da Silva Xavier Gabriela , Diraison Frederique , Zhao Chao , Varadi Aniko , Lopez-Casillas Fernando , Ferré Pascal , Foufelle Fabienne , Rutter Guy A.
Published: March 2007

Acetyl-CoA carboxylase I (ACCI) is a key lipogenic enzyme whose induction in islet β-cells may contribute to glucolipotoxicity. Here, we provide evidence that enhanced insulin release plays an important role in the activation of this gene by glucose. Glucose (30 vs. 3 mmol/l) increased ACCI mRNA levels ∼4-fold and stimulated ACCI (pII) promoter activity >30-fold in MIN6 cells. The latter effect was completely suppressed by blockade of insulin release or of insulin receptor signaling. However, added insulin substantially, but not completely, mimicked the effects of glucose, suggesting that intracellular metabolites of glucose may also contribute to transcriptional stimulation. Mutational analysis of the ACCI promoter, and antibody microinjection, revealed that the effect of glucose required sterol response element binding protein (SREBP)-1c. Moreover, adenoviral transduction with dominant-negative-acting SREBP1c blocked ACCI gene induction, whereas constitutively active SREBP1c increased ACCI mRNA levels. Finally, glucose also stimulated SREBP1c transcription, although this effect was independent of insulin release. These data suggest that glucose regulates ACCI gene expression in the β-cell by complex mechanisms that may involve the covalent modification of SREBP1c. However, overexpression of SREBP1c also decreased glucose-stimulated insulin release, implicating SREBP1c induction in β-cell lipotoxicity in some forms of type 2 diabetes.
Hepatocyte Growth Factor Prevents Endothelial Cell Death Through Inhibition of bax Translocation From Cytosol to Mitochondrial Membrane
Authors: Nakagami Hironori , Morishita Ryuichi , Yamamoto Kei , Taniyama Yoshiaki , Aoki Motokuni , Yamasaki Keita , Matsumoto Kunio , Nakamura Toshikazu , Kaneda Yasufumi , Ogihara Toshio
Published: March 2007

Injury of endothelial cells has been postulated to be an initial trigger of the progression of atherosclerosis in patients with diabetes. Previously, we demonstrated high d-glucose induced endothelial apoptosis through the bax-caspase pathway and the potential contribution of hepatocyte growth factor (HGF) to the pathogenesis of endothelial dysfunction. In this study, we analyzed the molecular mechanisms of the protective actions of HGF against endothelial cell death under high d-glucose conditions. High concentrations of d-glucose resulted in a significant increase in apoptosis and necrosis. In contrast, HGF attenuated high d-glucose-induced apoptosis and necrosis (P < 0.01). High d-glucose significantly increased bax protein, but not bcl-2, and activated caspase 3-like and 9, whereas HGF significantly increased bcl-2 expression without affecting bax level and attenuated the increase in caspase 3 and 9 activity. Interestingly, high d-glucose resulted in translocation of bax protein from cytosol to the mitochondrial membrane, whereas HGF inhibited the bax translocation. Importantly, this bax translocation was also completely blocked by overexpressed bcl-2. These findings suggest that HGF can activate bcl-2 expression and inhibit translocation of bax protein upstream of the mitochondria, thereby leading to the inhibition of caspase 3 and 9 activation. HGF may be an important factor in the maintenance of endothelial function.
Silent Coronary Atheromatosis in Type 1 Diabetic Patients and Its Relation to Long-Term Glycemic Control
Authors: Larsen Jakob , Brekke Magne , Sandvik Leiv , Arnesen Harald , Hanssen Kristian F. , Dahl-Jorgensen Knut
Published: March 2007

Type 1 diabetic patients have a pronounced risk of premature coronary artery disease and death. We sought to evaluate the prevalence of silent coronary atheromatosis and to evaluate the relation between coronary atheromatosis and glycemic control. Coronary atheromatosis was evaluated in type 1 diabetic patients with no symptoms of coronary artery disease by exercise electrocardiogram (ECG) in 39 patients and quantitative coronary angiography and by intravascular ultrasound (IVUS) examinations in 29 patients. The findings from the IVUS examinations were related to mean HbA1c collected prospectively over 18 years. Abnormal exercise ECGs were found in 15% of patients, and angiographic diameter stenosis of >50% in one or more of the main coronary arteries was found in 34% of patients. All patients examined with intracoronary ultrasound had developed atherosclerotic plaques with an increased intimal thickness (>0.5 mm) in one or more of the coronary arteries. Coronary artery plaque formation, as judged by ultrasound, was significantly related to mean HbA1c during 18 years (P < 0.05) after adjustment for total cholesterol and age. This study demonstrates a high prevalence of silent coronary atheromatosis in type 1 diabetic patients with no symptoms of coronary heart disease. Long-term glycemic control was shown to be associated with coronary atheromatosis.
Muscle Glucose Uptake Does Not Increase When Only Local Arterial Glucose Concentration Is Increased
Authors: Zierler Kenneth , Andres Reubin
Published: March 2007

Published models of mammalian whole-body glucose metabolism generally assume that glucose uptake is proportional to circulating glucose concentration at constant insulin concentration. One widely used model labels the increased whole-body glucose uptake seen with increased venous glucose concentration as “glucose effectiveness.” In 1956 and 1957, we found on average no change in forearm glucose uptake when we doubled, or more than doubled, local forearm arterial glucose concentration by close arterial infusion so that pancreatic arterial glucose concentration did not change. These experiments are being reported in extenso for the first time. Since that time, two other groups have found in glucose/insulin clamp experiments that whole-body glucose uptake was not proportional to hyperglycemic concentrations, although uptake did increase. It was hypothesized that perhaps some organs and tissues do increase uptake proportionally and other tissues not at all. Our results show that skeletal muscle is a tissue in which glucose uptake, at constant insulin, is not changed acutely by hyperglycemia; there is no forearm glucose effectiveness. We suppose that this constancy occurs because there are so few GLUTs on the sarcolemma surface in the basal state and that they are saturated even at euglycemia. We also report earlier experiments from 1954 to 1955 in which comparable hyperglycemia was reached by a large oral dose of glucose, with a 10-fold increase in glucose uptake. The arterial glucose time curve during hyperglycemia was 20–30 mg/dl higher than the forearm venous curve.
Isoform-Specific Regulation of 5′ AMP-Activated Protein Kinase in Skeletal Muscle From Obese Zucker (fa/fa) Rats in Response to Contraction
Authors: Barnes Brian R. , Ryder Jeffrey W. , Steiler Tatiana L. , Fryer Lee G.D. , Carling David , Zierath Juleen R.
Published: March 2007

Glucose transport can be activated in skeletal muscle in response to insulin via activation of phosphoinositide (PI) 3-kinase and in response to contractions or hypoxia, presumably via activation of 5′ AMP-activated protein kinase (AMPK). We determined the effects of insulin and muscle contraction/hypoxia on PI 3-kinase, AMPK, and glucose transport activity in epitrochlearis skeletal muscle from insulin-resistant Zucker (fa/ fa) rats. Insulin-stimulated glucose transport in isolated skeletal muscle was reduced 47% in obese versus lean rats, with a parallel 42% reduction in tyrosine-associated PI 3-kinase activity. Contraction and hypoxia elicited normal responses for glucose transport in skeletal muscle from insulin-resistant obese rats. Isoform-specific AMPK activity was measured in skeletal muscle in response to insulin, contraction, or hypoxia. Contraction increased AMPKα1 activity 2.3-fold in lean rats, whereas no effect was noted in obese rats. Hypoxia increased AMPKα1 activity to a similar extent (more than sixfold) in lean and obese rats. Regardless of genotype, contraction, and hypoxia, each increased AMPKα2 activity more than fivefold, whereas insulin did not alter either AMPKα1 or -α2 activity in skeletal muscle. In conclusion, obesity-related insulin resistance is associated with an isoform-specific impairment in AMPKα1 in response to contraction. However, this impairment does not appear to affect contraction-stimulated glucose transport. Activation of AMPKα2 in response to muscle contraction/ exercise is associated with a parallel and normal increase in glucose transport in insulin-resistant skeletal muscle.
Expression of Connective Tissue Growth Factor Is Increased in Injured Myocardium Associated With Protein Kinase C β2 Activation and Diabetes
Authors: Way Kerrie J. , Isshiki Keiji , Suzuma Kiyoshi , Yokota Tamotsu , Zvagelsky Dmitriy , Schoen Frederick J. , Sandusky George E. , Pechous Penelope A. , Vlahos Chris J. , Wakasaki Hisao , King George L.
Published: March 2007

Protein kinase C (PKC) β isoform activity is increased in myocardium of diabetic rodents and heart failure patients. Transgenic mice overexpressing PKCβ2 (PKCβ2Tg) in the myocardium exhibit cardiomyopathy and cardiac fibrosis. In this study, we characterized the expression of connective tissue growth factor (CTGF) and transforming growth factor β (TGFβ) with the development of fibrosis in heart from PKCβ2Tg mice at 4–16 weeks of age. Heart-to-body weight ratios of transgenic mice increased at 8 and 12 weeks, indicating hypertrophy, and ratios did not differ at 16 weeks. Collagen VI and fibronectin mRNA expression increased in PKCβ2Tg hearts at 4–12 weeks. Histological examination revealed myocyte hypertrophy and fibrosis in 4- to 16-week PKCβ2Tg hearts. CTGF expression increased in PKCβ2Tg hearts at all ages, whereas TGFβ increased only at 8 and 12 weeks. In 8-week diabetic mouse heart, CTGF and TGFβ expression increased two- and fourfold, respectively. Similarly, CTGF expression increased in rat hearts at 2–8 weeks of diabetes. This is the first report of increased CTGF expression in myocardium of diabetic rodents suggesting that cardiac injury associated with PKCβ2 activation, diabetes, or heart failure is marked by increased CTGF expression. CTGF could act independently or together with other cytokines to induce cardiac fibrosis and dysfunction.
Modulation of Glucocorticoid-Induced GAD Expression in Pancreatic β-Cells by Transcriptional Activation of the GAD67 Promoter and Its Possible Effect on the Development of Diabetes
Authors: Kim Kyung Soo , Kang Yup , Choi Sung E. , Kim Ju Hee , Kim Hyeon Man , Sun Beichen , Jun Hee-Sook , Yoon Ji-Won
Published: March 2007

GAD is a pancreatic β-cell autoantigen in humans and nonobese diabetic (NOD) mice. Modulation of GAD expression in pancreatic β-cells has been suggested to be associated with the development of autoimmune diabetes. Hormonal changes through environmental stimuli are considered to influence the expression of the disease. We determined whether steroid hormones would modulate the expression of GAD in pancreatic β-cells. We treated NOD mouse β-cells (MIN6N8a cells) with various steroids, including testosterone, estradiol, progesterone, and cortisol, and examined the expression of GAD67 mRNA. We found that only cortisol enhanced the expression of GAD67, whereas the other steroid hormones had no effect. When we treated MIN6N8a cells with a synthetic glucocorticoid, dexamethasone, we found that GAD67 mRNA expression was stimulated in a dose- and time-dependent manner. Cells treated with 100 nmol/l dexamethasone for 6 h showed a 10-fold increase in the expression of GAD67 mRNA and an increase in GAD67 protein. The upregulation of GAD67 expression in β-cells by dexamethasone was found to be due to the transcriptional activation of the GAD67 promoter. We then examined whether dexamethasone would influence the development of diabetes in NOD mice. Injection of dexamethasone into neonatal NOD mice resulted in a significant increase in the expression of GAD67 mRNA in pancreatic β-cells and the development of insulitis and diabetes. We conclude that glucocorticoid hormones can modulate GAD expression by the transcriptional activation of the GAD promoter and may influence the development of autoimmune diabetes in NOD mice.
Preservation of Pancreatic β-Cell Function and Prevention of Type 2 Diabetes by Pharmacological Treatment of Insulin Resistance in High-Risk Hispanic Women
Authors: Buchanan Thomas A. , Xiang Anny H. , Peters Ruth K. , Kjos Siri L. , Marroquin Aura , Goico Jose , Ochoa Cesar , Tan Sylvia , Berkowitz Kathleen , Hodis Howard N. , Azen Stanley P.
Published: March 2007

Type 2 diabetes frequently results from progressive failure of pancreatic β-cell function in the presence of chronic insulin resistance. We tested whether chronic amelioration of insulin resistance would preserve pancreatic β-cell function and delay or prevent the onset of type 2 diabetes in high-risk Hispanic women. Women with previous gestational diabetes were randomized to placebo (n = 133) or the insulin-sensitizing drug troglitazone (400 mg/day; n = 133) administered in double-blind fashion. Fasting plasma glucose was measured every 3 months, and oral glucose tolerance tests (OGTTs) were performed annually to detect diabetes. Intravenous glucose tolerance tests (IVGTTs) were performed at baseline and 3 months later to identify early metabolic changes associated with any protection from diabetes. Women who did not develop diabetes during the trial returned for OGTTs and IVGTTs 8 months after study medications were stopped. During a median follow-up of 30 months on blinded medication, average annual diabetes incidence rates in the 236 women who returned for at least one follow-up visit were 12.1 and 5.4% in women assigned to placebo and troglitazone, respectively (P < 0.01). Protection from diabetes in the troglitazone group 1) was closely related to the degree of reduction in endogenous insulin requirements 3 months after randomization, 2) persisted 8 months after study medications were stopped, and 3) was associated with preservation of β-cell compensation for insulin resistance. Treatment with troglitazone delayed or prevented the onset of type 2 diabetes in high-risk Hispanic women. The protective effect was associated with the preservation of pancreatic β-cell function and appeared to be mediated by a reduction in the secretory demands placed on β-cells by chronic insulin resistance.
Nitrosative Stress, Uric Acid, and Peripheral Nerve Function in Early Type 1 Diabetes
Authors: Hoeldtke Robert D. , Bryner Kimberly D. , McNeill Daniel R. , Hobbs Gerald R. , Riggs Jack E. , Warehime Sarah S. , Christie Ian , Ganser Gary , Van Dyke Knox
Published: March 2007

The present study was performed to determine whether nitric oxide overproduction is associated with deterioration in peripheral nerve function in type 1 diabetes. We measured peripheral nerve function and biochemical indicators of nitrosative stress annually for 3 years in 37 patients with type 1 diabetes. Plasma nitrite and nitrate (collectively NOx) were 34.0 ± 4.9 μmol/l in the control subjects and 52.4 ± 5.1, 50.0 ± 5.1, and 49.0 ± 5.2 in the diabetic patients at the first, second, and third evaluations, respectively (P < 0.01). Nitrotyrosine (NTY) was 13.3 ± 2.0 μmol/l in the control subjects and 26.8 ± 4.4, 26.1 ± 4.3, and 32.7 ± 4.3 in the diabetic patients (P < 0.01). Uric acid was suppressed by 20% in the diabetic patients (P < 0.001). Composite motor nerve conduction velocity for the median, ulnar, and peroneal nerves was decreased in patients with high versus low NTY (mean Z score −0.522 ± 0.25 versus 0.273 ± 0.22; P < 0.025). Patients with high NOx had decreased sweating, and those with suppressed uric acid had decreased autonomic function. In conclusion, nitrosative stress in early diabetes is associated with suppressed uric acid and deterioration in peripheral nerve function.
Type 1 And Type 2 Diabetic Patients Display Different Patterns of Cellular Microparticles
Authors: Sabatier Florence , Darmon Patrice , Hugel Benedicte , Combes Valery , Sanmarco Marielle , Velut Jean-Gabriel , Arnoux Dominique , Charpiot Phillipe , Freyssinet Jean-Marie , Oliver Charles , Sampol Jose , Dignat-George Francoise
Published: March 2007

The development of vasculopathies in diabetes involves multifactorial processes including pathological activation of vascular cells. Release of microparticles by activated cells has been reported in diseases associated with thrombotic risk, but few data are available in diabetes. The aim of the present work was to explore the number and the procoagulant activity of cell-derived microparticles in type 1 and 2 diabetic patients. Compared with age-matched control subjects, type 1 diabetic patients presented significantly higher numbers of platelet and endothelial microparticles (PMP and EMP), total annexin V-positive blood cell microparticles (TMP), and increased levels of TMP-associated procoagulant activity. In type 2 diabetic patients, only TMP levels were significantly higher without concomitant increase of their procoagulant activity. Interestingly, in type 1 diabetic patients, TMP procoagulant activity was correlated with HbA1c, suggesting that procoagulant activity is associated with glucose imbalance. These results showed that a wide vesiculation process, resulting from activation or apoptosis of several cell types, occurs in diabetes. However, diabetic patients differ by the procoagulant activity and the cellular origin of microparticles. In type 1 diabetic patients, TMP-procoagulant activity could be involved in vascular complications. Moreover, its correlation with HbA1c reinforces the importance of an optimal glycemic control in type 1 diabetes.
β-Cell Deficit and Increased β-Cell Apoptosis in Humans With Type 2 Diabetes
Authors: Butler Alexandra E. , Janson Juliette , Bonner-Weir Susan , Ritzel Robert , Rizza Robert A. , Butler Peter C.
Published: March 2007

Type 2 diabetes is characterized by impaired insulin secretion. Some but not all studies suggest that a decrease in β-cell mass contributes to this. We examined pancreatic tissue from 124 autopsies: 91 obese cases (BMI >27 kg/m2; 41 with type 2 diabetes, 15 with impaired fasting glucose [IFG], and 35 nondiabetic subjects) and 33 lean cases (BMI <25 kg/m2; 16 type 2 diabetic and 17 nondiabetic subjects). We measured relative β-cell volume, frequency of β-cell apoptosis and replication, and new islet formation from exocrine ducts (neogenesis). Relative β-cell volume was increased in obese versus lean nondiabetic cases (P = 0.05) through the mechanism of increased neogenesis (P < 0.05). Obese humans with IFG and type 2 diabetes had a 40% (P < 0.05) and 63% (P < 0.01) deficit and lean cases of type 2 diabetes had a 41% deficit (P < 0.05) in relative β-cell volume compared with nondiabetic obese and lean cases, respectively. The frequency of β-cell replication was very low in all cases and no different among groups. Neogenesis, while increased with obesity, was comparable in obese type 2 diabetic, IFG, or nondiabetic subjects and in lean type 2 diabetic or nondiabetic subjects. However, the frequency of β-cell apoptosis was increased 10-fold in lean and 3-fold in obese cases of type 2 diabetes compared with their respective nondiabetic control group (P < 0.05). We conclude that β-cell mass is decreased in type 2 diabetes and that the mechanism underlying this is increased β-cell apoptosis. Since the major defect leading to a decrease in β-cell mass in type 2 diabetes is increased apoptosis, while new islet formation and β-cell replication are normal, therapeutic approaches designed to arrest apoptosis could be a significant new development in the management of type 2 diabetes, because this approach might actually reverse the disease to a degree rather than just palliate glycemia.
Glucagon-Like Peptide 1 Induces Pancreatic β-Cell Proliferation Via Transactivation of the Epidermal Growth Factor Receptor
Authors: Buteau Jean , Foisy Sylvain , Joly Erik , Prentki Marc
Published: March 2007

We previously provided evidence that glucagon-like peptide 1 (GLP-1) induces pancreatic β-cell growth nonadditively with glucose in a phosphatidylinositol (PI) 3-kinase- and protein kinase C ζ-dependent manner. However, the exact mechanism by which the GLP-1 receptor (GLP-1R), a member of the G protein-coupled receptor (GPCR) superfamily, activates the PI 3-kinase signaling pathway to promote β-cell growth remains unknown. We hypothesized that the GLP-1R could activate PI 3-kinase and promote β-cell proliferation through transactivation of the epidermal growth factor (EGF) receptor (EGFR), an event possibly linked to GPCRs via activation of c-Src and the production of putative endogenous EGF-like ligands. Both the c-Src inhibitor PP1 and the EGFR-specific inhibitor AG1478 blocked GLP-1-induced [3H]thymidine incorporation in INS(832/13) cells as well as in isolated rat islets, while only AG1478 inhibited the proliferative action of betacellulin (BTC), an EGFR agonist. Both compounds also suppressed GLP-1-induced PI 3-kinase activation. A time-dependent increase in tyrosine phosphorylation of the EGFR in response to GLP-1 was observed in INS(832/13) cells. This transactivation of the EGFR was sensitive to both the pharmacological agents PP1 and AG1478. The action of GLP-1 and BTC on INS cell proliferation was found to be not additive. Overexpression of a dominant-negative EGFR in INS cells with a retroviral expression vector curtailed GLP-1-induced β-cell proliferation. GLP-1 treatment of INS cells caused a decrease in cell surface-associated BTC, as shown by FACS analysis. Also, the metalloproteinase inhibitor GM6001 and an anti-BTC neutralizing antibody suppressed the GLP-1 proliferative effect. Finally, coculturing the prostatic cancer cell line LNCaP that lacks GLP-1 responsiveness with INS cells increased LNCaP cell proliferation in the presence of GLP-1, thus revealing that INS cells secrete a growth factor in response to GLP-1. GM6001 and an anti-BTC neutralizing antibody suppressed increased LNCaP cell proliferation in the presence of GLP-1 in the coculture experiments. The results are consistent with a model in which GLP-1 increases PI 3-kinase activity and enhances β-cell proliferation via transactivation of the EGFR that would require the proteolytic processing of membrane-anchored BTC or other EGF-like ligands.
Effects of Acute Insulin Excess and Deficiency on Gluconeogenesis and Glycogenolysis in Type 1 Diabetes
Authors: Boden Guenther , Cheung Peter , Homko Carol
Published: March 2007

To determine whether insulin induces acute changes in endogenous glucose production (EGP) via changes in gluconeogenesis (GNG), glycogenolysis (GL), or both, we measured GNG (with 2H2O) and GL (EGP-GNG) in nine patients with type 1 diabetes during acute insulin excess produced by subcutaneous injection of insulin and during insulin deficiency which developed between 5 and 8 h after insulin injection. During insulin excess, free insulin concentration rose fivefold (from 36 to 180 pmol/l). Plasma glucose was maintained between 6.2 and 6.7 mmol/l for ∼4 h with IV glucose. EGP (with 6,6-2H glucose) decreased from 17.1 to 9.8 μmol · kg−1 · min−1 after 1 h. This decrease was almost completely accounted for by a decrease in GL (from 10.7 to 4.6 μmol · kg−1 · min−1). During insulin deficiency, plasma glucose rose from 6.2 to 10.5 mmol/l and EGP from 9.5 to 14.3 μmol/kg min. The increase in EGP again was accounted for by an increase in GL.

We conclude that in type 1 diabetes acute regulation of EGP by insulin is mainly via changes in GL while GNG changes little during the early hours of acute insulin excess or deficiency.
Apolipoprotein-E Influences Aspects of Intellectual Ability in Type 1 Diabetes
Authors: Ferguson Stewart C. , Deary Ian J. , Evans Julie C. , Ellard Sian , Hattersley Andrew T. , Frier Brian M.
Published: March 2007

The ε4 allele of the apolipoprotein-E (APOE) gene is associated with poor outcome following various cerebral insults. The relationship between APOE genotype and cognitive function in patients with type 1 diabetes is unknown. In a cross-sectional study of 96 people with type 1 diabetes, subjects were APOE genotyped, previous exposure to severe hypoglycemia was estimated by questionnaire, and cognition was assessed by neuropsychological testing. Cognitive abilities were compared using multivariate general linear modeling (multiple analysis of covariance, MANCOVA) in those with (n = 21) and without (n = 75) the APOE ε4 allele. APOE ε4 selectively influenced cognitive ability in a sex-specific manner (F = 2.3, P = 0.044, Eta2 = 0.15); women with APOE ε4 performed less well on tests of current, nonverbal intellectual ability (Wechsler Adult Intelligence Scale-Revised performance test score, P = 0.001, Eta2 0.26) and frontal lobe and executive function (Borkowski verbal fluency, P = 0.016, Eta2 = 0.15). Previous exposure to severe hypoglycemia did not interact with APOE ε4 to produce cognitive disadvantage. The APOE ε4 genotype is associated with specific cognitive disadvantage in young women with type 1 diabetes. APOE ε4 is unlikely to mediate susceptibility to hypoglycemia-induced cognitive disadvantage.
Elevated Free Fatty Acids Impair Glucose Metabolism in Women
Authors: Shah Pankaj , Vella Adrian , Basu Ananda , Basu Rita , Adkins Aron , Schwenk W. Frederick , Johnson C. Michael , Nair K. Sreekumaran , Jensen Michael D. , Rizza Robert A.
Published: March 2007

The present study sought to determine whether elevated plasma free fatty acids (FFAs) alter the splanchnic and muscle glucose metabolism in women. To do so, FFAs were increased in seven women by an 8-h Intralipid/heparin (IL/hep) infusion, and the results were compared with those observed in nine women who were infused with glycerol alone. Glucose was clamped at ∼8.3 mmol/l and insulin was increased to ∼300 pmol/l to stimulate both muscle and hepatic glucose uptake. Insulin secretion was inhibited with somatostatin. Leg and splanchnic glucose metabolism were assessed using a combined catheter and tracer dilution approach. The glucose infusion rates required to maintain target plasma glucose concentrations were lower (P < 0.01) during IL/hep than glycerol infusion (30.8 ± 2.6 vs. 65.0 ± 7.9 μmol · kg−1 · min−1). Whole-body glucose disappearance (37.0 ± 2.2 vs. 70.9 ± 8.7 μmol · kg−1 · min−1; P < 0.001) and leg glucose uptake (24.3 ± 4.2 vs. 59.6 ± 10.0 μmol · kg fat-free mass of the leg−1 · min−1; P < 0.02) were also lower, whereas splanchnic glucose production (8.2 ± 0.8 vs. 4.3 ± 0.7 μmol · kg−1 · min−1; P < 0.01) was higher during IL/hep than glycerol infusion. We conclude that in the presence of combined hyperinsulinemia and hyperglycemia, elevated FFAs impair glucose metabolism in women by inhibiting whole- body glucose disposal, muscle glucose uptake, and suppression of splanchnic glucose production.
Differential Regulation of Lipogenesis and Leptin Production by Independent Signaling Pathways and Rosiglitazone During Human Adipocyte Differentiation
Authors: Patel Nayan G. , Holder Julie C. , Smith Stephen A. , Kumar Sudesh , Eggo Margaret C.
Published: March 2007

Since leptin levels are independently correlated with risk of coronary heart disease, we have identified signaling pathways important in mediating leptin production and lipogenesis in human preadipocytes. We used inhibitors of p70S6 kinase, p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and phosphatidylinositol 3-kinase (PI3K). Human preadipocytes were induced to differentiate in insulin, dexamethasone, triiodothyronine, and 3-isobutyl-1-methylxanthine in the presence or absence of inhibitors and the peroxisome proliferator-activated receptor (PPAR)-γ activator rosiglitazone. Differentiation was assessed by measuring leptin secretion, lipid content, and lipogenic activity. Rosiglitazone increased cell protein by 15%, the lipid content of the cell layer was doubled, and the lipogenic activity increased sevenfold but did not stimulate leptin secretion. None of the inhibitors significantly inhibited protein content over 20 days, but lipid content and lipogenic activity were inhibited by p70S6 kinase and p38 MAPK inhibition but not by p42/44 MAPK or PI3K inhibition. All of the inhibitors significantly decreased leptin secretion, and these inhibitory effects were increased by coincubation with rosiglitazone. We conclude that PI3K and p42/44 MAPK pathways are not critical to the differentiation program leading to lipid accumulation, but stimulation of leptin secretion is dependent on these as well as the p70S6 kinase and p38 MAPK signaling pathways.
Purified Allogeneic Hematopoietic Stem Cell Transplantation Blocks Diabetes Pathogenesis in NOD Mice
Authors: Beilhack Georg F. , Scheffold Yolanda C. , Weissman Irving L. , Taylor Cariel , Jerabek Libuse , Burge Matthew J. , Masek Marilyn A. , Shizuru Judith A.
Published: March 2007

Purified hematopoietic stem cells (HSCs) were transplanted into NOD mice to test whether development of hyperglycemia could be prevented. Engraftment of major histocompatibility complex-mismatched HSCs was compared with bone marrow (BM) grafts. HSCs differed from BM because HSCs were more strongly resisted and HSC recipients retained significant levels of NOD T-cells, whereas BM recipients were full donor chimeras. Despite persistent NOD T-cells, all HSC chimeras were protected from hyperglycemia, and attenuation of islet lesions was observed. T-cell selection was altered in allogeneic HSC recipients as demonstrated by deletion of both donor and host superantigen-specific T-cells. Syngeneic and congenic hematopoietic cell transplants were also performed to differentiate the influence of the preparative regimen(s) versus the allografts. Unlike the allogeneic HSC transplantations, syngeneic or congenic grafts did not retard diabetes development. In a pilot study, overtly diabetic NOD mice were cured by co-transplantation of allogeneic HSCs and donor-matched islets. We conclude that allogeneic HSC transplants block allo- and autoimmunity, despite residual host T-cell presence. These data demonstrate for the first time that purified HSC grafts block development of autoimmune diabetes and illuminate how HSC grafts alter thymic and peripheral T-cell responses against auto- and alloantigens.