bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulitis-Caused Redistribution of Heat-Shock Protein HSP60 Inside β-cells Correlates With Induction of HSP60 Autoantibodies
Authors: Brudzynski Katrina
Published: September 2013

We recently reported that sera of NOD mice contain autoantibodies to the endogenous HSP60. Under physiological conditions, the HSP60 antigen was located to secretory granules and mitochondria of β-cells, but in islets affected by insulitis, the antigen accumulated rapidly in cytoplasm and on β-cell surface membranes. The relationship was examined between insulitis-dependent redistribution of the HSP60 inside β-cells and induction of the HSP60 autoantibodies. By use of quantitative immunoelectron microscopy and monoclonal antibodies to human HSP60, changes were monitored in levels of the cytoplasmic HSP60 with progression of insulitis. Development of insulitis was accompanied by reduction of the HSP60 levels in secretory granules and increased levels in the cytoplasm. Mean concentration of cytoplasmic HSP60 in 4-, 7-, and 13-wk-old NOD mice representing 0, 85, and 100% incidence of insulitis, gradually increased from 4.6 to 11.2 to 35.9 immunogold grains/μm2, respectively, and this increase was statistically significant (P < 0.001). Simultaneously, the time course of an appearance of HSP60 antibodies was established by testing sera of > 4- to 25-wk-old NOD mice, for their cross-reactivity to human recombinant HSP60 on Western blots. HSP60 antibodies developed in both sexes between 7 and 13 wk of age but were found with the highest frequency in sera of 10- to 15-wk-old mice. Antibodies declined rapidly with the onset of diabetes. HSP60 antibodies were not observed in sera of 4-wk-old NOD mice devoid of insulitis or in 7-wk-old males with delayed onset of insulitis. The cytoplasmic HSP60 levels in these cases were the same as in control mice. These data suggest that insulitis is a causative factor in the HSP60 redistribution, and that this HSP60 redistribution inside the β-cells is temporarily associated with the induction of the humoral autoimmune response to the HSP60.
Identification of a Simple Tandem Repeat DNA Polymorphism in the Human Glycogen Synthase Gene and Linkage to Five Markers on Chromosome 19q
Authors: Vionnet Nathalie , Bell Gtaeme I
Published: September 2013

We have identified a simple tandem repeat DNA polymorphism in the human glycogen synthase gene of the form (TG)n. This DNA polymorphism has 10 alleles and a heterozygosity of 0.82 and can be easily typed using the polymerase chain reaction. It has been localized within the framework genetic map of chromosome 19 and is located in the region of the apolipoprotein C-II and histidine-rich calcium-binding protein genes. This DNA polymorphism will facilitate genetic studies of the role of the glycogen synthase gene in the development of insulin resistance and NIDDM.
Pancreatic β-Cell–Selective Production of Tumor Necrosis Factor-α Induced by Interleukin-1
Authors: Yamada Kentaro , Takane Naoko , Otabe Shuichi , Inada Chizuko , Inoue Masahiro , Nonaka Kyohei
Published: September 2013

Cytokines have been regarded as effector molecules responsible for +-cell death and major histocompatibility complex hyperexpression in endocrine pancreas of type I diabetes. However, the mechanism that results in +-cell–selective destruction has not been elucidated. We demonstrated in this study, using cell lines of transformed mouse +-cells and α-cells, that only pancreatic +-cells but not α-cells produced tumor necrosis factor-α when exposed to interleukin-1 +. Northern blot analysis confirmed the +-cell-selective expression of tumor necrosis factor-α mRNA. Interleukin-1 + also provoked tumor necrosis factor-α mRNA expression in vitro by normal mouse islet cells. Because tumor necrosis factor-α has been shown to potentiate +-cell cytotoxicity of interleukin-1 and interferon-gamma, tumor necrosis factor-α produced in situ by +-cells might be self-destructive. In fact, a low dose of interleukin-1 + in combination with a low dose of interferon-gamma preferentially injured +-cells. Hence endogenous tumor necrosis factor-α production by +-cells may be involved in +-cell–selective destruction in type 1 diabetes.
Effects of Hyperinsulinemia on Muscle Fiber Composition and Capillarization in Rats
Authors: Holmäng Agneta , Brzezinska Zofia , Björntorp Per
Published: September 2013

Statistical studies repeatedly have shown an association between systemic insulin resistance and a preponderance of highly glycolytic, relatively insulin-insensitive muscle fibers as well as a low density of muscle capillaries. The nature of the relationship between these observations is, however, not clear. Female rats were made hyperinsulinemic for 7 days by implantation of osmotic minipumps. Elevated adrenergic activity and secretion of glucocorticoids were controlled by another minipump with propranolol and adrenalectomy was controlled with glucocorticoid substitution. This resulted in hyperinsulinemia and moderate hypoglycemia, the latter probably counteracted by overeating and increased glucagon secretion, as indicated by increased body weight and lower liver glycogen contents, respectively. Systemic insulin sensitivity was increased and measured with a hyperinsulinemic-euglycemic clamp technique. This was paralleled by an elevated glucose utilization estimated as uptake of 2-deoxyglucose in parametrial, retroperitoneal, and inguinal adipose tissues and the soleus and extensor digitorum longus muscles. Glycogen synthesis was also elevated in the soleus muscle. Muscle fiber composition changed with hyperinsulinemia and elevated 2-deoxyglucose uptake toward more fast-twitch, type II, particularly type IIb fibers, whereas the proportion of slow-twitch, type I fibers, diminished. Capillary density was elevated per unit muscle surface area as well as per muscle fiber. This was paralleled by increased insulin sensitivity systemically and in muscles. These results suggest that muscle fiber composition alterations may be a consequence rather than a cause of hyperinsulinemia and that capillarization rather than fiber composition is of importance for insulin sensitivity in muscle.
Association of HLA-A24 With Complete β-Cell Destruction in IDDM
Authors: Nakanishi Koji , Kobayashi Tetsuro , Murase Toshio , Nakatsuji Takako , Inoko Hidetoshi , Tsuji Kimiyoshi , Kosaka Kinori
Published: September 2013

A sensitive C-peptide immunoreactivity radioimmunoassay demonstrated the presence of subtle, but definite residual β-cell function in patients with IDDM of long duration. Although HLA antigens are known to influence susceptibility to IDDM, their contribution to the extent of pancreatic β-cell destruction has not yet been examined extensively. We studied the relationship between residual β-cell function and HLA class I and class II antigens in 111 unrelated Japanese IDDM patients. Using the sensitive C-peptide immunoreactivity radioimmunoassay, the presence or absence of residual β-cell function was evaluated by the C-peptide immunoreactivity response to a 100-g oral glucose load. DNA typing for HLA-DQA1 and HLA-DQB1 antigens was performed in addition to serological typing of HLA-A, HLA-B, HLA-C, and HLA-DR antigens. A C-peptide immunoreactivity response > 0.033 nM was regarded as an indication of the presence of residual β-cell function, not the assay error. Surprisingly, 35 of 37 (94.6%) patients without residual β-cell function had HLA-A24, whereas only 39 of 74 (52.7%) patients with residual β-cell function had this antigen (corrected P = 9.795 × 10(–6). Any other HLA antigens, including the DR and DQ loci, showed no difference in the frequency with regard to residual β-cell function. The duration of diabetes was similar between the groups with and without residual β-cell function. The duration of diabetes was similar between the groups with and without residual β-cell function. Even when the patients were stratified according to the duration of diabetes, HLA-A24 was more common in those with early complete loss of β-cell function (duration of diabetes <1 yr) (P = 0.035) and was less common in those with residual β-cell function despite a long duration of diabetes (>10 yr) (P = 0.001). The correlation between HLA-A24 positivity and complete β-cell loss also was confirmed in younger-onset (<30 yr old) and elder-onset (≥30 yr old) groups. The C-peptide immunoreactivity response in patients with HLA-A24 (0.09 ± 0.02 nM, mean ± SE, n = 74) was significantly lower than that in patients without HLA-A24 (0.19 ± 0.03 nM, n = 37, P < 5.0 × 10−5). Further typing of HLA-A24 by one-dimensional isoelectric focusing gel electrophoresis revealed that the isoelectric point of HLA-A24 was identical in charge in 17 of 18 patients and 7 normal control subjects (isoeletric point 6.32, HLA-A24.1). We conclude that a specific HLA class I antigen, HLA-A24, promotes pancreatic p-cell destruction in IDDM patients with other disease-susceptible HLA antigens.
Differential Regulation of GLUT1 and GLUT4 Glucose Transporters in Skeletal Muscle of a New Model of Type II Diabetes: The Obese SHR/N-cp Rat
Authors: Marette André , Atgié Claude , Liu Zhi , Bukowiecki Ludwik J , Klip Aamira
Published: September 2013

The obese diabetic SHR/N-cp rat is a newly developed strain that inherits obesity as an autosomal recessive trait. These rats display early-onset hyperinsulinemia and hyperglycemia, which are hallmarks of type II diabetes. This study was undertaken to determine the expression and the subcellular distribution of the GLUT1 and GLUT4 glucose transporters in skeletal muscle of obese diabetic SHR rats. D-glucose-protectable cytochalasin-B binding to subcellular membrane fractions of hindlimb muscles was used to determine glucose transporter number. GLUT1 and GLUT4 glucose transporter isotypes were detected using antibodies to the COOH-terminal region of the GLUT1 and GLUT4 proteins. Glucose transporter number was significantly lower (–40%) in crude unfractionated membranes of obese diabetic SHR than of lean SHR muscles. When crude membranes were fractionated to separate plasma membranes and the intracellular membranes containing glucose transporters, the number of cytochalasin-B binding sites was found to be markedly lower (–50%) in intracellular membranes and slightly but not significantly reduced (–20%) in plasma membranes of muscle from obese diabetic SHR compared with lean SHR rats. Western blot analysis revealed that a lower GLUT4 protein abundance (–40%) accounts for the reduced glucose transporter number in intracellular membranes of obese diabetic SHR compared with lean SHR muscles. GLUT4 protein content was also reduced by 50% in plasma membranes from obese SHR muscles relative to lean rat muscles. In contrast, GLUT1 glucose transporters were more abundant (40d%) in plasma membranes of obese diabetic SHR rats compared with their lean counterparts. The latter finding may explain the lack of phenotypic difference in plasma membrane total glucose transporter number as measured by cytochalasin-B binding. GLUT4 and GLUT1 mRNA levels were both found to be significantly decreased in obese SHR rats compared with their lean littermates. These results demonstrate that 1) GLUT4 transporter expression is reduced in skeletal muscle of obese diabetic SHR rats, a model of type II diabetes; 2) the decreased GLUT4 abundance was localized to both the sarcolemmal and intracellular membrane fractions; 3) in contrast to the GLUT4 glucose transporter, GLUT1 protein content was greater in plasma membranes of skeletal muscle from obese diabetic SHR rats, suggesting that these transporter isoforms are inversely regulated in muscle of these diabetic animals; and 4) posttranscriptional mechanisms appear to be responsible for the differential expression of GLUT1 and GLUT4 proteins in muscle of obese diabetic SHR rats.
Impaired Arteriolar Myogenic Reactivity in Early Experimental Diabetes
Authors: Hill Michael A , Meininger Gerald A
Published: September 2013

Hyperperfusion and an increase in capillary pressure has been implicated in the pathogenesis of diabetic microangiopathy. The existence of such alterations suggests that the myogenic response to increased intravascular pressure may be altered in diabetes. To examine this, in vivo studies were performed on the rat cremaster muscle microcirculation of age-matched control and STZ-induced (65 mg/kg) diabetic rats (3–4 wk of diabetes). Anesthetized rats were enclosed in an airtight Plexiglas box with the cremaster muscle exteriorized into an organ bath containing Krebs’ solution. To study myogenic responsiveness, box pressure was increased in steps of 10 mmHg from 0 to 30 mmHg for 2 min. Third-order arterioles of the control animals (lumen diameter 18 ± 2 μm) responded to increased pressure with a rapid onset vasoconstriction. In contrast, the rate of development of the constriction was markedly attenuated in similar vessels (15 ± 1 μm) of the diabetic animals, despite their ability to exhibit a similar maximal arteriolar constriction to that of the control animals. When 20 mmHg pressure steps were applied for only 10 s, arterioles of the diabetic animals constricted minimally, whereas those of the control animals constricted to 75% of the maximal response expected for that pressure increase (P < 0.01). Second-order arterioles of both groups of animals responded with a primarily passive distension to increased intravascular pressure suggesting that the impaired responsiveness of the third-order arterioles is not compensated for by an increase in the myogenic responsiveness of upstream vessels. Basal intravascular pressures, measured in first-, second-, and third-order arterioles, were similar in control and diabetic animals. These data suggest that experimental diabetes is associated with an impairment in the rate of development of myogenic vasoconstriction in precapillary arterioles that may predispose the capillary bed to transient episodes of elevated pressure.
Relationship of Proinsulin and Insulin to Cardiovascular Risk Factors in Nondiabetic Subjects
Authors: Haffner Steven M , Mykkänen Leena , Stern Michael P , Valdez Rodolfo A , Heisserman Judith A , Bowsher Ronald R
Published: September 2013

Recent data suggest that proinsulin is strongly associated with cardiovascular risk factors in diabetic subjects. However, this relationship has not been examined in nondiabetic subjects. Therefore, we examined the relation of proinsulin to lipids, obesity (body mass index), and waist-to-hip ratio in 260 nondiabetic individuals from the San Antonio Heart Study, a population-based study of diabetes and cardiovascular disease. Proinsulin was measured by radioimmunoassay, and insulin was measured by a Linco radioimmunoassay that does not cross-react with proinsulin. Fasting insulin was significantly associated with body mass index (0.42), waist-to-hip ratio (r = 0.30), triglyceride (r = 0.29), high-density lipoprotein cholesterol (r = −0.20), and systolic blood pressure (r = 0.16) but not significantly related to diastolic blood pressure (r = 0.11). Fasting proinsulin was significantly associated with body mass index (r = 0.19), waist-to-hip ratio (r = 0.25), triglyceride (r = 0.41), systolic blood pressure (r = 0.19), and diastolic blood pressure (r = 0.15). Proinsulin was more strongly related to increased triglyceride than insulin despite its weaker relationship to obesity. In multivariate analyses, proinsulin continued to be significantly related to triglyceride concentrations (explaining 23.1% of the variance) and to systolic blood pressure (explaining 4.0% of the variance), even after adjusting for insulin. These observations suggest that proinsulin should be measured in addition to insulin in epidemiological studies. Proinsulin may be a marker for metabolic decompensation in prediabetic subjects.
Inositol Glycan Phosphate Derived from Human Erythrocyte Acetylcholinesterase Glycolipid Anchor and Inositol Cyclic 1,2-Phosphate Antagonize Glucagon Activation of Glycogen Phosphorylase
Authors: Deeg Mark A , Brass Eric P , Rosenberry Terrone L
Published: September 2013

In this study we examine the hypothesis that an inositol glycan phosphate can act similarly to insulin on intact cells. The inositol glycan phosphate used in this study (glycan α) was isolated previously from the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase and was shown to have the structure glycine-ethanolamine-PO4-Man-Man-(N,N-dimethylethanolamine-PO4)Man- (N,N-dimethyl)GlcN-inositol-PO4. The cellular response investigated was the glucagon-stimulated activation of glycogen phosphorylase in rat hepatocytes. When hepatocytes were incubated with 20 nM glucagon for 4 min, the ratio of phosphorylase a activity to total phosphorylase increased from a basal value of 0.49 ± 0.02 to 0.82 ± 0.03 (mean ± SE, n = 15). Inclusion of either 100 nM insulin or 3–10 μM glycan α during the glucagon incubation significantly decreased the glucagon-stimulated activity ratio to 0.74 ± 0.03 for either agent. Furthermore, hepatocyte preparations differed in their response to insulin and were divided into insulin-responsive and -resistant groups. Glycan α had a significant effect only in the insulin-responsive group for which the observed activity ratio for 10 microM glycan α plus glucagon (0.68 ± 0.05) compared closely with that for insulin plus glucagon (0.70 ± 0.04). For the insulin-resistant group, the activity ratio in the presence of 10 μM glycan α was 0.81 ± 0.03, unchanged from the control with glucagon alone. Because glycan α contains an inositol phosphate group, the effect of inositol cyclic 1,2-phosphate on the glucagon-stimulated activity ratio was determined. Inositol cyclic 1,2-phosphate at 1–10 μM significantly decreased the activity ratio to the same extent as insulin in seven insulin-responsive preparations. Glycan a and inositol cyclic 1,2-phosphate differed from insulin in eliciting no decrease in the 8-bromoadenosine cyclic 3′-5′-monophosphatestimulated phosphorylase activity ratio in hepatocytes. The results indicate that glycan α and inositol cyclic 1,2-phosphate can mimic some effects of insulin on intact hepatocytes.
Pre-Exposure to Glucosamine Induces Insulin Resistance of Glucose Transport and Glycogen Synthesis in Isolated Rat Skeletal Muscles: Study of Mechanisms in Muscle and in Rat-1 Fibroblasts Overexpressing the Human Insulin Receptor
Authors: Robinson Kathrine A , Sens Donald A , Buse Maria G
Published: September 2013

Increased routing of glucose through the hexosamine-biosynthetic pathway has been implicated in the development of glucose-induced insulin resistance of glucose transport in cultured adipocytes. Because both glucosamine and glucose enter this pathway as glucosamine-6-phosphate, we examined the effects of preincubation with glucosamine in isolated rat diaphragms and in fibroblasts overexpressing the human insulin receptor (HIR-cells). In muscles, pre-exposure to glucosamine inhibited subsequent basal and, to a greater extent, insulin-stimulated glucose transport in a time- and dose-dependent manner and abolished the stimulation by insulin of glycogen synthesis. Insulin receptor number, activation of the insulin receptor tyrosine kinase in situ and after solubilization, and the total pool of glucose transporters (GLUT4) were unaffected, and glycogen synthase was activated by glucosamine pretreatment. In HIR-cells, which express GLUT1 and not GLUT4, basal and insulin-stimulated glucose transport were unaffected by glucosamine, but glycogen synthesis was markedly inhibited. Insulin-stimulated activation of protein kinases (MAP and S6) was unaffected, and the fractional velocity and apparent total activity of glycogen synthase was increased in glucosamine-treated HIR-cells. In pulse-labeling studies, addition of glucosamine during the chase prolonged processing of insulin proreceptors to receptors and altered the electrophoretic mobility of proreceptors and processed α-subunits, consistent with altered glycosylation. Glucosamine-induced insulin resistance of glucose transport appears to be restricted to GLUT4-expressing cells, i.e., skeletal muscle and adipocytes; it may reflect impaired translocation of GLUT4 to the plasmalemma. The glucosamine-induced imbalance in UDP sugars, i.e., increased UDP-N-acetylhexosamines and decreased UDP-glucose, may alter glycosylation of critical proteins and limit the flux of glucose into glycogen.
Activation of Protein Kinase C in Glomerular Cells in Diabetes: Mechanisms and Potential Links to the Pathogenesis of Diabetic Glomerulopathy
Authors: Derubertis Frederick R , Craven Patricia A
Published: September 2013

Protein kinase C (PKC) is activated in rat renal glomerulus within a week of induction of experimental diabetes. Studies in isolated glomeruli and in cultured endothelial and mesangial cells have demonstrated that high ambient concentrations of glucose activate PKC and thus implicate hyperglycemia per se as a mediator of PKC activation in glomerular cells in diabetes. High glucose concentrations activate PKC by increasing cellular levels of diacylglycerol (DAG), the major endogenous modulator of this signalling system. In contrast to physiological extracellular stimuli of PKC that increase cellular DAG levels by receptor-mediated enhancement of membrane inositol phospholipid hydrolysis, in glomerular cells high concentrations of glucose increase DAG by de novo synthesis from glycolytic intermediates. Activation of PKC by glucose or other agonists increases the permeability of endothelial cells to albumin and stimulates matrix protein synthesis in mesangial cells; it thereby may be involved in the pathogenesis of both the functional and structural alterations of the glomerulus in diabetes. Recent studies in isolated glomeruli from diabetic rats have also implicated activation of PKC in suppression of nitric oxide (NO)-mediated increases in glomerular cGMP generation in response to cholinergic stimuli. In mesangial cells, cGMP suppresses PKC-mediated increases in matrix protein synthesis. Thus, impaired NO-mediated cGMP generation in glomeruli of diabetic individuals may amplify matrix protein synthesis in response to hyperglycemia and other stimuli of PKC. These and other observations suggest that activation of the PKC system by hyperglycemia may represent an important pathway by which glucotoxicity is transduced in susceptible cells in diabetes.
A New Transgenic Mouse Model of Chronic Hyperglycemia
Authors: Schaefer Erik M , Viard Valérie , Morin Joëlle , Ferré Pascal , Pénicaud Luc , Ramos Purita , Maika Shanna D , Ellis Leland , Hammer Robert E
Published: September 2013

Expression under the control of the mouse transferrin promoter of a transgene encoding a soluble secreted derivative of the ectodomain of the human insulin receptor in transgenic mice results in the accumulation of this high-affinity insulin-binding protein in the plasma. Alterations of glucose homeostasis are observed including postabsorptive hyperglycemia concomitant with increased hepatic glucose production and hyperinsulinemia. Thus, this is the first transgenic animal model of chronic hyperglycemia with alterations in glucose homeostasis that are produced without a targeted alteration of pancreatic function. These mice provide a new experimental model to follow the progression and long-term consequences of chronic hyperglycemia.
Associations of Anti-GAD Antibodies with Islet Cell Antibodies and Insulin Autoantibodies in First-Degree Relatives of Type I Diabetic Patients
Authors: Roll Ursula , Christie Michael R , Standl Eberhard , Ziegler Anette-G
Published: September 2013

Sera from 114 first-degree relatives of insulin-dependent diabetes mellitus (type I diabetes) patients and 81 healthy individuals living in Germany were analyzed for antibodies to rat brain glutamic acid decarboxylase (GAD-ab) using an immunoprecipitation assay. The determination of GAD-ab in the 81 islet cell antibody (ICA) and insulin autoantibody (IAA) negative healthy individuals established a normal range (mean ± 2 SD); 2 healthy individuals (2.5%) possessed GAD-ab levels above this range, but became negative on follow-up. None of 86 ICA−/IAA− first-degree relatives had GAD-ab; whereas, 42.9% of 28 ICA+ and/or IAA+ relatives were positive for GAD-ab. Presence of GAD-ab was negatively correlated with IAA (P = 0.02) and positively with ICA (P = 0.0006). Follow-up samples were obtained from 25 of 28 ICA+ and/or IAA+ relatives with a mean (± SD) follow-up period of 20.6 ± 12.1 months. In these 25 relatives, GAD-ab were positive in 70% ICA+/IAA−, 0% ICA−/IAA+, and 57.1% ICA+/IAA+ relatives in the first sample and in 57.1% ICA+/IAA−, 0% ICA−/IAA+, and 70% ICA+/IAA+ relatives in the last sample. GAD-ab, once detected, persisted in 9 of 11 GAD-ab+ relatives. Of the relatives, 2 converted to GAD-positivity, concomitant with the appearance of ICA, and 2 others lost GAD-ab during follow-up. Of the 28 ICA+ and/or IAA+ relatives, 6 progressed to overt type I diabetes on follow-up, and GAD-ab were detectable in 4 of these relatives. In conclusion, this study indicates that GAD-ab in first-degree relatives are significantly correlated with ICA, are independent of the appearance of IAA, and are present in 66.7% of relatives who subsequently developed type I diabetes.
GAD Autoantibodies in IDDM, Stiff-Man Syndrome, and Autoimmune Polyendocrine Syndrome Type I Recognize Different Epitopes
Authors: Björk Elisabeth , Velloso Licio A , Kämpe Olle , Karlsson F Anders
Published: September 2013

Glutamic acid decarboxylase (GAD) is a major islet cell autoantigen in insulin-dependent diabetes mellitus (IDDM), and autoantibodies are found in high frequencies in patients with recent-onset IDDM, stiff-man syndrome (SMS), and autoimmune polyendocrine syndrome type I (APS I). Antigens in autoimmune disorders are often enzymes, and autoantibody binding frequently inhibit their activity. In this study, we examined the reactivity of anti-GAD–containing sera from 7 patients with IDDM, 4 patients with SMS, and 5 patients with APS I. All sera immunoprecipitated GAD from [35S]methionine-labeled rat islet lysates and the sera from patients with SMS and APS I, but none of the IDDM patients’ sera, identified the GAD protein in Western blots. Two of four SMS patients’ sera and 5 of 5 APS I patients’ sera, in contrast to 0 of 7 IDDM patients’ sera, inhibited the enzymatic activity of GAD. When the various sera were tested with the GAD65 and GAD67 isoforms, produced separately by transient expression in COS cells, the enzymatic activity of GAD65 was inhibited by sera from patients with SMS and APS I, whereas no effect on the GAD67 activity was observed. Taken together, the results demonstrate that the GAD autoantibodies in these three disorders display marked differences in epitope recognition and indicate that, during the development of the diseases, the autoantigen is being presented to the immune system through separate pathogenetic mechanisms.
ADP Ribosylation by Cholera Toxin Identifies Three G-Proteins That Are Activated by the Galanin Receptor: Studies With RINm5F Cell Membranes
Authors: Gillison Susan L , Sharp Geoffrey W G
Published: September 2013

Inhibition of insulin secretion by galanin is pertussis toxin (PTX) sensitive, suggesting the activation of one or more heterotrimeric (α, β, γ) G-proteins (Gi/Go). Multiple effectors, including the K+ATP and L-type Ca2+ channels, adenylyl cyclase, and an as yet unidentified system at a site close to exocytosis, are modulated by galanin. Therefore, it is necessary to delineate the particular G-proteins activated by the galanin receptor as a first step to understanding its net cellular response. During specific conditions, cholera toxin (CTX) can ADP-ribosylate the αi/αo-subunits of the PTX-sensitive substrates but only during receptor/G-protein interaction. Therefore, we used CTX-catalyzed A DP ribosylation to identify galanin receptor-associated G-protein α-subunits in RINmSF cells. Galanin enhanced the A DP ribosylation of membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in two bands at 39,000 and 42,000 Mr This labeling was blocked in membranes prepared from PTX-treated cells, enhanced by Mg2+, and showed a biphasic dependence on exogenous guanine nucleotides. Identification of the CTX ADP-ribosylated G-proteins by immunoprecipitation with selective antisera indicate activation by the galanin receptor of αi1, and αi3, which have the same mobility on SDS-PAGE (42,000 Mr), and αi2 (39,000 Mr). These studies provide evidence for the activation of multiple G-proteins by receptors for galanin in RINmSF cells.
Growth Hormone Induces Resistance to the Mitogenic Action of Insulin Through Local IGF-I: Studies in Normal and Pygmy T-Cell Lines
Authors: Geffner Mitchell E , Bersch Noelle , Bailey Robert C , Golde David W
Published: September 2013

Growth hormone (GH) and insulin have both mitogenic and metabolic actions. The growth-promoting effects of GH in vivo are thought to be mediated by insulin-like growth factor-I (IGF-I), whereas the metabolic effects of GH are thought to be either direct or mediated by factors other than IGF-I. In previous studies using HTLV-II-transformed T-lymphoblast cell lines established from normal individuals, we have shown that GH preincubation induces resistance to the growth-promoting (mitogenic) action of insulin. In this study, using T-cell lines from 3 American control subjects, 1 African control subject, and 1 African Pygmy (the latter previously shown to be resistant to the growth-promoting actions of both IGF-I and GH), we examined the role of local IGF-I in the mediation of GH-induced resistance to the mitogenic action of insulin. In these studies, we quantified the stimulation of T-cell colony formation in response to insulin in the presence and absence of either GH or IGF-I. We found that 1) GH or IGF-I preincubation of normal T-cell lines completely blunts mitogenic responsiveness to subsequent stimulation with insulin (all P < 0.001 versus either no GH or no IGF-I); 2) pretreatment with a monoclonal antibody against the IGF-I receptor blocks both GH- and IGF-I-induced resistance to the mitogenic action of insulin in normal T-cell lines (both P = NS versus insulin alone); and 3) neither GH nor IGF-I preincubation induces resistance to the growth-promoting action of insulin in the Pygmy T-cell line (P = 0.37 and P = 0.74, respectively, versus insulin alone). We conclude from these data that 1) GH-induced resistance to the mitogenic action of insulin is mediated by local IGF-I, and 2) the Pygmy T-cell line does not develop insulin resistance in response to GH or IGF-I treatment because of a primary variation in IGF-I receptor function.
Autoantibodies to Glutamic Acid Decarboxylase in Patients with IDDM and Autoimmune Thyroid Disease
Authors: Kawasaki Eiji , Takino Hirofumi , Yano Mayumi , Uotani Shigeo , Matsumoto Kazunari , Takao Yukio , Yamaguchi Yoshihiko , Akazawa Shoichi , Nagataki Shigenobu
Published: September 2013

Autoantibodies to glutamic acid decarboxylase (GAD), previously reported to be the 64,000-Mr (64K) islet cell protein, were measured by a radioimmunoassay using purified pig brain GAD in 29 insulin-dependent diabetes mellitus (IDDM) patients with autoimmune thyroid disease (AITD) and in 29 sex- and disease duration-matched IDDM patients without AITD. Islet cell antibodies (ICAs) and 64K antibodies were also determined. In IDDM patients with short-duration diabetes (<1 year), the prevalence and levels of GAD antibodies were 100% (8 of 8) and 609 ± 166 U (means ± SE), respectively, in IDDM patients with AITD and 81.8% (9 of 11) and 90 ± 51 U, respectively, in patients without AITD. In patients with long-standing IDDM (3–22 years), the prevalence and levels of GAD antibodies were 76.2% (16 of 21) and 193 ± 66 U, respectively, in patients with AITD and 50.0% (9 of 18) and 36 ± 14 U, respectively, in patients without AITD. For up to 6 years after the onset of IDDM, the levels of GAD antibodies in IDDM patients with AITD were significantly higher than in IDDM patients without AITD. A close and significant correlation was found between GAD antibodies and ICA or 64K antibodies in IDDM patients with AITD. Our results demonstrate that high levels of GAD antibodies were present in IDDM patients with AITD. The observed differences in GAD immunoreactivity between IDDM patients with and without AITD might help evaluate the role of GAD antibodies in IDDM.
Role of Endothelium-Derived Nitric Oxide in the Pathogenesis of the Renal Hemodynamic Changes of Experimental Diabetes
Authors: Komers Radko , Allen Terri J , Cooper Mark E
Published: September 2013

To evaluate the role of nitric oxide (NO) in diabetic hyperfiltration, renal hemodynamic changes and changes in urinary excretion of NO2/NO3 in response to the NO inhibitor nitro-L-arginine methyl ester (L-NAME) and the NO-donating agent glyceryl trinitrate (GTN) were investigated in conscious streptozocin-induced diabetic (D) and age-matched control (C) rats. In all experiments, D rats demonstrated increased glomerular filtration rate (GFR), renal plasma flow (RPF), polyuria, and an increased urinary sodium excretion when compared with C rats. An intravenous bolus of low-dose L-NAME (1 mg/kg body wt) increased modestly systolic blood pressure (sBP) in C rats but had no effect on sBP in D rats. L-NAME induced a marked decrease in GFR and RPF in D rats with no change in filtration fraction (FF). In C rats, no change in GFR was observed, and RPF decreased, resulting in a rise in FF. A supramaximal dose of L-NAME (10 mg/kg body wt) increased sBP in C and D rats to a similar degree. With high-dose L-NAME, GFR decreased in D but not in C rats. There was a greater decrease in RPF in D rats when compared with C animals. An intravenous infusion of GTN induced a modest decrease in sBP in both C and D rats (P < 0.01). There were no changes in GFR and RPF in D rats, but in the C group, GTN increased RPF (P < 0.05) with a tendency for a rise in GFR (P = 0.09). Basal urinary NO2/NO3 excretion was increased in D rats in all experiments. A decrease in urinary NO2/NO3 levels was observed after low-dose L-NAME in D rats, with a similar trend after high-dose L-NAME. NO such changes were observed in C rats. GTN infusion had no effect on urinary NO2/NO3 excretion in D rats but increased urinary NO2/NO3 levels in C rats to levels that were not statistically different from those in D rats. These studies suggest that increased renal production and/or sensitivity to endothelium-derived relaxing factor/NO may play a role in the genesis of diabetic hyperfiltration.
Ambulatory Blood Pressure in the Transition from Normo- to Microalbuminuria: A Longitudinal Study in IDDM Patients
Authors: Poulsen Per L , Hansen Klavs W , Mogensen Carl E
Published: September 2013

To describe the development in blood pressure (BP) in relation to urinary albumin excretion (UAE) more exactly, 44 initially normoalbuminuric type I diabetic patients and 21 healthy individuals were included in a 3.1-year follow-up study by using ambulatory BP (AMBP) monitoring. Six patients developed microalbuminuria according to accepted criteria (progressors; UAE at follow-up was >20 μg/min). Initial UAE was higher in this group (9.0 ×/÷ by 1.4 μg/min) compared with both the nonprogressors (5.2 ×/÷ by 1.6 μg/min) and the control subjects (3.9 ×/÷ by 1.6 μg/min), P < 0.01. The values were almost identical for initial 24-h AMBP between the progressors and the two other groups. The transition to microalbuminuria (31.7 ×/÷ by 1.8 μg/min) was associated with an increase in 24-h systolic AMBP of 11.5 ± 8.3 mmHg, which was significantly higher than the increase in the nonprogressors (3.1 ± 7.7 mmHg) and the control subjects (2.2 ± 6.1 mmHg, P = 0.02). Significant correlations were detected between development in UAE and development in systolic and diastolic 24-h AMBP (r = 0.39, r = 0.41, P < 0.01). In addition, an increase in UAE, even including increases within the normoalbuminuric range, was always associated with an increase in 24-h AMBP (P < 0.01). Ordinary clinical measurements did not reveal any of these differences or correlations. In conclusion, a close association between increases in UAE and 24-h AMBP emerges in this study. Initial BP was not increased in the progressors. microalbuminuria is associated with significant BP increases but only when AMBP monitoring is analyzed.
Normalization of Diacylglycerol-Protein Kinase C Activation by Vitamin E in Aorta of Diabetic Rats and Cultured Rat Smooth Muscle Cells Exposed to Elevated Glucose Levels
Authors: Kunisaki Makoto , Bursell Sven-Erik , Umeda Fumio , Nawata Hajime , King George L
Published: September 2013

Hyperglycemia and diabetes have been shown to increase diacylglycerol (DAG) level and activate protein kinase C (PKC) activity in the vascular tissues, possibly altering vascular function. We have characterized the effects of D-α-tocopherol (vitamin E) on PKC activities and DAG levels in rat aortic smooth muscle cells (ASMCs) cultured with elevated glucose levels as well as in the vascular tissues obtained from control and diabetic rats. In ASMCs, the specific PKC activity from the membraneous fraction and total DAG level were increased by 31 ± 4% (P < 0.05) and 50 ± 7% (P < 0.05), respectively, when the glucose levels were changed from 5.5 to 22 mmol/1. The addition of D-α-tocopherol and another lipophilic antioxidant, probucol, prevented the glucose-stimulated increases in DAG level and PKC activity. By immunoblotting studies, D-α-tocopherol treatment was able to reduce the enhancement of PKC (ill isoform in the membraneous fraction isolated from ASMCs. Comparing streptozotocin-induced diabetic rats with their nondiabetic controls, both membraneous-specific PKC activities and total cellular DAG levels were increased in aorta by 162% (P < 0.05) and 60% (P < 0.05), respectively. Intraperitoneal injection of D-α-tocopherol (40 mg/kg) every other day prevented the increases in membraneous-specific PKC activities and total DAG levels in parallel with a significant increase of D-α-tocopherol contents in the aorta and plasma. These findings have demonstrated that D-α-tocopherol can prevent the activation of PKC activities in the vascular cells and tissues induced by hyperglycemia by lowering DAG levels, possibly via its antioxidant effect.
Metabolic Effects of Troglitazone on Fructose-Induced Insulin Resistance in the Rat
Authors: Lee Moon-Kyu , Miles Philip D G , Khoursheed Mousa , Gao Ke-Ming , Moossa A R , Olefsky Jerrold M
Published: September 2013

Troglitazone is a new orally active hypoglycemic agent that has been shown to reduce insulin resistance and hyperinsulinemia in both diabetic animal models and non-insulin-dependent diabetes mellitus (NIDDM) subjects. To determine whether this drug could prevent the development of fructose-induced insulin resistance and related abnormalities, we studied the effects of troglitazone on the insulin resistance induced by fructose feeding in rats. Normal male Sprague-Dawley rats were fed a high-fructose diet for 3 weeks with and without troglitazone as a food admixture (0.2%) or were fed normal chow to serve as a control group. In vivo insulin resistnace was measured by the euglycemic hyperinsulinemic clamp technique at two different insulin infusion rates, 29 (submaximal stimulation) and 290 (maximal stimulation) pmol.kg−1 · min−1. Fructose feeding markedly reduced submaximal glucose disposal rate (GDR) (113.8 ± 8.3 vs. 176.0 ± 5.6 µmol.kg−1 · min−1 P < 0.05) and maximal GDR (255.9 ± 5.6 vs. 313.6 ± 10.5 µmol.kg−1 · min−1 P < 0.05), reduced the suppressibility of submaximal hepatic glucose production (HGP; 45.5 ± 5.0 vs. 11.7 ± 5.0 µmol.kg−1 · min−1 P < 0.05), and resulted in hypertriglyceridemia and hypertension. Troglitazone treatment completely restored the GDR (submaximal 158.2 ± 5.6, maximal 305.3 ± 6.1 µmol.kg−1 · min−1) and submaximal HGP (9.4 ± 2.8 µmol.kg−1 · min−1) to control levels and also normalized the elevated plasma triglyceride concentration and systolic blood pressure levels in fructose-fed rats. These results suggest that troglitazone treatment could completely prevent the insulin resistance, hypertension, and hypertriglyceridemia induced by a diet high in fructose and that the drug might prove useful in the treatment and/or prevention of nonhyperglycemic insulin-resistant states as well as in the treatment of established NIDDM.
Relationship Between Hepatic Glucose Production and Fasting Plasma Glucose Concentration in Patients With NIDDM
Authors: Jeng Chii-Yuan , Sheu Wayne H-H , Fuh Martin M-T , Chen Y-D Ida , Reaven Gerald M
Published: September 2013

This study was initiated to reevaluate the changes in basal hepatic glucose production (HGP) rate that occur in patients with non-insulin-dependent diabetes mellitus (NIDDM). Measurements were made in 51 volunteers: 18 with normal glucose tolerance and 33 with newly diagnosed NIDDM of varying degrees of severity. To avoid the methodological problems associated with quantifying HGP over short time periods, using non-steady-state isotopic kinetics, radiolabeled glucose was infused for a 12-h period, from 10 P.M. to 10 A.M. with HGP quantified from 9 to 10 A.M.. The results showed that fasting plasma glucose (FPG) concentration and HGP were significantly correlated (r = 0.68, P < 0.001) in patients with NIDDM. However, when the 33 patients with NIDDM were divided into three groups of 11 each on the basis of FPG concentration, it became clear that the relationship between FPG and HGP was complex. Thus, values for HGP in patients with NIDDM and FPG < 180 mg/dl were not higher than in the normal population (1.67 ± 0.07 vs. 1.69 ± 0.04 mg.kg−1 · min−1, NS). Significant increases (P < 0.01) in HGP above normal were seen in the 11 patients with NIDDM and FPG concentrations between 180 and 250 mg/dl (2.05 ± 0.07 mg.kg−1 · min−1), as well as in those with FPG > 250 mg/dl (2.18 ± 0.13 mg.kg−1 · min−1). Although those with the highest FPG concentrations tended to have the greatest values for HGP, the difference between the latter two groups of patients with NIDDM was not statistically significant. Finally, HGP rates in the 11 patients with FPG concentrations > 250 mg/dl were only 29% higher than values in the control population. These data indicate that HGP rates determined under steady-state isotopic conditions are not higher than normal until FPG concentration is >180 mg/dl, and that the increase in HGP is still relatively modest in magnitude (∼29%) in patients with the most severe degree of fasting hyperglycemia (FPG >250 mg/dl).
Prevalence of Mutations in the Insulin Receptor Gene in Subjects With Features of the Type A Syndrome of Insulin Resistance
Authors: Moller David E , Cohen Ohad , Yamaguchi Yoshihiko , Assiz Richardo , Grigorescu Florin , Eberle Andrea , Morrow Linda A , Moses Aorrow C , Flier Jeffrey S
Published: September 2013

Mutations of the insulin receptor gene are a cause of the type A syndrome of extreme insulin resistance. This study assessed the prevalence of such mutations in women with clinical features of the type A syndrome including ovarian hyperandrogenism, moderate-to-severe degrees of insulin resistance, and acanthosis nigricans. We studied 22 unrelated women with insulin resistance (fasting insulin >300 pM [50 μU/ml] and/or peak during an oral glucose tolerance test (OGTT) > 1,800 pM [300 μU/ml]), acanthosis nigricans, and the polycystic ovary syndrome (hyperandrogenemia, oligoamenorrhea, and hirsutism). Two insulin-resistant probands with congenital generalized lipodystrophy and one male proband with severe insulin resistance also were included in the study. Southern blotting experiments were performed to exclude gross gene deletions, insertions, or rearrangements. Exons 2–22 of the insulin receptor gene were polymerase chain reaction (PCR) amplified from genomic DNA and screened for nucleotide variation using single-strand conformation polymorphism (SSCP). No nucleotide variation between study subjects was detected in exons 4–6, 10–12, 15, 16, 18, 19, or 21. Sequencing of amplified DNA revealed that SSCP variants in exons 2, 3, 8, 9, and 17 corresponded to known silent polymorphisms within the coding region. Variants in exons 2, 9, 13, and 14 were caused by novel silent polymorphisms; variants in exons 7 and 22 were caused by nucleotide substitutions in flanking introns. One proband was found to have a heterozygous point mutation in exon 20 (CGG→CAG, Arg1174→Gln) that involves the intracellular receptor β-subunit. Gin1174 is a novel mutant of the insulin receptor tyrosine kinase domain and is a likely cause of dominantly inherited insulin resistance. The mutation was present in an affected sister but was absent in the unaffected mother and 64 normal alleles. Two paternal aunts also are reportedly affected. The results of this study suggest that mutations at the insulin receptor locus are uncommon in insulin-resistant women with acanthosis nigricans and ovarian hyperandrogenism.
Insulin Degradation In Vitro and In Vivo: A Comparative Study in Men: Evidence That Immunoprecipitable, Partially Rebindable Degradation Products Are Released From Cells and Circulate in Blood
Authors: Benzi Luca , Cecchetti Paolo , Ciccarone Aannamaria , Pilo Alessandro , Di Cianni Graziano , Navalesi Renzo
Published: September 2013

The products of insulin metabolism generated in vitro and in vivo were compared in this study. Monocytes from 10 control subjects were incubated with 125IA14-labeled insulin, acid washed, and solubilized or reincubated in insulin-free binding buffer to study both intracellular radioactivity or radioactivity released from cells to medium. To evaluate in vivo insulin metabolism, labeled insulin (100–120 μCi) was injected as a single intravenous bolus in 5 of the 10 subjects. Cellular and plasma radioactivity was characterized by high-performance liquid chromatography (HPLC). The results of the study show the following: 1) Products with superimposable HPLC elution profiles are found within cells and in medium. Two new labeled products are observed in the latter, suggesting that a membrane degradation process exists in monocytes. 2) Intermediates found within monocytes, in medium from monocytes, and in plasma have identical elution profiles, supporting the possibility that insulin is metabolized in various cells by a common pathway. 3) Insulin metabolism produces intermediates that bind well to anti-insulin antibody. The presence in plasma of these products induces a significant difference in the value of the metabolic clearance rate of insulin when HPLC or immunoprecipitation is used to detect intact insulin. 4) Immunoprecipitable products maintain, in part, the capacity to bind to insulin receptors and to be internalized into monocytes.
Search for a Third Susceptibility Gene for Maturity-Onset Diabetes of the Young: Studies With Eleven Candidate Genes
Authors: Vaxillaire Martine , Vionnet Nathalie , Vigouroux Corinne , Sun Fang , Espinosa Rafael , Lebeau Michelle M , Stoffel Markus , Lehto Markku , Beckmann Jacques S , Detheux Michel , Passa Philippe , Cohen Daniel , Schaftingen Emile Van , Velho Gilberto , Bell Graeme I , Froguel Philippe
Published: September 2013

Maturity-onset diabetes of the young (MODY) is a model for genetic studies of non-insulin-dependent diabetes mellitus. We have identified 15 MODY families in which diabetes is not the result of mutations in the glucokinase gene. This cohort of families will be useful for identifying other diabetes-susceptibility genes. Nine other candidate genes potentially implicated in insulin secretion or insulin action have been tested for linkage with MODY in these families, including glucokinase regulatory protein, hexokinase II, insulin receptor substrate 1, fatty acid–binding protein 2, glucagon-like peptide-1 receptor, apolipoprotein C-II, glycogen synthase, adenosine deaminase (a marker for the MODY gene on chromosome 20), and phosphoenolpyruvate carboxykinase. None of these loci showed evidence for linkage with MODY, implying that mutations in these genes do not make a major genetic contribution to the development of MODY. In addition to these linkage analyses, one or two affected subjects from each family were screened for the presence of the A to G mutation at nucleotide 3,243 of the mitochondrial tRNALeu(UUR) gene. This mutation was not found in any of these subjects. Finally, we report the localization of the gene encoding the regulatory protein of glucokinase to chromosome 2, band p22.3 and the identification of a restriction fragment length polymorphism at this locus.