bluuebunny/crossref_metadata_2025 huggingface dataset:
Free Radicals and the Pathogenesis of Type 1 Diabetes
Authors: Tabatabaie Tahereh , Vasquez-Weldon Angelica , Moore Danny R. , Kotake Yashige
Published: March 2007

Free radical formation evoked by proinflammatory cytokines has been suggested to be involved in the destruction of β-cells in the course of type 1 diabetes development. However, there is no direct evidence to support this hypothesis. In this study, we used electron paramagnetic resonance spectroscopy in conjunction with spin-trapping methodology to directly determine whether cytokines give rise to free radical formation in the islets. Our results demonstrate that direct, in vivo administration of tumor necrosis factor-α (1,000 units), interleukin-1β (1,000 units), and interferon-γ (2,000 units) into the rat pancreas through a bile duct cannula leads to the formation of lipid-derived free radicals in this tissue. These free radicals most likely are generated by the β-cells because previous depletion of these cells by streptozotocin abolished the cytokine-induced free radical formation. Furthermore, macrophage depletion was found to decrease the production of free radicals. Inhibition of the enzyme inducible cyclooxygenase (COX-2) and the transcription factor nuclear factor-κB (NF-κB) significantly diminished the free radicals’ signal intensity, implicating these factors in the formation of free radicals. We have also demonstrated that cytokine treatment leads to the activation of NF-κB in the pancreatic islets of the rats.
KATP Channels and Pancreatic Islet Blood Flow in Anesthetized Rats
Authors: Jansson Leif , Kullin Mikael , Karlsson F. Anders , Bodin Birgitta , Hansen John Bondo , Sandler Stellan
Published: March 2007

KATP channels are important for insulin secretion and depolarization of vascular smooth muscle. In view of the importance of drugs affecting KATP channels in the treatment of diabetes, we investigated the effects of these channels on splanchnic blood perfusion in general and pancreatic islet blood flow in particular. We treated anesthetized Sprague-Dawley rats with the KATP channel openers diazoxide or NNC 55-0118 or the KATP channel closer glipizide. Both diazoxide and NNC 55-0118 dose-dependently increased total pancreatic and islet blood flow in the presence of moderate hyperglycemia, but had no effects on the blood perfusion of other splanchnic organs. Diazoxide markedly lowered the mean arterial blood pressure and thus increased vascular conductance in all organs studied. NNC 55-0118 had much smaller effects on the blood pressure. Glipizide did not affect total pancreatic blood flow, but decreased islet blood flow by 50% in the presence of hypoglycemia. We conclude that KATP channels actively participate in the blood flow regulation of the pancreatic islets and that substances affecting such channels may also influence islet blood flow.
Cortisol Elevations Comparable to Those That Occur During Hypoglycemia Do Not Cause Hypoglycemia-Associated Autonomic Failure
Authors: Raju Bharathi , McGregor Veronica P. , Cryer Philip E.
Published: March 2007

The concept of hypoglycemia-associated autonomic failure (HAAF) in diabetes posits that recent antecedent iatrogenic hypoglycemia causes both defective glucose counterregulation (by reducing the epinephrine response in the setting of an absent glucagon response) and hypoglycemia unawareness (by reducing the autonomic-sympathetic neural and adrenomedullary response and the resulting neurogenic [autonomic] symptom responses) and thus causes a vicious cycle of recurrent hypoglycemia. To assess the suggestion that it is the cortisol response to antecedent hypoglycemia that mediates HAAF, we tested the hypothesis that plasma cortisol elevations during euglycemia that are comparable to those that occur during hypoglycemia reduce sympathoadrenal and neurogenic symptom responses to subsequent hypoglycemia. To do this, 12 healthy subjects were studied with hyperinsulinemic-stepped hypoglycemic clamps the day after saline or cortisol (1.3 ± 0.2 μg · kg−1 · min−1) infusions from 0930 to 1200 and from 1330 to 1600. Compared with saline, antecedent cortisol elevations did not reduce the sympathoadrenal (e.g., final plasma epinephrine levels of 674 ± 84 vs. 606 ± 80 pg/ml and final plasma norepinephrine levels of 332 ± 26 vs. 304 ± 26 pg/ml) or neurogenic symptom (e.g., final scores of 9.3 ± 1.1 vs. 13.2 ± 1.3) responses to subsequent hypoglycemia. Thus, these data do not support the suggestion that cortisol mediates HAAF.
Expression of Constitutively Active cGMP-Dependent Protein Kinase Prevents Glucose Stimulation of Thrombospondin 1 Expression and TGF-β Activity
Authors: Wang Shuxia , Wu Xing , Lincoln Thomas M. , Murphy-Ullrich Joanne E.
Published: March 2007

Hyperglycemia is a crucial factor in the development of diabetic nephropathy. We previously showed that high glucose upregulates thrombospondin 1 (TSP1)-dependent transforming growth factor (TGF)-β activation by altering cGMP-dependent protein kinase (PKG) activity as a result of decreased nitric oxide signaling. In the present study, we showed that high glucose concentrations significantly reduced endogenous PKG activity. To further examine the mechanisms by which PKG regulates TSP1 expression and TSP1-dependent TGF-β activation, we generated stably transfected rat mesangial cells (RMCs) with inducible expression tetracycline-induced gene expression of the catalytic domain of PKG. After tetracycline induction, the catalytic domain of PKG is expressed as a cGMP-independent active kinase. Expression of the catalytic domain prevented high glucose-mediated increases in transcription of the TSP1 gene with no alteration in TSP1 mRNA stability. Glucose stimulation of TSP1 protein expression and TGF-β bioactivity were also downregulated. TGF-β-dependent fibronectin and type IV collagen expression under high glucose conditions were significantly reduced upon catalytic domain expression in transfected RMCs. These results show that constitutively active PKG inhibits the fibrogenic potential of high glucose through repression of TSP1-dependent TGF-β bioactivity, suggesting that gene transfer of the catalytic domain of PKG might provide a new strategy for treatment of diabetic renal fibrosis.
Genetic Modifiers of the Age at Diagnosis of Diabetes (MODY3) in Carriers of Hepatocyte Nuclear Factor-1α Mutations Map to Chromosomes 5p15, 9q22, and 14q24
Authors: Kim Sung-Hoon , Ma Xiaowei , Klupa Tomasz , Powers Christine , Pezzolesi Marcus , Warram James H. , Rich Stephen S. , Krolewski Andrzej S. , Doria Alessandro
Published: March 2007

Mutations in hepatocyte nuclear factor (HNF)-1α (MODY3) account for the largest proportion of maturity-onset diabetes of the young (MODY) cases in the U.S. This form of diabetes is characterized by impaired insulin secretion in response to glucose, but wide variability exists in the severity of hyperglycemia and in the age at which it becomes clinically manifest. We have previously shown that the age at onset of diabetes in MODY3 families is influenced by familial factors (including modifying genes) and exposure to diabetes in utero. To identify genes influencing the onset of MODY3, we conducted a genome scan in 13 extended MODY families in which diabetes segregates with an HNF-1α mutation. Linkage with age at onset of diabetes was assessed by genetic variance component analysis using SOLAR. The locus with the strongest evidence of linkage was on chromosome 14q24 (D14S588; logarithm of odds [LOD] = 2.58, P = 0.0004). This location overlaps with IDDM11 and includes SEL1L, a negative regulator of the Notch pathway that may control islet development. Linkage evidence also supported loci on 5p15 (D5S817; LOD = 2.44, P = 0.0004) and 9q22 (D9S910; LOD = 2.02, P = 0.0018). The latter matches a region linked to 2-h insulin levels in Pima Indians. Less strong linkage evidence was observed at three other regions: chromosomes 3p24 (LOD = 1.44), 7q21 (1.20), and 16q23 (1.51). Our data are consistent with the existence of multiple loci that contribute to the expression of the MODY3 phenotype. Identification of these genes will offer new insights into the pathophysiology of MODY that may, in turn, increase our understanding of the cellular events underlying more common forms of diabetes.
Regulation and Function of the Muscle Glycogen-Targeting Subunit of Protein Phosphatase 1 (GM) in Human Muscle Cells Depends on the COOH-Terminal Region and Glycogen Content
Authors: Lerín Carlos , Montell Eulàlia , Nolasco Teresa , Clark Cathy , Brady Matthew J. , Newgard Christopher B. , Gómez-Foix Anna M.
Published: March 2007

GM, the muscle-specific glycogen-targeting subunit of protein phosphatase 1 (PP1) targeted to the sarcoplasmic reticulum, was proposed to regulate recovery of glycogen in exercised muscle, whereas mutation truncation of its COOH-terminal domain is known to be associated with type 2 diabetes. Here, we demonstrate differential effects of GM overexpression in human muscle cells according to glycogen concentration. Adenovirus-mediated delivery of GM slightly activated glycogen synthase (GS) and inactivated glycogen phosphorylase (GP) in glycogen-replete cells, causing an overaccumulation of glycogen and impairment of glycogenolysis after glucose deprivation. Differently, in glycogen-depleted cells, GM strongly increased GS activation with no further enhancement of early glycogen resynthesis and without affecting GP. Effects of GM on GS and GP were abrogated by treatment with dibutyryl cyclic AMP. Expression of a COOH-terminal deleted-mutant (GMΔC), lacking the membrane binding sequence to sarcoplasmic reticulum, failed to activate GS in glycogen-depleted cells, while behaving similar to native GM in glycogen-replete cells. This is explained by loss of stability of the GMΔC protein following glycogen-depletion. In summary, GM promotes glycogen storage and inversely regulates GS and GP activities, while, specifically, synthase phosphatase activity of GM-PP1 is inhibited by glycogen. The conditional loss of function of the COOH-terminal deleted GM construct may help to explain the reported association of truncation mutation of GM with insulin resistance in human subjects.
Molecular Mechanisms of Insulin Resistance in IRS-2-Deficient Hepatocytes
Authors: Valverde Angela M. , Burks Deborah J. , Fabregat Isabel , Fisher Tracey L. , Carretero José , White Morris F. , Benito Manuel
Published: March 2007

To assess the role of insulin receptor (IR) substrate (IRS)-2 in insulin action and resistance in the liver, immortalized neonatal hepatocyte cell lines have been generated from IRS-2−/−, IRS-2+/−, and wild-type mice. These cells maintained the expression of the differentiated liver markers albumin and carbamoyl phosphate synthetase, as well as bear a high number of IRs. The lack of IRS-2 did not result in enhanced IRS-1 tyrosine phosphorylation or IRS-1-associated phosphatidylinositol (PI) 3-kinase activity on insulin stimulation. Total insulin-induced PI 3-kinase activity was decreased by 50% in IRS-2−/− hepatocytes, but the translocation of PI-3,4,5-trisphosphate to the plasma membrane in these cells was almost completely abolished. Downstream PI 3-kinase, activation of Akt, glycogen synthase kinase (GSK)-3 (α and β isoforms), Foxo1, and atypical protein kinase C were blunted in insulin-stimulated IRS-2−/− cells. Reconstitution of IRS-2−/− hepatocytes with adenoviral IRS-2 restored activation of these pathways, demonstrating that IRS-2 is essential for functional insulin signaling in hepatocytes. Insulin induced a marked glycogen synthase activity in wild-type and heterozygous primary hepatocytes; interestingly, this response was absent in IRS-2−/− cells but was rescued by infection with adenoviral IRS-2. Regarding gluconeogenesis, the induction of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase by dibutyryl cAMP and dexamethasone was observed in primary hepatocytes of all genotypes. However, insulin was not able to suppress gluconeogenic gene expression in primary hepatocytes lacking IRS-2, but when IRS-2 signaling was reconstituted, these cells recovered this response to insulin. Suppression of gluconeogenic gene expression in IRS-2-deficient primary hepatocytes was also restored by infection with dominant negative Δ256Foxo1.
Critical Roles of Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand in Type 1 Diabetes
Authors: Lamhamedi-Cherradi Salah-Eddine , Zheng Shijun , Tisch Roland M. , Chen Youhai H.
Published: March 2007

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis of tumor cells but not most normal cells. Its roles in normal nontransformed tissues are not clear. To explore the potential roles of TRAIL in type 1 diabetes, we examined the consequences of TRAIL blockade or TRAIL deficiency in two animal models of autoimmune diabetes. In the first model, NOD mice received an injection of a soluble TRAIL receptor to block TRAIL function. This significantly accelerated the diabetes and increased the degree of autoimmune inflammation in both pancreatic islets and salivary glands. The GAD65-specific immune responses were also significantly enhanced in animals that received the soluble TRAIL receptor. In the second model, we treated normal and TRAIL-deficient C57BL/6 mice with multiple low-dose streptozotocin to induce diabetes. We found that both the incidence and the degree of islet inflammation were significantly enhanced in TRAIL-deficient animals. On the basis of these observations, we conclude that TRAIL deficiency accelerates autoimmune diabetes and enhances autoimmune responses.
The Metabolic Syndrome and Antioxidant Concentrations
Authors: Ford Earl S. , Mokdad Ali H. , Giles Wayne H. , Brown David W.
Published: March 2007

Oxidative stress may play a role in the pathophysiology of diabetes and cardiovascular disease, but little is known about antioxidant status among individuals with the metabolic syndrome who are at high risk for developing these conditions. Using data from the Third National Health and Nutrition Examination Survey (1988–1994), we compared circulating concentrations of vitamins A, C, and E; retinyl esters; five carotenoids; and selenium in 8,808 U.S. adults aged ≥20 years with and without the metabolic syndrome. After adjusting for age, sex, race or ethnicity, education, smoking status, cotinine concentration, physical activity, fruit and vegetable intake, and vitamin or mineral use, participants with the metabolic syndrome had significantly lower concentrations of retinyl esters, vitamin C, and carotenoids, except lycopene. With additional adjustment for serum lipid concentrations, vitamin E concentrations were significantly lower in participants with the metabolic syndrome than those without the syndrome. Retinol concentrations were similar between the two groups. After excluding participants with diabetes, the results were very similar. Consumption of fruits and vegetables was also lower among people with the metabolic syndrome. Adults with the metabolic syndrome have suboptimal concentrations of several antioxidants, which may partially explain their increased risk for diabetes and cardiovascular disease.
Effects of Rosuvastatin on Nitric Oxide–Dependent Function in Aorta and Corpus Cavernosum of Diabetic Mice
Authors: Nangle Matthew R. , Cotter Mary A. , Cameron Norman E.
Published: March 2007

Elevated plasma lipids contribute to neurovascular dysfunction in diabetes. Statins have lipid-lowering properties and can modulate endothelial nitric oxide (NO) bioavailability. The aim was to assess the impact of these factors on autonomic nitrergic nerve and endothelial function. Thus, the effects of diabetes and treatment with the HMG-CoA reductase inhibitor rosuvastatin (RSV) were examined on corpus cavernosum and aorta from streptozotocin-induced diabetic mice in a 4-week prevention study and a 2-week intervention study, following 4 weeks of untreated diabetes. Cotreatment with mevalonate was used to assess the dependence of RSV’s effects on HMG-CoA reductase blockade. Diabetes caused a 25% reduction in NO-mediated endothelium-dependent relaxation to acetylcholine for aorta and cavernosum. Relaxations of cavernosum were in the nondiabetic range following prevention or reversal treatment. The aortic deficit was completely prevented and 60% reversed by RSV. Maximum NO-dependent nonadrenergic, noncholinergic nerve-mediated relaxations of cavernosum were reduced 25–33% by diabetes. RSV treatment prevented 75% and reversed 71% of this diabetic deficit. Cotreatment with mevalonate inhibited the beneficial actions of RSV on aorta and cavernosum. Total plasma cholesterol was unaltered by diabetes or treatment. Thus, RSV corrected defective NO-mediated nerve and vascular function in diabetic mice independent of cholesterol lowering but via effects dependent on cholesterol biosynthesis pathway inhibition.
Clinical and Molecular Characterization of a Dominant Form of Congenital Hyperinsulinism Caused by a Mutation in the High-Affinity Sulfonylurea Receptor
Authors: Thornton Paul S. , MacMullen Courtney , Ganguly Arupa , Ruchelli Eduardo , Steinkrauss Linda , Crane Ana , Aguilar-Bryan Lydia , Stanley Charles A.
Published: March 2007

Recessive mutations of sulfonylurea receptor 1 (SUR1) and potassium inward rectifier 6.2 (Kir6.2), the two adjacent genes on chromosome 11p that comprise the β-cell plasma membrane ATP-sensitive K+ (KATP) channels, are responsible for the most common form of congenital hyperinsulinism in children. The present study was undertaken to identify the genetic defect in a family with dominantly inherited hyperinsulinism affecting five individuals in three generations. Clinical tests were carried out in three of the patients using acute insulin responses (AIRs) to intravenous stimuli to localize the site of defect in insulin regulation. The affected individuals showed abnormal positive calcium AIR, normal negative leucine AIR, subnormal positive glucose AIR, and impaired tolbutamide AIR. This AIR pattern suggested a KATP channel defect because it resembled that seen in children with recessive hyperinsulinism due to two common SUR1 mutations, g3992-9a and delPhe1388. Genetic linkage to the KATP locus was established using intragenic polymorphisms. Mutation analysis identified a novel trinucleotide deletion in SUR1 exon 34 that results in the loss of serine 1387. Studies of delSer1387 in COSm6 cells confirmed that the expressed mutant protein assembles with Kir6.2 and trafficks to the plasma membrane, but it had no 86Rb efflux ion transport activity. These results indicate that hyperinsulinism in this family is caused by a SUR1 mutation that is expressed dominantly rather than recessively.
Genome-Wide Linkage Analysis of Serum Adiponectin in the Pima Indian Population
Authors: Lindsay Robert S. , Funahashi Tohru , Krakoff Jonathan , Matsuzawa Yuji , Tanaka Sachiyo , Kobes Sayuko , Bennett Peter H. , Tataranni P. Antonio , Knowler William C. , Hanson Robert L.
Published: March 2007

Adiponectin is a circulating protein secreted by adipocytes and is thought to have insulin-sensitizing effects. We present genetic analysis of adiponectin levels in 517 Pima Indians without diabetes (from 162 families, 750 sib-pairs). Adiponectin concentrations were heritable, with 39% of the variance of age- and sex-adjusted adiponectin potentially accounted for by additive genetic influences in this population. In genome-wide linkage analyses, suggestive linkage (logarithm of odds [LOD] = 3.0) of adiponectin adjusted for age and sex was found on chromosome 9p at 18 cM. Linkage was also present after inclusion of adiponectin concentrations of siblings with type 2 diabetes not treated pharmacologically (total siblings 582, 182 families, 860 sib-pairs: LOD = 3.5). Tentative evidence of linkage was also found on chromosomes 2 (LOD = 1.7 at 89 cM), 3 (LOD = 1.9 at 124 cM), and 10 (LOD = 1.7 at 70 cM), offering some support to findings of a previous genome-wide scan of adiponectin. Our data suggest that quantitative trait loci on chromosomes 2, 3, 9, and 10 may influence circulating adiponectin concentrations in the Pima population.
Induction of β-Cell Proliferation and Retinoblastoma Protein Phosphorylation in Rat and Human Islets Using Adenovirus-Mediated Transfer of Cyclin-Dependent Kinase-4 and Cyclin D1
Authors: Cozar-Castellano Irene , Takane Karen K. , Bottino Rita , Balamurugan A.N. , Stewart Andrew F.
Published: March 2007

The major regulator of the gap-1/synthesis phase (G1/S) cell cycle checkpoint is the retinoblastoma protein (pRb), and this is regulated in part by the activities of cyclin-dependent kinase (cdk)-4 and the D cyclins. Surprisingly, given the potential importance of β-cell replication for islet replacement therapy, pRb presence, phosphorylation status, and function have not been explored in β-cells. Here, adenoviruses expressing cdk-4 and cyclin D1 were used to explore rat and human pRb phosphorylation and β-cell cycle control. pRb is present in rat and human islets, and overexpression of cyclin D1/cdk-4 led to strikingly enhanced pRb phosphorylation in both species. Combined overexpression of both cdk-4 and cyclin D1 caused a threefold increase in [3H]thymidine incorporation. This increase in proliferation was confirmed independently using insulin and bromodeoxyuridine immunohistochemistry, where human β-cell replication rates were increased 10-fold. Cdk-4 or cyclin D1 overexpression did not adversely effect β-cell differentiation or function. The key cell cycle regulatory protein, pRb, can be harnessed to advantage using cyclin D1/cdk-4 for the induction of human and rodent β-cell replication, enhancing replication without adversely affecting function or differentiation. This approach will allow detailed molecular study of the cellular mechanisms regulating the cell cycle in β-cells, β-cell lines, and stem cell-derived β-cells.
Mode of Onset of Type 2 Diabetes from Normal or Impaired Glucose Tolerance
Authors: Ferrannini Ele , Nannipieri Monica , Williams Ken , Gonzales Clicerio , Haffner Steve M. , Stern Michael P.
Published: March 2007

Fasting plasma glucose concentrations (FPG) predict development of type 2 diabetes. Whether hyperglycemia evolves from normoglycemia gradually over time or as a step increase is not known. We measured plasma glucose and insulin levels during oral glucose testing in 35- to 64-year-old men and nonpregnant women from a population-based survey (Mexico City Diabetes Study) at baseline (n = 2,279) and after 3.25 (n = 1,740) and 7 years (n = 1,711) of follow-up. In subjects with normal glucose tolerance (NGT) on all three occasions (nonconverters; n = 911), FPG increased only slightly (0.23 ± 0.79 mmol/l, mean ± SD; P < 0.0001) over 7 years. In contrast, conversion to diabetes among NGT subjects (n = 98) was marked by a large step-up in FPG regardless of time of conversion (3.06 ± 2.57 and 2.94 ± 3.11 mmol/l, respectively, at 3.25 and 7 years; P < 0.0001 vs. nonconverters). Likewise, in subjects who converted to diabetes from impaired glucose tolerance (n = 75), FPG rose by 3.14 ± 3.83 and 3.12 ± 3.61 mmol/l (P < 0.0001 vs. nonconverters). Three-quarters of converters had increments in FPG above the 90th percentile of the corresponding increments in nonconverters. Converters had higher baseline BMI (30.4 ± 4.9 vs. 27.3 ± 4.0 kg/m2; P < 0.001) and fasting plasma insulin values (120 ± 78 vs. 84 ± 84 pmol/l; P < 0.02) than nonconverters; however, no consistent change in either parameter had occurred before conversion. In contrast, changes in 2-h postglucose insulin levels between time of conversion and preceding measurement were significantly (P < 0.0001) related to the corresponding changes in FPG in an inverse manner. We conclude that, within a 3-year time frame, the onset of diabetes is very often rapid rather than gradual and is in part explained by a fall in glucose-stimulated insulin response.
Endothelial Progenitor Cell Dysfunction
Authors: Loomans Cindy J.M. , de Koning Eelco J.P. , Staal Frank J.T. , Rookmaaker Maarten B. , Verseyden Caroline , de Boer Hetty C. , Verhaar Marianne C. , Braam Branko , Rabelink Ton J. , van Zonneveld Anton-Jan
Published: March 2007

Type 1 diabetes is associated with reduced vascular repair, as indicated by impaired wound healing and reduced collateral formation in ischemia. Recently, endothelial progenitor cells (EPCs) have been identified as important regulators of these processes. We therefore explored the concept that EPCs are dysfunctional in diabetes. The number of EPCs obtained from type 1 diabetic patients in culture was 44% lower compared with age- and sex-matched control subjects (P < 0.001). This reduction was inversely related to levels of HbA1c (R = −0.68, P = 0.01). In addition, we demonstrated that patient EPCs were also impaired in function using an in vitro angiogenesis assay. Conditioned media from patient EPCs were significantly reduced in their capacity to support endothelial tube formation in comparison to control EPCs. Therefore, despite culturing the EPCs under normoglycemic conditions, functional differences between patient and control EPCs were maintained. Our findings demonstrate that adverse metabolic stress factors in type 1 diabetes are associated with reduced EPC numbers and angiogenicity. We hypothesize that EPC dysfunction contributes to the pathogenesis of vascular complications in type 1 diabetes.
Exercise-Induced Protein Kinase C Isoform-Specific Activation in Human Skeletal Muscle
Authors: Perrini Sebastio , Henriksson Jan , Zierath Juleen R. , Widegren Ulrika
Published: March 2007

We determined whether protein kinase C (PKC) isoforms are redistributed and phosphorylated in response to acute exercise in skeletal muscle. Muscle biopsies were obtained from six healthy subjects (four women, two men; age 25 ± 1 years) before, during, and after 60 min of one-leg cycle ergometry at ∼70% Vo2peak. Exercise for 30 and 60 min was associated with a three- and fourfold increase in PKC-ζ/λ abundance and a four- and threefold increase in phosphorylation, respectively, in total membranes (P < 0.05) and a decrease in PKC-ζ/λ phosphorylation in cytosolic fractions. During exercise recovery, PKC-ζ/λ abundance and phosphorylation remained elevated. PKC-ζ/λ abundance and phosphorylation were increased in nonexercised muscle upon cessation of exercise, indicating a systemic response may contribute to changes in PKC abundance and phosphorylation. Exercise did not change PKC-δ or -ε abundance or phosphorylation in either the cytosolic or total membrane fraction. In conclusion, exercise is associated with an isoform-specific effect on PKC. PKC-ζ/λ are candidate PKC isoforms that may play a role in the regulation of exercise-related changes in metabolic and gene-regulatory responses.
Familial Hyperinsulinemic Hypoglycemia Caused by a Defect in the SCHAD Enzyme of Mitochondrial Fatty Acid Oxidation
Authors: Molven Anders , Matre Guri E. , Duran Marinus , Wanders Ronald J. , Rishaug Unni , Njølstad Pål R. , Jellum Egil , Søvik Oddmund
Published: March 2007

Inappropriately elevated insulin secretion is the hallmark of persistent hyperinsulinemic hypoglycemia of infancy (PHHI), also denoted congenital hyperinsulinism. Causal mutations have been uncovered in genes coding for the β-cell’s ATP-sensitive potassium channel and the metabolic enzymes glucokinase and glutamate dehydrogenase. In addition, one hyperinsulinemic infant was recently found to have a mutation in the gene encoding short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), an enzyme participating in mitochondrial fatty acid oxidation. We have studied a consanguineous family with severe neonatal hypoglycemia due to increased insulin levels and where well-established genetic causes of hyperinsulinism had been eliminated. A genome-wide, microsatellite-based screen for homozygous chromosomal segments was performed. Those regions that were inherited in accordance with the presupposed model were searched for mutations in genes encoding metabolic enzymes. A novel, homozygous deletion mutation was found in the gene coding for the SCHAD enzyme. The mutation affected RNA splicing and was predicted to lead to a protein lacking 30 amino acids. The observations at the molecular level were confirmed by demonstrating greatly reduced SCHAD activity in the patients’ fibroblasts and enhanced levels of 3-hydroxybutyryl-carnitine in their blood plasma. Urine metabolite analysis showed that SCHAD deficiency resulted in specific excretion of 3-hydroxyglutaric acid. By the genetic explanation of our family’s cases of severe hypoglycemia, it is now clear that recessively inherited SCHAD deficiency can result in PHHI. This finding suggests that mitochondrial fatty acid oxidation influences insulin secretion by a hitherto unknown mechanism.
Dehydroepiandrosterone Stimulates Glucose Uptake in Human and Murine Adipocytes by Inducing GLUT1 and GLUT4 Translocation to the Plasma Membrane
Authors: Perrini Sebastio , Natalicchio Annalisa , Laviola Luigi , Belsanti Gaetana , Montrone Carmela , Cignarelli Angelo , Minielli Vincenza , Grano Maria , De Pergola Giovanni , Giorgino Riccardo , Giorgino Francesco
Published: March 2007

Dehydroepiandrosterone (DHEA) has been shown to modulate glucose utilization in humans and animals, but the mechanisms of DHEA action have not been clarified. We show that DHEA induces a dose- and time-dependent increase in glucose transport rates in both 3T3-L1 and human adipocytes with maximal effects at 2 h. Exposure of adipocytes to DHEA does not result in changes of total GLUT4 and GLUT1 protein levels. However, it does result in significant increases of these glucose transporters in the plasma membrane. In 3T3-L1 adipocytes, DHEA increases tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 and stimulates IRS-1- and IRS-2-associated phosphatidylinositol (PI) 3-kinase activity with no effects on either insulin receptor or Akt phosphorylation. In addition, DHEA causes significant increases of cytosolic Ca2+ concentrations and a parallel activation of protein kinase C (PKC)-β2. The effects of DHEA are abrogated by pretreatment of adipocytes with PI 3-kinase and phospholipase Cγ inhibitors, as well as by inhibitors of Ca2+-dependent PKC isoforms, including a specific PKC-β inhibitor. Thus, DHEA increases glucose uptake in both human and 3T3-L1 adipocytes by stimulating GLUT4 and GLUT1 translocation to the plasma membrane. PI 3-kinase, phospholipase Cγ, and the conventional PKC-β2 seem to be involved in DHEA effects.
Metabolic Pathways That Mediate Inhibition of Hypothalamic Neurons by Glucose
Authors: Yang Xue-Jun , Kow Lee-Ming , Pfaff Donald W. , Mobbs Charles V.
Published: March 2007

Neurons in the ventromedial hypothalamus mediate some counterregulatory responses to hypoglycemia and 2-deoxyglucose, but the mechanisms that mediate these responses to glucose are unclear. In the present study, ventromedial hypothalamus neurons were identified on the basis of their inhibition by the transition from 5 to 20 mmol/l glucose. Tolbutamide, which activates glucose-stimulated neurons, failed to inhibit or activate glucose-inhibited neurons. Inhibitors of glucose transport and glycolysis, in particular by the glucokinase inhibitor glucosamine, blocked the effect of glucose on glucose-inhibited neurons. Furthermore, the glucose-inhibited neurons were activated by 2-deoxyglucose, which also activates counterregulatory responses. Conversely, glucose-inhibited neurons were inhibited by glycolytic metabolites, including lactate, but not by pyruvate. These data indicate that hypoglycemia induces electrical activity in glucose-inhibited neurons by attenuating glycolysis in those neurons. Thus, counterregulatory failure could be due to relatively enhanced glycolysis in glucose-stimulated neurons during hypoglycemia and attenuation of glycolysis in glucose-inhibited neurons might reverse counterregulatory failure.
Hypothalamic ATP-sensitive K + Channels Play a Key Role in Sensing Hypoglycemia and Triggering Counterregulatory Epinephrine and Glucagon Responses
Authors: Evans Mark L. , McCrimmon Rory J. , Flanagan Daniel E. , Keshavarz Tara , Fan Xiaoning , McNay Ewan C. , Jacob Ralph J. , Sherwin Robert S.
Published: March 2007

It has been postulated that specialized glucose-sensing neurons in the ventromedial hypothalamus (VMH) are able to detect falling blood glucose and trigger the release of counterregulatory hormones during hypoglycemia. The molecular mechanisms used by glucose-sensing neurons are uncertain but may involve cell surface ATP-sensitive K+ channels (KATP channels) analogous to those of the pancreatic β-cell. We examined whether the delivery of sulfonylureas directly into the brain to close KATP channels would modulate counterregulatory hormone responses to either brain glucopenia (using intracerebroventricular 5-thioglucose) or systemic hypoglycemia in awake chronically catheterized rats. The closure of brain KATP channels by global intracerebroventricular perfusion of sulfonylurea (120 ng/min glibenclamide or 2.7 μg/min tolbutamide) suppressed counterregulatory (epinephrine and glucagon) responses to brain glucopenia and/or systemic hypoglycemia (2.8 mmol/l glucose clamp). Local VMH microinjection of a small dose of glibenclamide (0.1% of the intracerebroventricular dose) also suppressed hormonal responses to systemic hypoglycemia. We conclude that hypothalamic KATP channel activity plays an important role in modulating the hormonal counterregulatory responses triggered by decreases in blood glucose. Our data suggest that closing of KATP channels in the VMH (much like the β-cell) impairs defense mechanisms against glucose deprivation and therefore could contribute to defects in glucose counterregulation.
A Salen-Manganese Catalytic Free Radical Scavenger Inhibits Type 1 Diabetes and Islet Allograft Rejection
Authors: Olcott Angelica P. , Tocco Georges , Tian Jide , Zekzer Dan , Fukuto Jon , Ignarro Louis , Kaufman Daniel L.
Published: March 2007

Reactive oxygen species, such as superoxide, and nitrogen oxides, such as peroxynitrite, are thought to contribute to β-cell destruction during the disease process that leads to type 1 diabetes. EUK-8 is a member of a new class of synthetic salen-manganese compounds with low toxicity that possess catalytic superoxide dismutase, peroxidase, and catalase activity that can inactivate superoxide and nitrogen oxides (e.g., peroxynitrite and nitrogen dioxide). We observed that EUK-8 administration inhibited the adoptive transfer of type 1 diabetes to NOD mice. In addition, administration of EUK-8 to NOD mice with established autoimmunity completely prevented the development of type 1 diabetes for up to 1 year in age, even though the treatment was discontinued after 35 weeks of age. EUK-8 treatment also prolonged the survival of islet allografts in newly diabetic NOD mice. Thus, reactive oxygen and nitrogen species contribute to the pathoetiology of both spontaneous type 1 diabetes and allograft rejection. In cultures of NIT-1 cells, EUK-8 inhibited cytotoxicity caused by superoxide as well as nitric oxide. Collectively, our findings implicate a greater role for nitrogen oxides (other than peroxynitrite) in β-cell damage. Antioxidants designed to prevent the formation of both cytotoxic reactive oxygen and nitrogen species may effectively protect β-cells from spontaneous autoimmunity and alloresponses.
Promotion of β-Cell Differentiation by Conophylline in Fetal and Neonatal Rat Pancreas
Authors: Ogata Takeki , Li Lei , Yamada Satoko , Yamamoto Yoritsuna , Tanaka Yuji , Takei Izumi , Umezawa Kazuo , Kojima Itaru
Published: March 2007

Conophylline is a vinca alkaloid extracted from the tropical plant Ervatamia microphylla and has been shown to induce differentiation of pancreatic AR42J cells. In the present study, we investigated the effect of conophylline on the differentiation of pancreatic precursor cells. In the rat pancreatic rudiment in organ culture, conophylline inhibited the formation of cystic structure and increased the number of insulin-positive cells. Conophylline also markedly increased the expression of mRNA for insulin and the number of pancreatic duodenal homeobox-1–positive cells. These effects of conophylline were similar to those of activin A. We also examined the effect of conophylline on neonatal rats treated with streptozotocin, a model of type 2 diabetes. Treatment with conophylline significantly reduced the plasma glucose concentration and improved glucose tolerance in response to glucose loading. The insulin content and the β-cell mass at 2 months were significantly increased by conophylline. The number of islet-like cell clusters and pancreatic duodenal homeobox-1–positive ductal cells was greater in conophylline-treated rats. These results suggest that conophylline induces differentiation of pancreatic precursor cells and increases the formation of β-cells.
Hepatic Insulin Resistance Precedes the Development of Diabetes in a Model of Intrauterine Growth Retardation
Authors: Vuguin Patricia , Raab Elisabeth , Liu Bing , Barzilai Nir , Simmons Rebecca
Published: March 2007

Intrauterine growth retardation (IUGR) has been linked to the development of type 2 diabetes in adulthood. We developed an IUGR model in rats whereby at age 3–6 months the animals develop a diabetes that is associated with insulin resistance. Hyperinsulinemic-euglycemic clamp studies were performed at age 8 weeks, before the onset of obesity and diabetes. Basal hepatic glucose production (HGP) was significantly higher in IUGR than in control rats (14.6 ± 0.4 vs. 12.3 ± 0.3 mg · kg−1 · min−1; P < 0.05). Insulin suppression of HGP was blunted in IUGR versus control rats (10.4 ± 0.6 vs. 6.5 ± 1.0 mg · kg−1 · min−1; P < 0.01); however, rates of glucose uptake and glycogenolysis were similar between the two groups. Insulin-stimulated insulin receptor substrate 2 and Akt-2 phosphorylation were significantly blunted in IUGR rats. PEPCK and glucose-6-phosphatase mRNA levels were increased at least threefold in liver of IUGR compared with control rats. These studies suggest that an aberrant intrauterine milieu permanently impairs insulin signaling in the liver so that gluconeogenesis is augmented in the IUGR rat. These processes occur early in life, before the onset of hyperglycemia, and indicate that uteroplacental insufficiency causes a primary defect in gene expression and hepatic metabolism that leads to the eventual development of overt hyperglycemia.
Thiazolidinediones Reduce Endothelial Expression of Receptors for Advanced Glycation End Products
Authors: Marx Nikolaus , Walcher Daniel , Ivanova Nina , Rautzenberg Kirstin , Jung Annelie , Friedl Reinhard , Hombach Vinzenz , de Caterina Raffaele , Basta Giuseppina , Wautier Marie-Paule , Wautiers Jean-Luc
Published: March 2007

Advanced glycation end products (AGEs) are critically involved in atherogenesis in diabetes by binding to receptors for AGE (RAGEs) in vascular cells, thus inducing the expression of proinflammatory mediators. In animal models, interruption of the AGE-RAGE interaction reduces lesion size and plaque development. Therefore, limiting RAGE expression might be an intriguing concept to modulate vascular disease in diabetic patients. The present study investigated whether thiazolidinediones (TZDs), antidiabetic agents clinically used to treat patients with type 2 diabetes, might modulate endothelial RAGE expression. Stimulation of human endothelial cells with rosiglitazone or pioglitazone decreased basal as well as tumor necrosis factor-α–induced RAGE cell surface and total protein expression. In addition, TZDs reduced RAGE mRNA expression in endothelial cells. These effects on RAGE expression were caused by an inhibition of nuclear factor-κB (NF-κB) activation at the proximal NF-κB site of the RAGE promoter. The functional relevance of reduced RAGE expression was demonstrated by showing that pretreatment of endothelial cells with TZDs decreased AGE- as well as β-amyloid–induced monocyte chemoattractant protein-1 expression. In conclusion, TZDs reduce RAGE expression in human endothelial cells, thus limiting the cells’ susceptibility toward proinflammatory AGE effects. These data provide new insight on how TZDs, in addition to their metabolic effects, might modulate the development of vascular dysfunction in diabetic patients.
Melanocortin 4 Receptor Gene Variation Is Associated With Severe Obesity in Pima Indians
Authors: Ma Lijun , Tataranni P. Antonio , Bogardus Clifton , Baier Leslie J.
Published: March 2007

Heterozygous coding mutations in the melanocortin 4 receptor gene (MC4R) are the most common genetic causes of severe human obesity identified to date. To determine whether MC4R has a role in causing severe obesity in Pima Indians, we sequenced the coding region of this gene in 426 full-heritage, non–first-degree related, adult Pima Indians (300 severely obese and 126 nondiabetic nonobese control subjects). Three coding variations were detected as heterozygotes only in severely obese subjects. One variation, detected in three obese subjects, was a novel single-base insertion (A) at nucleotide 100, and it predicted a frameshift and premature STOP at codon 37. The second variant, detected in 10 obese subjects, predicted a previously identified arginine-to-glutamine substitution at codon 165, and the third variant, detected in one obese subject, predicted a novel glycine-to-serine substitution at codon 231. Three polymorphisms were also identified in the 5′ untranslated region, but these variants were detected in both obese and lean subjects and had similar allele frequencies. We conclude that variations in MC4R may account for a small portion of obesity in Pima Indians, but they do not explain the overall high prevalence of obesity in this Native American population.
Analysis of the Vitamin D Receptor Gene Sequence Variants in Type 1 Diabetes
Authors: Nejentsev Sergey , Cooper Jason D. , Godfrey Lisa , Howson Joanna M.M. , Rance Helen , Nutland Sarah , Walker Neil M. , Guja Cristian , Ionescu-Tirgovişte Constantin , Savage David A. , Undlien Dag E. , Rønningen Kjersti S. , Tuomilehto-Wolf Eva , Tuomilehto Jaakko , Gillespie Kathleen M. , Ring Susan M. , Strachan David P. , Widmer Barry , Dunger David , Todd John A.
Published: March 2007

Vitamin D is known to modulate the immune system, and its administration has been associated with reduced risk of type 1 diabetes. Vitamin D acts via its receptor (VDR). Four single nucleotide polymorphisms (SNPs) of the VDR gene have been commonly studied, and evidence of association with type 1 diabetes has been reported previously. We sequenced the VDR gene region and developed its SNP map. Here we analyzed association of the 98 VDR SNPs in up to 3,763 type 1 diabetic families. First, we genotyped all 98 SNPs in a minimum of 458 U.K. families with two affected offspring. We further tested eight SNPs, including four SNPs associated with P < 0.05 in the first set and the four commonly studied SNPs, in up to 3,305 additional families from the U.K., Finland, Norway, Romania, and U.S. We only found weak evidence of association (P = 0.02–0.05) of the rs4303288, rs12721366, and rs2544043 SNPs. We then tested these three SNPs in an independent set of 1,587 patients and 1,827 control subjects from the U.K. and found no evidence of association. Overall, our results indicate that common sequence variation in the VDR gene has no major effect in type 1 diabetes in the populations tested.
Physiological Hyperinsulinemia in Dogs Augments Access of Macromolecules to Insulin-Sensitive Tissues
Authors: Ellmerer Martin , Kim Stella P. , Hamilton-Wessler Marianthe , Hücking Katrin , Kirkman Erlinda , Bergman Richard N.
Published: March 2007

Pharmacological doses of insulin increase limb blood flow and enhance tissue recruitment for small solutes such as glucose. We investigated whether elevating insulin within the physiological range (68 ± 6 vs. 425 ± 27 pmol/l) can influence tissue recruitment of [14C]inulin, an inert diffusionary marker of molecular weight similar to that of insulin itself. During hyperinsulinemic-euglycemic clamps, transport parameters and distribution volumes of [14C]inulin were determined in conscious dogs by applying a three-compartment model to the plasma clearance data of intravenously injected [14C]inulin (0.8 μCi/kg). In a second set of experiments in anesthetized dogs with direct cannulation of the hindlimb skeletal muscle lymphatics, we measured a possible effect of physiological hyperinsulinemia on the response of the interstitial fluid of skeletal muscle to intravenously injected [14C]inulin and compared this response with the model prediction from plasma data. Physiological hyperinsulinemia caused a 48 ± 10% (P < 0.005) and a 35 ± 15% (P < 0.05) increase of peripheral and splanchnic interstitial distribution volumes for [14C]inulin. Hindlimb lymph measurements directly confirmed the ability of insulin to enhance the access of macromolecules to the peripheral interstitial fluid compartment. The present results show that physiological hyperinsulinemia will enhance the delivery of a substance of similar molecular size to insulin to previously less intensively perfused regions of insulin-sensitive tissues. Our data suggest that the delivery of insulin itself to insulin-sensitive tissues could be a mechanism of insulin action on cellular glucose uptake independent of and possibly synergistic with either enhanced blood flow distribution or GLUT4 transporter recruitment to enhance glucose utilization. Because of the differences between inulin and insulin itself, whether delivery of the bioactive hormone is increased remains speculative.
Interplay of Glucagon-Like Peptide-1 and Transforming Growth Factor-β Signaling in Insulin-Positive Differentiation of AR42J Cells
Authors: Yew Kok-Hooi , Prasadan Krishna L. , Preuett Barry L. , Hembree Mark J. , McFall Christopher R. , Benjes Christina L. , Crowley Amanda R. , Sharp Susan L. , Li Zhixing , Tulachan Sidhartha S. , Mehta Sheilendra S. , Gittes George K.
Published: March 2007

The differentiation of pancreatic exocrine AR42J cells into insulin-expressing endocrine cells has served as an important model for both endogenous in vivo β-cell differentiation as well as potential application to β-cell engineering of progenitor cells. Exogenous activin, possibly working through intracellular smad 2 and/or smad 3, as well as exogenous exendin-4 (a long-acting glucagon-like peptide-1 agonist) have both been shown to induce insulin-positive/endocrine differentiation in AR42J cells. In this study, we present evidence of significant interplay and interdependence of these two pathways as well as potential synergy between the pathways. In particular, insulin-positive differentiation seems to entail an exendin-4–induced drop in smad 2 and elevation in smad 3 in RNA levels. The latter appears to be dependent on endogenous transforming growth factor (TGF)-β isoform release by the AR42J cells and may serve as a mechanism to promote β-cell maturation. The drop in smad 2 may mediate early endocrine commitment. The coapplication of exogenous exendin-4 and, specifically, low-dose exogenous TGF-β1 led to a dramatic 20-fold increase in insulin mRNA levels, supporting a novel synergistic and codependent relationship between exendin-4 signaling and TGF-β isoform signaling.