bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin II type 1 receptor gene polymorphism and the response to hyperglycemia in early type 1 diabetes.
Authors: Miller J A , Thai K , Scholey J W
Published: March 2007

Recent studies suggest that there is an association between the A1166-->C polymorphism of the angiotensin II type 1 receptor (AGT1R), glycemic control, and the risk of diabetic nephropathy in subjects with type 1 diabetes. Because hypertension and renal hemodynamic function are also related to the risk of diabetic nephropathy and because hyperglycemia can activate the renin angiotensin system, we sought to determine if there is an association between the AGT1R polymorphism, baseline renal and peripheral hemodynamic function, and pressor response to high glucose in subjects with early uncomplicated type 1 diabetes. There were 39 diabetic subjects genotyped for the AGT1R polymorphism by polymerase chain reaction and segregated into 2 groups: those with and those without the C1166 allele (AA and AC/CC). The average age was 27 +/- 1 years, and the mean duration of diabetes was 3.5 +/- 0.6 years. HbA(1c) values were <10% in all subjects and were similar in the 2 groups (8.2 +/- 0.3 vs. 9.1 +/- 0.4%). After a 7-day controlled diet (150 mmol sodium, 1.5-2.0 g x kg(-1) x day(-1) protein), renal hemodynamic function was assessed by inulin and para-aminohippurate clearance during clamped euglycemic conditions (4-6 mmol/l). Mean values for glomerular filtration rates did not differ between groups during euglycemia. In contrast, mean values for renal plasma flow and renal blood flow were significantly greater in the AC/CC group compared with the AA group. Values for mean arterial pressure were similar in the 2 groups, whereas renal vascular resistance was significantly reduced in the AC/CC group. In 20 subjects (10 from each genotype subgroup), hemodynamic function was assessed on a second occasion during controlled clamped hyperglycemia (9-11 mmol/l) after a similar preparatory period. In response to high glucose, plasma renin activity increased in both genotype groups to the same extent, but a pressor response was noted only in subjects with the C1166 allele. Mean arterial pressure increased significantly in the AC/CC subgroup and remained unchanged in the AA subgroup. We conclude that there is an association between the AGT1R A1166-->C polymorphism and renal hemodynamic function in early type 1 diabetes. But more importantly, the pressor response to hyperglycemia is augmented in those diabetic patients with the C1166 allele and may represent a factor that predisposes them to renal injury during periods of inadequate glucose control.
Hypoglycemia Activates Orexin Neurons and Selectively Increases Hypothalamic Orexin-B Levels
Authors: Cai Xue J. , Evans Martyn L. , Lister Carolyn A. , Leslie Ron A. , Arch Jonathan R. S. , Wilson Shelagh , Williams Gareth
Published: March 2007

Orexins are novel appetite-stimulating peptides expressed in the lateral hypothalamic area (LHA), and their expression is stimulated by hypoglycemia in fasted rats. We investigated activation of orexin and other neurons during insulin-induced hypoglycemia using the immediate early gene product Fos. Insulin (50 U/kg) lowered plasma glucose by >50% after 5 h and stimulated feeding sixfold compared with saline-injected controls. Hypoglycemic rats allowed to feed and normoglycemic controls both showed sparse Fos-positive (Fos+) neurons in the LHA and the paraventricular nucleus (PVN) and arcuate nucleus (ARC) and showed none in the nucleus of the solitary tract (NTS), which relays visceral feeding signals to the LHA. In the LHA, total numbers of Fos+ neurons were comparable in fed hypoglycemic and control groups (60 ± 6 vs. 52 ± 4 cells/mm2, P > 0.05), as were Fos+ neurons immunoreactive for orexin (1.4 ± 0.4 vs. 0.6 ± 0.4 cells/mm2, P > 0.05). By contrast, hypoglycemic rats that were fasted showed significantly more Fos+ nuclei in the LHA (96 ± 10 cells/mm2, P < 0.05, vs. both other groups) and Fos+ orexin neurons (8.4 ± 3.3 cells/mm2, P < 0.001, vs. both other groups). They also showed two- to threefold more Fos+ nuclei (P < 0.001) in the PVN and ARC than both fed hypoglycemic rats and controls and showed strikingly abundant Fos+ neurons in the NTS and dorsal motor nucleus of the vagus. In parallel studies, whole hypothalamic orexin-A levels were not changed in hypoglycemic rats, whether fasted or freely fed, whereas orexin-B levels were 10-fold higher in hypoglycemic fasted rats than in control and hypoglycemic fed groups. These data support our hypothesis that orexin neurons are stimulated by falling glucose levels but are readily inhibited by signals related to nutrient ingestion and suggest that they may functionally link with neuronal activity in the NTS. Orexin-A and -B may play specific roles in behavioral or neuroendocrine responses to hypoglycemia.
Oral Glucose Tolerance Test Minimal Model Indexes of β-Cell Function and Insulin Sensitivity
Authors: Breda Elena , Cavaghan Melissa K. , Toffolo Gianna , Polonsky Kenneth S. , Cobelli Claudio
Published: March 2007

The simultaneous assessment of quantitative indexes of insulin secretion and action in a single individual is important when quantifying their relative role in the evolution of glucose tolerance in different physiopathological states. Available methods quantify these indexes in relatively nonphysiological conditions, e.g., during glucose clamps or intravenous glucose tolerance tests. Here, we present a method based on a physiological test applicable to large-scale genetic and epidemiologic studies—the oral glucose tolerance test (OGTT). Plasma C-peptide, insulin, and glucose data from a frequently sampled OGTT with 22 samples throughout 300 min (FSOGTT300-22) were analyzed in 11 subjects with various degrees of glucose tolerance. In each individual, two indexes of pancreatic sensitivity to glucose (Φs [109 min-1] and Φd [109]) and the insulin sensitivity index (SI) (105 dl/kg per min per pmol/l) were estimated by using the minimal model of C-peptide secretion and kinetics originally proposed for intravenous graded glucose infusion and the minimal model approach recently proposed for meal/OGTTs. The indexes obtained from FSOGTT300-22 were used as a reference for internal validation of OGTT protocols with reduced sampling schedules. Our results show that 11 samples in a 300-min period (OGTT300-11) is the test of choice because the indexes it provides (Φs = 36 ± 3 [means ± SE]; Φd = 710 ± 111; SI = 10.2 ± 2.4) show excellent correlation and are not statistically different from those of FSOGTT300-22 (Φs = 33 ± 3; Φd = 715 ± 120; SI = 10.1 ± 2.3). In conclusion, OGTT300-11, interpreted with C-peptide and glucose minimal models, provides a quantitative description of β-cell function and insulin sensitivity in a single individual while preserving the important clinical classification of glucose tolerance provided by the standard 120-min OGTT.
The Stimulation-Induced Increase in Skeletal Muscle Glycogen Synthase Content Is Impaired in Carriers of the Glycogen Synthase XbaI Gene Polymorphism
Authors: St-Onge Josée , Joanisse Denis R. , Simoneau Jean-Aimé
Published: March 2007

Associations between glycogen synthase gene (GYS1) polymorphism and states of insulin resistance and type 2 diabetes have been reported. The purpose of this study was to establish if the GYS1 genotype impacts on the content of glycogen synthase (GS) protein in muscle measured under basal and stimulated conditions. To examine this, GYS1 XbaI and Met416Val polymorphisms and thigh muscle GYS1 protein content were determined at rest, both before and after several weeks of neuromuscular electrical stimulation in carriers and noncarriers of the mutations. The allelic frequency was 0.086 for the XbaI mutation (A2) and 0.006 for the Met416Val in our cohort of French-Canadian subjects. When measured at rest, the GS protein content in muscle was similar among carriers and noncarriers of the XbaI variant. However, the stimulation-induced increase (23%) in the amount of GS muscle protein normally seen in wild-type individuals was impaired in those carrying the XbaI mutation. These data demonstrate that some individuals, because of their genetic background, are unable to stimulate the process of GS protein accumulation in skeletal muscle. These results could explain why some individuals appear to be genetically predisposed to developing skeletal muscle insulin resistance when exposed to unfavorable metabolic environments.
Physiological Differences in Insulin-Like Growth Factor Binding Protein-1 (IGFBP-1) Phosphorylation in IGFBP-1 Transgenic Mice
Authors: Sakai Keiji , D'Ercole A. Joseph , Murphy Liam J. , Clemmons David R.
Published: March 2007

Insulin-like growth factor binding protein (IGFBP)-1 has been shown to alter cellular responses to insulin-like growth factor 1 (IGF-1). Human IGFBP-1 undergoes serine phosphorylation, and this enhances both its affinity for IGF-1 by six- to eightfold and its capacity to inhibit IGF-1 actions. To investigate the physiological role of IGFBP-1 in vivo, transgenic mice have been generated using either the human IGFBP-1 or rat IGFBP-1 transgene. Both lines of mice expressed high concentrations of IGFBP-1 in serum and tissues; however, human IGFBP-1 transgenic mice did not show glucose intolerance and exhibited no significant intrauterine growth retardation, whereas rat IGFBP-1 transgenic mice showed fasting hyperglycemia and intrauterine growth restriction. The aim of this study was to investigate the physiological differences in the phosphorylation state of human IGFBP-1 and rat IGFBP-1 in these transgenic mice. The phosphorylation status of IGFBP-1 in transgenic mouse serum was analyzed by nondenaturing PAGE. Almost all of the IGFBP-1 in serum from the human IGFBP-1 transgenic mice was present as a nonphosphorylated form. Most of the rat IGFBP-1 in the serum of the mice expressing the rat IGFBP-1 was phosphorylated. Immunoprecipitation showed that mouse hepatoma (Hepa 1-6) cells (exposed to [32P]H3PO4) secrete 32P-labeled IGFBP-1. When the human IGFBP-1 transgene was transfected into Hepa 1-6 cells, all of the IGFBP-1 was secreted in the nonphosphorylated form. However, when the rat IGFBP-1 transgene was transfected into these cells, phosphorylated forms of IGFBP-1 were secreted. To confirm this result, the mouse hepatoma cell protein kinase was partially purified. This kinase activity phosphorylated mouse and rat IGFBP-1 in vitro, but it did not phosphorylate human IGFBP-1. Scatchard analysis showed that the affinity of phosphorylated rat IGFBP-1 for IGF-1 was 3.9-fold higher than that of nonphosphorylated human IGFBP-1. We conclude that the mouse IGFBP-1 kinase activity cannot phosphorylate human IGFBP-1, whereas it can phosphorylate rat IGFBP-1. The phosphorylation state of human IGFBP-1 may account for part of the phenotypic differences noted in the two studies of transgenic mice, and it is an important determinant of the capacity of human IGFBP-1 to inhibit IGF-1 actions in vivo.
Glucagon-Like Peptide 1 Stimulates Lipolysis in Clonal Pancreatic β-Cells (HIT)
Authors: Yaney Gordon C. , Civelek Vildan N. , Richard Ann-Marie , Dillon Joseph S. , Deeney Jude T. , Hamilton James A. , Korchak Helen M. , Tornheim Keith , Corkey Barbara E. , Boyd Aubrey E.
Published: March 2007

Glucagon-like peptide 1 (GLP-1) is the most potent physiological incretin for insulin secretion from the pancreatic β-cell, but its mechanism of action has not been established. It interacts with specific cell-surface receptors, generates cAMP, and thereby activates protein kinase A (PKA). Many changes in pancreatic β-cell function have been attributed to PKA activation, but the contribution of each one to the secretory response is unknown. We show here for the first time that GLP-1 rapidly released free fatty acids (FFAs) from cellular stores, thereby lowering intracellular pH (pHi) and stimulating FFA oxidation in clonal β-cells (HIT). Similar changes were observed with forskolin, suggesting that stimulation of lipolysis was a function of PKA activation in β-cells. Triacsin C, which inhibits the conversion of FFAs to long-chain acyl CoA (LC-CoA), enhanced basal FFA efflux as well as GLP-1-induced acidification and efflux of FFAs from the cell. Increasing the concentration of the lipase inhibitor orlistat progressively and largely diminished the increment in secretion caused by forskolin. However, glucose-stimulated secretion was less inhibited by orlistat and only at the highest concentration tested. Because the acute addition of FFAs also increases glucose-stimulated insulin secretion, these data suggest that the incretin function of GLP-1 may involve a major role for lipolysis in cAMP-mediated potentiation of secretion.
Identification of an Interactive Effect of β3- and α2b-Adrenoceptor Gene Polymorphisms on Fat Mass in Caucasian Women
Authors: Dionne Isabelle J. , Turner Amy N. , Tchernof André , Pollin Toni I. , Avrithi Dionyssia , Gray Daniel , Shuldiner Alan R. , Poehlman Eric T.
Published: March 2007

Several adrenoceptor subtypes are expressed in adipocytes, which together exert their influence on adipocyte metabolism. Therefore, we specifically examined the interactive effect of Trp64Arg (β3) and Glu12/Glu9 (α2b) adrenoceptor (AR) polymorphisms on energy metabolism and body composition in healthy women with a wide range of body habitus. We genotyped 909 unrelated women (age 55 ± 12 [mean ± SD] years, range 19-87; body weight 88 ± 22 kg, range 40-167; and BMI 33 ± 8 kg/m2, range 16-64) for Trp64Arg β3AR and Glu12/Glu9 α2bAR variants. We examined the independent effect of the Glu12/Glu9 α2bAR variant on body composition and energy balance, in a large cohort of Caucasian women (n = 909). A second goal was to examine the interaction effect of Glu12/Glu9 α2bAR and Trp64Arg β3AR on the same phenotypes. The obesity-related phenotypes studied were as follows: body weight, BMI, fat mass, visceral fat, fat-free mass, resting metabolic rate (RMR), Vo2max, leisure time physical activity, and daily energy intake. Body composition and body fat distribution were measured by dual-energy X-ray absorptiometry and radiographic imagery, Vo2max by a treadmill test to exhaustion, and RMR by indirect calorimetry. An analysis of covariance indicated that in the entire cohort, there was no significant difference between Glu12/Glu9 α2bAR carriers and control subjects for any of the obesity-related phenotypes that were examined. However, we observed a significant interaction effect of the Trp64Arg and Glu12/Glu9 variants on fat mass (P = 0.009) and percent fat (P = 0.016). Age, height, body weight, BMI, fat-free mass, visceral fat, energy expenditure, respiratory quotient, physical fitness, and energy intake were not different among groups. Collectively, these findings support an interaction effect of the two adrenoceptor variants on body fatness in Caucasian women, although the physiological mechanism by which they exert this effect remains to be determined.
Effects of Leptin Deficiency and Short-Term Repletion on Hepatic Gene Expression in Genetically Obese Mice
Authors: Ferrante Anthony W. , Thearle Marie , Liao Ted , Leibel Rudolph L.
Published: March 2007

By supplying most organs of the body with metabolic substrates, the liver plays a central role in maintaining energy balance. Hepatic metabolism of glucose, fatty acids, and lipoproteins is disrupted in the leptin-deficient obese (Lepob/Lepob) mouse, leading to hyperglycemia, steatosis, and hypercholesterolemia. Microarray expression profiles were used to identify transcriptional perturbations that underlie the altered hepatic physiology of Lepob/Lepob mice. A wide variety of genes involved in fatty acid metabolism are altered in expression, which suggests that both fatty acid synthesis and oxidation programs are activated in obese mice. The expression of a small subset of genes is upregulated by leptin deficiency, not modulated by caloric restriction, and markedly suppressed by short-term leptin treatment. Among these leptin-regulated genes, apolipoprotein A-IV is a strong candidate for mediating the atherogenic-resistant phenotype of Lepob/Lepob mice.
Analysis of the Coding and Promoter Regions of the Autoantigen IA-2 in Subjects With and Without Autoantibodies to IA-2
Authors: Cai Tao , Xie Jingping , She Jin-Xiong , Notkins Abner Louis
Published: March 2007

Despite extensive studies on HLA polymorphism, there have been few, if any, studies on allelic forms or mutations in proteins that serve as autoantigens. The present experiments were designed to look for alterations in the coding and promoter regions of the autoantigen IA-2 in type one (insulin-dependent) diabetic patients with autoantibodies to IA-2 as compared with siblings without diabetes or autoantibodies to IA-2. Genomic DNA was used as a template and was amplified by polymerase chain reaction, with pairs of primers encompassing the promoter region and the 23 exons of the coding region of IA-2. A total of nine nucleotide changes were found in the coding region of the six type 1 diabetic patients; four were silent and five were missense changes, but all occurred in the extracellular domain of IA-2 to which autoantibodies are not directed. Few, if any, changes were found in the 5′ upstream (−706 to +135) promoter region. The results of the experiments support the null hypothesis that differences among individuals in the nucleotide and amino acid sequences of the promoter and coding regions of IA-2, respectively, do not account for why some individuals develop autoantibodies to IA-2 and others do not.
Skeletal Muscle Insulin Resistance in Normoglycemic Subjects With a Strong Family History of Type 2 Diabetes Is Associated With Decreased Insulin-Stimulated Insulin Receptor Substrate-1 Tyrosine Phosphorylation
Authors: Pratipanawatr Wilailak , Pratipanawatr Thongchai , Cusi Kenneth , Berria Rachele , Adams John M. , Jenkinson Christopher P. , Maezono Katsumi , DeFronzo Ralph A. , Mandarino Lawrence J.
Published: March 2007

Normoglycemic subjects with a strong family history of type 2 diabetes are insulin resistant, but the mechanism of insulin resistance in skeletal muscle of such individuals is unknown. The present study was undertaken to determine whether abnormalities in insulin-signaling events are present in normoglycemic, nonobese subjects with a strong family history of type 2 diabetes. Hyperinsulinemic-euglycemic clamps with percutaneous muscle biopsies were performed in eight normoglycemic relatives of type 2 diabetic patients (FH+) and eight control subjects who had no family history of diabetes (FH−), with each group matched for age, sex, body composition, and ethnicity. The FH+ group had decreased insulin-stimulated glucose disposal (6.64 ± 0.52 vs. 8.45 ± 0.54 mg · kg−1 fat-free mass · min−1; P < 0.05 vs. FH−). In skeletal muscle, the FH+ and FH− groups had equivalent insulin stimulation of insulin receptor tyrosine phosphorylation. In contrast, the FH+ group had decreased insulin stimulation of insulin receptor substrate (IRS)-1 tyrosine phosphorylation (0.522 ± 0.077 vs. 1.328 ± 0.115 density units; P < 0.01) and association of PI 3-kinase activity with IRS-1 (0.299 ± 0.053 vs. 0.466 ± 0.098 activity units; P < 0.05). PI 3-kinase activity was correlated with the glucose disposal rate (r = 0.567, P = 0.02). In five subjects with sufficient biopsy material for further study, phosphorylation of Akt was 0.266 ± 0.061 vs. 0.404 ± 0.078 density units (P < 0.10) and glycogen synthase activity was 0.31 ± 0.06 vs. 0.50 ± 0.12 ng · min−1 · mg−1 (P < 0.10) for FH+ and FH− subjects, respectively. Therefore, despite normal insulin receptor phosphorylation, postreceptor signaling was reduced and was correlated with glucose disposal in muscle of individuals with a strong genetic background for type 2 diabetes.
Characterization of Allelic and Nucleotide Variation Between the RAGE Gene on Chromosome 6 and a Homologous Pseudogene Sequence to Its 5′ Regulatory Region on Chromosome 3
Authors: Hudson Barry I. , Stickland Max H. , Grant Peter J. , Futers T. Simon
Published: March 2007

Activation of the receptor for advanced glycation end products (RAGE) appears to be a key mechanism in the pathogenesis of diabetic vascular disease, making RAGE a candidate gene for investigation. RAGE is located in the major histocompatibility complex locus on chromosome 6, which contains a multitude of overlapping and duplicated genes involved predominantly in inflammatory and immune responses. The RAGE 5′ flanking region from −505 in a 5′ direction overlaps with PBX2, a gene that has a pseudogene copy on chromosome 3, making any studies of polymorphisms in this duplicated region potentially fraught with error. In this study we have addressed these issues by confirming RAGE as a predominantly single-copy gene and PBX2 to have two gene copies in the haploid human genome. We have characterized the gene:pseudogene differences between RAGE/PBX2 on chromosome 6 and ΨPBX2 on chromosome 3, which include a change from C to A at position −1139 RAGE/+2298 PBX2, previously reported as a polymorphism. Single chromosome–specific DNA amplification of the duplicated region has clarified five polymorphisms to be on chromosome 3 and one (at −1202 RAGE/+2234 PBX2) to be on chromosome 6. In conclusion, this study provides essential data for the study of RAGE and its genetics.
Convergence of Pre- and Postsynaptic Influences on Glucosensing Neurons in the Ventromedial Hypothalamic Nucleus
Authors: Song Zhentao , Levin Barry E. , McArdle Joseph J. , Bakhos Nader , Routh Vanessa H.
Published: March 2007

Glucosensing neurons in the ventromedial hypothalamic nucleus (VMN) were studied using visually guided slice-patch recording techniques in brain slices from 14- to 21-day-old male Sprague-Dawley rats. Whole-cell current-clamp recordings were made as extracellular glucose levels were increased (from 2.5 to 5 or 10 mmol/l) or decreased (from 2.5 to 0.1 mmol/l). Using these physiological conditions to define glucosensing neurons, two subtypes of VMN glucosensing neurons were directly responsive to alterations in extracellular glucose levels. Another three subtypes were not directly glucose-sensing themselves, but rather were presynaptically modulated by changes in extracellular glucose. Of the VMN neurons, 14% were directly inhibited by decreases in extracellular glucose (glucose-excited [GE]), and 3% were directly excited by decreases in extracellular glucose (glucose-inhibited [GI]). An additional 14% were presynaptically excited by decreased glucose (PED neurons). The other two subtypes of glucosensing neurons were either presynaptically inhibited (PIR; 11%) or excited (PER; 8%) when extracellular glucose was raised to >2.5 mmol/l. GE neurons sensed decreased glucose via an ATP-sensitive K+ (KATP) channel. The inhibitory effect of increased glucose on PIR neurons appears to be mediated by a presynaptic γ-aminobutyric acid–ergic glucosensing neuron that probably originates outside the VMN. Finally, all types of glucosensing neurons were both fewer in number and showed abnormal responses to glucose in a rodent model of diet-induced obesity and type 2 diabetes.
PPAR-α–Null Mice Are Protected From High-Fat Diet–Induced Insulin Resistance
Authors: Guerre-Millo Michèle , Rouault Christine , Poulain Philippe , André Jocelyne , Poitout Vincent , Peters Jeffrey M. , Gonzalez Frank J. , Fruchart Jean-Charles , Reach Gérard , Staels Bart
Published: March 2007

Peroxisome proliferator–activated receptor (PPAR)-α controls the expression of genes involved in lipid metabolism. PPAR-α furthermore participates to maintain blood glucose during acute metabolic stress, as shown in PPAR-α–null mice, which develop severe hypoglycemia when fasted. Here, we assessed a potential role for PPAR-α in glucose homeostasis in response to long-term high-fat feeding. When subjected to this nutritional challenge, PPAR-α–null mice remained normoglycemic and normoinsulinemic, whereas wild-type mice became hyperinsulinemic (190%; P < 0.05) and slightly hyperglycemic (120%; NS). Insulin tolerance tests (ITTs) and glucose tolerance tests (GTTs) were performed to evaluate insulin resistance (IR). Under standard diet, the response to both tests was similar in wild-type and PPAR-α–null mice. Under high-fat diet, however, the efficiency of insulin in ITT was reduced and the amount of hyperglycemia in GTT was increased only in wild-type and not in PPAR-α–null mice. The IR index, calculated as the product of the areas under glucose and insulin curves in GTT, increased fourfold in high-fat–fed wild-type mice, whereas it remained unchanged in PPAR-α–null mice. In contrast, PPAR-α deficiency allowed the twofold rise in adiposity and blood leptin levels elicited by the diet. Thus, the absence of PPAR-α dissociates IR from high-fat diet–induced increase in adiposity. The effects of PPAR-α deficiency on glucose homeostasis seem not to occur via the pancreas, because glucose-stimulated insulin secretion of islets was not influenced by the PPAR-α genotype. These data suggest that PPAR-α plays a role for the development of IR in response to a Western-type high-fat diet.
Plasma Glucose–Lowering Effect of Tramadol in Streptozotocin-Induced Diabetic Rats
Authors: Cheng Juei-Tang , Liu I-Min , Chi Tzong-Cherng , Tzeng Thing-Fong , Lu Feng-Hwa , Chang Chih Jen
Published: March 2007

The effect of tramadol on the plasma glucose level of streptozotocin (STZ)-induced diabetic rats was investigated. A dose-dependent lowering of plasma glucose was seen in the fasting STZ-induced diabetic rats 30 min after intravenous injection of tramadol. This effect of tramadol was abolished by pretreatment with naloxone or naloxonazine at doses sufficient to block opioid μ-receptors. However, response to tramadol was not changed in STZ-induced diabetic rats receiving p-chlorophenylalanine at a dose sufficient to deplete endogenous 5-hydroxytrptamine (5-HT). Therefore, mediation of 5-HT in this action of tramadol is ruled out. In isolated soleus muscle, tramadol enhanced the uptake of radioactive glucose in a concentration-dependent manner. The stimulatory effects of tramadol on glycogen synthesis were also seen in hepatocytes isolated from STZ-induced diabetic rats. The blockade of these actions by naloxone and naloxonazine indicated the mediation of opioid μ-receptors. The mRNA and protein levels of the subtype 4 form of glucose transporter in soleus muscle were increased after repeated treatments for 4 days with tramadol in STZ-induced diabetic rats. Moreover, similar repeated treatments with tramadol reversed the elevated mRNA and protein levels of phosphoenolpyruvate carboxykinase in the liver of STZ-induced diabetic rats. These results suggest that activation of opioid μ-receptors by tramadol can increase the utilization of glucose and/or decrease hepatic gluconeogenesis to lower plasma glucose in diabetic rats lacking insulin.
Genome-Wide Linkage Analysis Assessing Parent-of-Origin Effects in the Inheritance of Type 2 Diabetes and BMI in Pima Indians
Authors: Lindsay Robert S. , Kobes Sayuko , Knowler William C. , Bennett Peter H. , Hanson Robert L.
Published: March 2007

We examined the hypothesis that imprinted genes may affect the propensity to type 2 diabetes and obesity in Pima Indians. Multipoint variance component methods were used to assess linkage of BMI (kg/m2) and age-adjusted diabetes to loci derived from either father (LODFA) or mother (LODMO) in a genome-wide scan. Tentative evidence of loci where imprinted genes might be acting was found for diabetes with maternally derived alleles on chromosomes 5 (LODMO = 1.5) and 14 (LODMO = 1.6). Evidence of linkage of BMI to maternally derived alleles was found on chromosome 5 (LODMO = 1.7) and to paternally derived alleles on chromosome 10p (LODFA = 1.7). Additional analyses of sibling pairs who were affected by diabetes and younger than 25 years of age showed an increase of sharing of maternally derived alleles on chromosome 6 (LODMO = 3.0). We also examined sites of a priori interest where action of imprinted genes has been proposed in diabetes or obesity. We found no evidence of parent-specific linkage (of either diabetes or BMI) on chromosome 11p, a region that contains several imprinted genes, but observed weak evidence of linkage of diabetes to paternally derived alleles (LODFA = 0.9) in the region of chromosome 6q, believed to contain an exclusively paternally expressed gene or genes that cause transient neonatal diabetes mellitus. In conclusion, we determined regions of interest on chromosomes 5, 6, and 10 where imprinted genes might be affecting the risk of type 2 diabetes or obesity in Pima Indians.
PKCδ Activation
Authors: Shen Shlomzion , Alt Addy , Wertheimer Efrat , Gartsbein Marina , Kuroki Toshio , Ohba Motoi , Braiman Liora , Sampson Sanford R. , Tennenbaum Tamar
Published: March 2007

Insulin and insulin-like growth factor-1 (IGF-1) are members of the family of the insulin family of growth factors, which activate similar cellular downstream pathways. In this study, we analyzed the effects of insulin and IGF-1 on the proliferation of murine skin keratinocytes in an attempt to determine whether these hormones trigger the same signaling pathways. Increasing doses of insulin and IGF-1 promote keratinocyte proliferation in an additive manner. We identified downstream pathways specifically involved in insulin signaling that are known to play a role in skin physiology; these include activation of the Na+/K+ pump and protein kinase C (PKC). Insulin, but not IGF-1, stimulated Na+/K+ pump activity. Furthermore, ouabain, a specific Na+/K+ pump inhibitor, abolished the proliferative effect of insulin but not that of IGF-1. Insulin and IGF-1 also differentially regulated PKC activation. Insulin, but not IGF-1, specifically activated and translocated the PKCδisoform to the membrane fraction. There was no effect on PKC isoforms α,η, ϵ, and ζ, which are expressed in skin. PKCδoverexpression increased keratinocyte proliferation and Na+/K+ pump activity to a degree similar to that induced by insulin but had no affect on IGF-1—induced proliferation. Furthermore, a dominant negative form of PKCδ abolished the effects of insulin on both proliferation and Na+/K+ pump activity but did not abrogate induction of keratinocyte proliferation induced by other growth factors. These data indicate that though insulin or IGF-1 stimulation induce keratinocyte proliferation, only insulin action is specifically mediated via PKCδ and involves activation of the Na+/K+ pump.
Prolonged Islet Graft Survival in NOD Mice by Blockade of the CD40-CD154 Pathway of T-Cell Costimulation
Authors: Molano R. Damaris , Berney Thierry , Li Hua , Cattan Pierre , Pileggi Antonello , Vizzardelli Caterina , Kenyon Norma S. , Ricordi Camillo , Burkly Linda C. , Inverardi Luca
Published: March 2007

Allorejection and recurrence of autoimmunity are the major barriers to transplantation of islets of Langerhans for the cure of type 1 diabetes in humans. CD40-CD154 (CD40 ligand) interaction blockade by the use of anti-CD154 monoclonal antibody (mAb) has shown efficacy in preventing allorejection in several models of organ and cell transplantation. Here we report the beneficial effect of the chronic administration of a hamster anti-murine CD154 mAb, MR1, in prolonging islet graft survival in NOD mice. We explored the transplantation of C57BL/6 islets into spontaneously diabetic NOD mice, a combination in which both allogeneic and autoimmune components are implicated in graft loss. Recipients were treated either with an irrelevant control antibody or with MR1. MR1 administration was effective in prolonging allograft survival, but did not provide permanent protection from diabetes recurrence. The autoimmune component of graft loss was studied in spontaneously diabetic NOD mice that received syngeneic islets from young male NOD mice. In this combination, a less dramatic yet substantial delay in diabetes recurrence was observed in the MR1-treated recipients when compared with the control group. Finally, the allogeneic component was explored by transplanting C57BL/6 islets into chemically induced diabetic male NOD mice. In this setting, long-term graft survival (> 100 days) was achieved in MR1-treated mice, whereas control recipients rejected their grafts within 25 days. In conclusion,chronic blockade of CD154 results in permanent protection from allorejection and significantly delays recurrence of diabetes in NOD mice.
Physiological Increase in Plasma Leptin Markedly Inhibits Insulin Secretion In Vivo
Authors: Cases Jane A. , Gabriely Ilan , Ma Xiao Hui , Yang Xiao Man , Michaeli Tamar , Fleischer Norman , Rossetti Luciano , Barzilai Nir
Published: March 2007

The demonstration of leptin receptors on the pancreatic β-cells suggests the possibility of direct actions of leptin on insulin secretion. In vitro studies on islets or perfused pancreas and β-cell lines produced inconsistent results. We performed an in vivo study to distinctly examine whether leptin has an effect on glucose-stimulated insulin secretion. Young chronically catheterized Sprague-Dawley rats (n = 28) were subjected to a 4-h hyperglycemic clamp study (∼ 11 mmol/l). At minute 120 to 240,rats were assigned to receive either saline or leptin (0.1, 0.5, and 5 μg· kg-1 · min) infusion. Leptin decreased plasma insulin levels abruptly, and an approximately twofold decrease in plasma insulin levels compared with saline control was sustained over the 2 h of the study (14.8 ± 5.8 vs. 34.8 ± 2.6 ng/ml with leptin and saline infusion, respectively, P < 0.001). Moreover, a dose-dependent decrease in plasma insulin levels was noted (r = -0.731, P< 0.01). Since milrinone, an inhibitor of cAMP phosphodiesterase (PDE) 3,did not reverse the effect of leptin on glucose-induced insulin secretion, its action may be independent of PDE3. These findings suggest that acute physiological increase in plasma leptin levels acutely and significantly inhibits glucose-stimulated insulin secretion in vivo. The site of leptin effects on insulin secretion remains to be determined.
Cyclic Stretch and Hypertension Induce Retinal Expression of Vascular Endothelial Growth Factor and Vascular Endothelial Growth Factor Receptor—2
Authors: Suzuma Izumi , Hata Yasuaki , Clermont Allen , Pokras Frank , Rook Susan L. , Suzuma Kiyoshi , Feener Edward P. , Aiello Lloyd Paul
Published: March 2007

Systemic hypertension exacerbates diabetic retinopathy and other coexisting ocular disorders through mechanisms that remain largely unknown. Increased vascular permeability and intraocular neovascularization characterize these conditions and are complications primarily mediated by vascular endothelial growth factor (VEGF). Because systemic hypertension increases vascular stretch, we evaluated the expression of VEGF, VEGF-R2 (kinase insert domain-containing receptor [KDR]), and VEGF-R1 (fms-like tyrosine kinase[Flt]) in bovine retinal endothelial cells (BRECs) undergoing clinically relevant cyclic stretch and in spontaneously hypertensive rat (SHR) retina. A single exposure to 20% symmetric static stretch increased KDR mRNA expression 3.9 ± 1.1-fold after 3 h (P = 0.002), with a gradual return to baseline within 9 h. In contrast, BRECs exposed to cardiac-profile cyclic stretch at 60 cpm continuously accumulated KDR mRNA in a transcriptionally mediated, time-dependent and stretch-magnitude—dependent manner. Exposure to 9% cyclic stretch increased KDR mRNA expression 8.7 ±2.9-fold (P = 0.011) after 9 h and KDR protein concentration 1.8± 0.3-fold (P = 0.005) after 12 h. Stretched-induced VEGF responses were similar. Scatchard binding analysis demonstrated a 180 ±40% (P = 0.032) increase in high-affinity VEGF receptor number with no change in affinity. Cyclic stretch increased basal thymidine uptake 60± 10% (P < 0.001) and VEGF-stimulated thymidine uptake by 2.6 ± 0.2-fold (P = 0.005). VEGF-NAb reduced cyclic stretch—induced thymidine uptake by 65%. Stretched-induced KDR expression was not inhibited by AT1 receptor blockade using candesartan. Hypertension increased retinal KDR expression 67 ± 42% (P <0.05) in SHR rats compared with normotensive WKY control animals. When hypertension was reduced using captopril or candesartan, retinal KDR expression returned to baseline levels. VEGF reacted similarly, but Flt expression did not change. These data suggest a novel molecular mechanism that would account for the exacerbation of diabetic retinopathy by concomitant hypertension, and may partially explain the principal clinical manifestations of hypertensive retinopathy itself. Furthermore, these data imply that anti-VEGF therapies may prove therapeutically effective for hypertensive retinopathy and/or ameliorating the deleterious effects of coexistent hypertension on VEGF-associated disorders such as diabetic retinopathy.
beta-cell genes and diabetes: quantitative and qualitative differences in the pathophysiology of hepatic nuclear factor-1alpha and glucokinase mutations.
Authors: Pearson E R , Velho G , Clark P , Stride A , Shepherd M , Frayling T M , Bulman M P , Ellard S , Froguel P , Hattersley A T
Published: March 2007

Mutations in the beta-cell genes encoding the glycolytic enzyme glucokinase (GCK) and the transcription factor hepatocyte nuclear factor (HNF)-1alpha are the most common causes of maturity-onset diabetes of the young (MODY). Studying patients with mutations in these genes gives insights into the functions of these two critical beta-cell genes in humans. We studied 178 U.K. and French MODY family members, including 45 GCK mutation carriers and 40 HNF-1alpha mutation carriers. Homeostasis model assessment of fasting insulin and glucose showed reduced beta-cell function in both GCK (48% controls, P<0.0001) and HNF-1alpha (42% controls, P<0.0001). Insulin sensitivity was similar to that of control subjects in the GCK subjects (93% controls, P = 0.78) but increased in the HNF-1alpha subjects (134.5% controls, P = 0.005). The GCK patients showed a similar phenotype between and within families with mild lifelong fasting hyperglycemia (fasting plasma glucose [FPG] 5.5-9.2 mmol/l, interquartile [IQ] range 6.6-7.4), which declined slightly with age (0.017 mmol/l per year) and rarely required pharmacological treatment (17% oral hypoglycemic agents, 4% insulin). HNF-1alpha patients showed far greater variation in fasting glucose both between and within families (FPG 4.1-18.5 mmol/l, IQ range 5.45-10.4), with a marked deterioration with age (0.06 mmol/l per year), and 59% of patients required treatment with tablets or insulin. Proinsulin-to-insulin ratios are increased in HNF-1alpha subjects (29.5%) but not in GCK (18.5%) subjects. In an oral glucose tolerance test, the 0- to 120-min glucose increment was small in GCK patients (2.4+/-1.8 mmol/l) but large in HNF-1alpha patients (8.5+/-3.0 mmol/l, P< 0.0001). This comparison shows that the clear clinical differences in these two genetic subgroups of diabetes reflect the quantitative and qualitative differences in beta-cell dysfunction. The defect in GCK is a stable defect of glucose sensing, whereas the HNF-1alpha mutation causes a progressive defect that alters beta-cell insulin secretion directly rather than the sensing of glucose.
Sialylated form of the neural cell adhesion molecule (NCAM): a new tool for the identification and sorting of beta-cell subpopulations with different functional activity.
Authors: Bernard-Kargar C , Kassis N , Berthault M F , Pralong W , Ktorza A
Published: March 2007

To clarify the relationship between variations in beta-cell mass and pancreatic function, we investigated the possibility to analyze, quantify, and sort beta-cell subpopulations with different functional maturity. To this aim, we tested the reliability of the sialylated form of neural cell adhesion molecule (NCAM) (PSA-NCAM) as a marker of beta-cell functional activity. Islet cells isolated from adult rats were analyzed for their PSA-NCAM abundance using an anti-PSA-NCAM antibody. We found that PSA-NCAM is expressed only in beta-cells. The PSA-NCAM labeling was also studied with a fluorescence-activated cell sorter. We showed that the beta-cell population is heterogeneous for PSA-NCAM labeling. To directly determine the relationship between PSA-NCAM labeling and beta-cell activity, in vitro insulin secretion studies were performed on sorted beta-cell subpopulations using a perifusion technique. Two beta-cell subpopulations were analyzed: one that was highly labeled for PSA-NCAM and another that was poorly labeled. Insulin secretion from high PSA-NCAM-labeled beta-cells was significantly higher than that in low PSA-NCAM-labeled beta-cells. This differential expression in the beta-cell population was well correlated with differences in glucose responsiveness. PSA-NCAM seems thus suitable for use as a tool to identify beta-cell subpopulations according to their glucose responsiveness.
Effects of serum from patients with type 1 diabetes on primary cerebellar granule cells.
Authors: Chandra J , Yang S N , Köhler M , Zaitsev S , Juntti-Berggren L , Berggren P O , Zhivotovsky B , Orrenius S
Published: March 2007

Type 1 diabetes is an autoimmune disease of unknown etiology. Our previous work has shown that a factor present in serum from type 1 diabetic patients causes increased Ca2+ channel activity and apoptotic DNA fragmentation in pancreatic beta-cells. Here we examined the effects of type 1 diabetic serum on primary cerebellar granule cells (CGCs). In CGCs, exposure to type 1 diabetic serum did not cause increased apoptosis or changes in Ca2+ channel activity. However, patient serum did cause modulation of Ca2+ signals in a cell type with triangular soma that exhibited low voltage-gated Ca2+ currents. This cell was present primarily in cultures exposed to type 1 diabetic serum. The presence of low voltage-gated Ca2+ currents and long neuronal dendrites indicated that this unique cell was of neuronal origin and not of glial origin.
Efficient Ex Vivo Transduction of Pancreatic Islet Cells With Recombinant Adeno-Associated Virus Vectors
Authors: Flotte Terence , Agarwal Anupam , Wang Jianming , Song Sihong , Fenjves Elizabeth S. , Inverardi Luca , Chesnut Kye , Afione Sandra , Loiler Scott , Wasserfall Clive , Kapturczak Matthias , Ellis Tamir , Nick Harry , Atkinson Mark
Published: March 2007

The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islet cells may allow enhanced posttransplantation survival of islet allografts and inhibition of recurrent autoimmune destruction of these cells in type 1 diabetes. However, transient transgene expression and the tendency to induce host inflammatory responses have limited previous gene delivery studies using viral transfer vectors. We demonstrate here that recombinant adeno-associated virus (rAAV) serotype 2, a vector that can overcome these limitations, effectively transduces both human and murine pancreatic islet cells with reporter genes as well as potentially important immunoregulatory cytokine genes (interleukin-4, interleukin-10), although a very high multiplicity of infection (10,000 infectious units/islet equivalent) was required. This requirement was alleviated by switching to rAAV serotype 5, which efficiently transduced islets at a multiplicity of infection of 100. Although adenovirus (Ad) coinfection was required for efficient ex vivo expression at early time points, islets transduced without Ad expressed efficiently when they were transplanted under the renal capsule and allowed to survive in vivo. The rAAV-delivered transgenes did not interfere with islet cell insulin production and were expressed in both β- and non–β-cells. We believe rAAV will provide a useful tool to deliver therapeutic genes for modulating immune responses against islet cells and markedly enhance long-term graft survival.
Multipotential Nestin-Positive Stem Cells Isolated From Adult Pancreatic Islets Differentiate Ex Vivo Into Pancreatic Endocrine, Exocrine, and Hepatic Phenotypes
Authors: Zulewski Henryk , Abraham Elizabeth J. , Gerlach Melissa J. , Daniel Philip B. , Moritz Wolfgang , Müller Beat , Vallejo Mario , Thomas Melissa K. , Habener Joel F.
Published: March 2007

The endocrine cells of the rat pancreatic islets of Langerhans, including insulin-producing β-cells, turn over every 40–50 days by processes of apoptosis and the proliferation and differentiation of new islet cells (neogenesis) from progenitor epithelial cells located in the pancreatic ducts. However, the administration to rats of islet trophic factors such as glucose or glucagon-like peptide 1 for 48 h results in a doubling of islet cell mass, suggesting that islet progenitor cells may reside within the islets themselves. Here we show that rat and human pancreatic islets contain a heretofore unrecognized distinct population of cells that express the neural stem cell–specific marker nestin. Nestin-positive cells within pancreatic islets express neither the hormones insulin, glucagon, somatostatin, or pancreatic polypeptide nor the markers of vascular endothelium or neurons, such as collagen IV and galanin. Focal regions of nestin-positive cells are also identified in large, small, and centrolobular ducts of the rat pancreas. Nestin-positive cells in the islets and in pancreatic ducts are distinct from ductal epithelium because they do not express the ductal marker cytokeratin 19 (CK19). After their isolation, these nestin-positive cells have an unusually extended proliferative capacity when cultured in vitro (∼8 months), can be cloned repeatedly, and appear to be multipotential. Upon confluence, they are able to differentiate into cells that express liver and exocrine pancreas markers, such as α-fetoprotein and pancreatic amylase, and display a ductal/endocrine phenotype with expression of CK19, neural-specific cell adhesion molecule, insulin, glucagon, and the pancreas/duodenum specific homeodomain transcription factor, IDX-1. We propose that these nestin-positive islet-derived progenitor (NIP) cells are a distinct population of cells that reside within pancreatic islets and may participate in the neogenesis of islet endocrine cells. The NIP cells that also reside in the pancreatic ducts may be contributors to the established location of islet progenitor cells. The identification of NIP cells within the pancreatic islets themselves suggest possibilities for treatment of diabetes, whereby NIP cells isolated from pancreas biopsies could be expanded ex vivo and transplanted into the donor/recipient.
Glucokinase Gene Locus Transgenic Mice Are Resistant to the Development of Obesity-Induced Type 2 Diabetes
Authors: Shiota Masakazu , Postic Catherine , Fujimoto Yuka , Jetton Thomas L. , Dixon Kathryn , Pan Danhua , Grimsby Joseph , Grippo Joseph F. , Magnuson Mark A. , Cherrington Alan D.
Published: March 2007

Transgenic mice that overexpress the entire glucokinase (GK) gene locus have been previously shown to be mildly hypoglycemic and to have improved tolerance to glucose. To determine whether increased GK might also prevent or diminish diabetes in diet-induced obese animals, we examined the effect of feeding these mice a high-fat high–simple carbohydrate low-fiber diet (HF diet) for 30 weeks. In response to this diet, both normal and transgenic mice became obese and had similar BMIs (5.3 ± 0.1 and 5.0 ± 0.1 kg/m2 in transgenic and nontransgenic mice, respectively). The blood glucose concentration of the control mice increased linearly with time and reached 17.0 ± 1.3 mmol/l at the 30th week. In contrast, the blood glucose of GK transgenic mice rose to only 9.7 ± 1.2 mmol/l at the 15th week, after which it returned to 7.6 ± 1.0 mmol/l by the 30th week. The plasma insulin concentration was also lower in the GK transgenic animals (232 ± 79 pmol/l) than in the controls (595 ± 77 pmol/l), but there was no difference in plasma glucagon concentrations. Together, these data indicate that increased GK levels dramatically lessen the development of both hyperglycemia and hyperinsulinemia associated with the feeding of an HF diet.