bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial FAD-Glycerophosphate Dehydrogenase and G-Protein—Coupled Inwardly Rectifying K+ Channel: No Evidence for Linkage in Maturity-Onset Diabetes of the Young or NIDDM
Authors: Warren-Perry Margaret G , Stoffel Markus , Saker Philip J , Zhang Yun , Brown Louise J , MacDonald Michael J , Turner Robert C
Published: September 2013

Two genes that have potentially important regulatory roles in insulin secretion are both located on chromosome 2q24.1. G-protein–coupled muscarinic potassium channel (GIRK1) is an inwardly rectifying K+ channel that helps to maintain the resting potential and excitability of cells. Mitochondrial FAD-linked glycerophosphate dehydrogenase (m-GDH) catalyzes a rate-limiting step of the glycerol phosphate shuttle in pancreatic islets. Reduced m-GDH activity has been demonstrated in islets isolated from diabetic subjects compared with islets from nondiabetic control subjects and from the diabetic GK rat. To study the relationship between these candidate genes and NIDDM, we have examined a simple tandem-repeat polymorphism (STRP) close to both the KCN J3 (GIRK1) locus and the m-GDH locus. In a linkage study of three maturity-onset diabetes of the young (MODY) pedigrees, not linked to MODY1, MODY2, or MODY3, a cumulative score of −9.6 at a recombination fraction of θ = 0 excluded linkage. In a population-association study, no linkage disequilibrium for the STRP was found between 190 unselected NIDDM patients and 60 geographically and age-matched white nondiabetic subjects (χ2 = 1.51 on 3 df, P = 0.68). Thus, mutations involving the genes for GIRK1 or FAD-glycerophosphate dehydrogenase are unlikely to cause MODY, and a common mutation in either gene is unlikely to contribute to NIDDM in whites. These data do not exclude mutations in some families or other ethnic groups.
Absence of Mutations in the Human OB Gene in Obese/Diabetic Subjects
Authors: Maffei Margherita , Stoffel Markus , Barone Marisa , Moon Byoung , Dammerman Marilyn , Ravussin Eric , Bogardus Clifton , Ludwig David S , Flier Jeffrey S , Talley Matchugo , Auerbach Stephen , Friedman Jeffrey M
Published: September 2013

The product of the obese (ob) gene, leptin, is a secreted protein that is important in the regulation of body weight. Mice with mutations in the ob gene are obese and diabetic and manifest reduced physical as well as metabolic activity. In this study, we tested the possibility that mutations in the OB gene may contribute to human obesity. We report the isolation and partial sequence of the human OB gene and the screening of 105 obese patients for mutations in the protein coding sequence using the technique of single-strand conformational polymorphism. No coding sequence polymorphism was found, suggesting that mutations in the coding sequence of the OB gene do not constitute a common cause of increased body weight in humans. We also identified a highly polymorphic simple dinucleotide repeat DNA polymorphism in this gene that will be useful for genetic studies.
Extreme Obesity May Be Linked to Markers Flanking the Human OB Gene
Authors: Reed Danielle R , Ding Yuan , Xu Weizhen , Cather Corinne , Green Eric D , Price R Arlen
Published: September 2013

Mice with mutations of the ob gene are extremely obese, and the human homologue (OB) has been cloned and physically mapped. The protein product of the ob gene (leptin) reduces body fat in mice when given exogenously, and leptin has been proposed to provide a lipostatic signal that regulates adiposity. Variation in the OB gene may be one genetically determined cause of obesity in human populations. To test this hypothesis, we genotyped siblings from 78 families at markers flanking the human OB gene. Pairs of siblings with extreme obesity (BMI ≥ 40; n = 59) shared haplotypes identical-by-descent for the region containing the OB gene at greater than chance levels (corrected P = 0.04). Furthermore, one haplotype containing the OB gene was transmitted by heterozygous parents to extremely obese (BMI ≥40) offspring more frequently than expected by chance, indicting significant allelic disequilibrium (corrected P = 0.027). One explanation for these linkage findings is that some individuals with extreme obesity have an allelic variant of the OB gene, although other nearby genes could contribute to obesity in these families.
Plasma Leptin and Insulin Relationships in Obese and Nonobese Humans
Authors: Dagogo-Jack Samuel , Fanelli Carmine , Paramore Deanna , Brothers Joy , Landt Michael
Published: September 2013

Hyperinsulinemia is associated with an overexpression of mRNA for the ob protein leptin in rodent models of genetic obesity, and insulin has been reported to directly stimulate leptin mRNA in rat adipocytes. Human obesity is also associated with increased leptin mRNA as well as plasma levels, but there have been no reports of the effect of insulin on leptin secretion. We, therefore, tested the hypothesis that insulin stimulates leptin secretion in humans. Using a newly developed leptin assay, immunoreactive leptin was measured in fasting and postprandial plasma samples from 27 healthy adults and in samples before and during euglycemic-hyperinsulinemic then stepped hypoglycemic (hourly steps at 85, 75, 65, 55, and 45 mg/dl) clamps from 10 healthy subjects and 11 patients with IDDM. Plasma leptin was correlated (r = 0.84, P = 0.0005) with BMI in obese but not nonobese subjects and with fasting (r = 0.75, P = 0.008) but not postprandial plasma insulin levels. (Leptin levels did not change postprandially.) Euglycemic hyperinsulinemia did not alter leptin levels, nor did hyperinsulinemic hypoglycemia. Thus, because circulating leptin levels are not increased during postprandial hyperinsulinemia or during euglycemic (or hypoglycemic) hyperinsulinemia, we conclude that, at least in the short term, insulin does not increase leptin secretion in humans and that hyperleptinemia in obese individuals is not likely the result of hyperinsulinemia.
Increased Insulin Resistance and Insulin Secretion in Nondiabetic African-Americans and Hispanics Compared With Non-Hispanic Whites: The Insulin Resistance Atherosclerosis Study
Authors: Haffner Steven M , Ralph D'Agostino , Saad Mohammed F , Rewers Marian , Mykkänen Leena , Selby Joseph , Howard George , Savage Peter J , Hamman Richard F , Wegenknecht Lynne E , Bergman Richard N
Published: September 2013

The etiology of NIDDM is still controversial, with both insulin resistance and decreased insulin secretion postulated as potential important factors. African-Americans and Hispanics have a two- to threefold excess risk of developing NIDDM compared with non-Hispanic whites. Yet little is known concerning the prevalence of insulin resistance and secretion defects in minorities, especially in African-Americans in population-based studies. Fasting and 2-h post–glucose load glucose and insulin levels, insulin-mediated glucose disposal (insulin sensitivity index) (SI), glucose effectiveness (SG), and first-phase insulin response (acute insulin response [AIR]) were determined in nondiabetic African-Americans (n = 288), Hispanics (n = 363), and non-Hispanic whites (n = 435) as part of the Insulin Resistance Atherosclerosis Study. Subjects received a standard 2-h oral glucose tolerance test on the first day and an insulin-modified frequently sampled intravenous glucose tolerance test on the second day. African-Americans and Hispanics were more obese than non-Hispanic whites. Both African-Americans and Hispanics had higher fasting and 2-h insulin concentrations and AIR but lower SI than non-Hispanic whites. No ethnic difference was observed in SG. After further adjustments for obesity, body fat distribution, and behavioral factors, African-Americans continued to have higher fasting and 2-h insulin levels and AIR, but lower SI, than non-Hispanic whites. In contrast, after adjustment for these covariates, no significant ethnic differences in SI or fasting insulin levels were observed between Hispanics and non-Hispanic whites. Hispanics continued to havehigher 2-h insulin levels and AIRs than those in non-Hispanic whites. In this report, the association between SI and upper body adiposity (waist-to-hip ratio) was similar in each ethnic group. Both nondiabetic African-Americans and Hispanics have increased insulin resistance and higher AIR than nondiabetic non-Hispanic whites, suggesting that greater insulin resistance may be in large part responsible for the higher prevalence of NIDDM in these minority groups. However, in Hispanics, the greater insulin resistance may be due to greater adiposity and other behavioral factors.
Glucagon-Like Peptide I Receptors in the Subfornical Organ and the Area Postrema Are Accessible to Circulating Glucagon-Like Peptide I
Authors: Ørskov Cathrine , Poulsen Steen S , Mørten Morten , Holst Jens J
Published: September 2013

The intestinal incretin hormone glucagon-like peptide I (GLP-I) inhibits gastric motility and secretion in normal, but not in vagotomized subjects, pointing to a centrally mediated effect. Therefore, our aim was to study the availability of rat brain GLP-I receptors to peripherally injected 125I-labeled GLP-I. The specificity of the binding was tested by co-injection of excess amounts of unlabeled GLP-I. Using light microscopical autoradiography of rat brain sections, we found specific 125I-GLP-I binding exclusively in the subfornical organ and the area postrema. This binding was abolished when an excess amount of unlabeled GLP-I was co-injected with the labeled GLP-I. We conclude that cells in the subfornical organ and the area postrema could be responsive to blood-borne GLP-I. The observed binding of peripherally administered GLP-I to the subfornical organ and the area postrema, which both have close neuroanatomical connections with hypothalamic areas involved in water and appetite homeostasis, is consistent with the potential roles of circulating GLP-I in the central regulation of appetite and autonomic functions.
Effects of a Physiological Insulin Concentration on the Endothelin-Sensitive Ca2+ Store in Porcine Coronary Artery Smooth Muscle
Authors: Dick Gregory M , Sturek Michael
Published: September 2013

The effect of insulin to attenuate the Ca2+ and contractile response of vascular smooth muscle to a number of agonists has been described previously, but the Ca2+ regulatory mechanisms of insulin action remain unclear. We determined the effect of a physiological insulin concentration (300 pmol/L) on the Ca2+ response of vascular smooth muscle cells of the porcine right coronary artery to endothelin 1 (ET-1); furthermore, we examined the cellular Ca2+ stores affected by insulin (i.e., Ca2+ stores releasable by inositol 1,4,5-trisphosphate, caffeine, and ionomycin). We measured the Ca2+ responses of acutely isolated single smooth muscle cells with the fluorescent Ca2+ indicator Fura-2. Acute insulin exposure (20 min) significantly attenuated the Ca2+ response of single smooth muscle cells to 10 nmol/L ET-1. This inhibitory effect of insulin was observed both in the presence and absence of extracellular Ca2+. In contrast with the effects on ET-1-induced Ca2+ responses, insulin did not inhibit the Ca2+ response to 5 mmol/l caffeine, an agent that directly releases sarcoplasmic reticulum Ca2+ stores. Insulin was also without effect on the total cellular Ca2+ store released by 1 µmol/l ionomycin, a Ca2+-transporting ionophore. When ET-1 and caffeine were given in succession, a sizable caffeine-sensitive Ca2+ store could be released from insulin-treated cells but not control cells, indicating that the sarcoplasmic reticulum Ca2+ store of insulin-treated cells was not depleted by ET-1. Generalized depletion of the sarcoplasmic reticulum Ca2+ store is not one of the cellular mechanisms involved in the effect of insulin on coronary smooth muscle; instead, the effect may be due to an inhibitory influence on transmembrane signal transduction, such as diminished ET-1-induced inositol 1,4,5-trisphosphate production or reduced ability of this phosphoinositol to release stored Ca2+.
Effects of an Engineered Human Anti–TNF-α Antibody (CDP571) on Insulin Sensitivity and Glycemic Control in Patients With NIDDM
Authors: Ofei Francis , Hurel Steve , Newkirk José , Sopwith Mark , Taylor Roy
Published: September 2013

Inhibition of tumor necrosis factor (TNF)-α action has recently been shown to reverse insulin resistance dramatically and to improve glycemic control in obese rodents. This double-blind study was designed to assess the effects of a recombinant-engineered human TNF-α-neutralizing antibody (CDP571) on glucose homeostasis in obese NIDDM patients. Glycemic control and insulin sensitivity were monitored in 21 NIDDM subjects for a 2-week run-in and then for 6 weeks after treatment in a randomized fashion with a single intravenous dose of either CDP571 (5 mg/kg) or an equivalent volume of normal saline. The prolonged half-life of the antibody ensured adequate plasma levels as measured throughout the study. Concentrations of fasting glucose (CDP571: 10.0 ± 0.8, 10.1 ± 0.8, 10.0 ± 1.0; placebo: 8.5 ± 0.6, 8.1 ± 0.5, 8.7 ± 0.8 mmol/l at baseline, day 1, and week 4, respectively), fasting serum insulin (CDP571:21.2 ± 2.8,21.0 ± 2.8,24.8 ± 3.3; placebo: 19.0 ± 2.8, 20.8 ± 2.9, 17.5 ± 2.2 pmol/l, respectively), and C-peptide remained unaffected by the type of treatment throughout the study. The percentage rate of glucose clearance per minute (KITT) during intravenous insulin sensitivity tests was identical in the CDP571 and placebo groups at baseline and also at 1 and 4 weeks after treatment (mean ± SE; CDP571: 1.33 ± 0.21,1.44 ± 0.25,1.26 ± 0.18; placebo: 1.38 ± 0.15,1.47 ± 0.20, 1.52 ± 0.20;P=0.85, 0.93, and 0.36, respectively). TNF-α neutralization over a period of 4 weeks had no effect on insulin sensitivity in obese NIDDM subjects.Diabetes45:881–885, 1996
Prediction of Type I Diabetes in First-Degree Relatives Using a Combination of Insulin, GAD, and ICA512bdc/IA-2 Autoantibodies
Authors: Verge Charles F , Gianani Roberto , Kawasaki Eiji , Yu Liping , Pietropaolo Massimo , Chase H. Peter , Eisenbarth George S. , Jackson Richard A.
Published: September 2013

Islet cell antibodies (ICAs) are predictive of type I diabetes in first-degree relatives, but this immunohistochemical assay has proven difficult to standardize. As an alternative, we assessed the use of radioassays for antibodies against three molecularly characterized islet autoantigens, including ICA512bdc (amino acid residues 256–979 of the IA-2 molecule, incorporating the intracellular domain). We measured insulin autoantibodies (IAAs), GAD autoantibodies (GAAs), and ICA512bdc autoantibodies (ICA512bdcAAs) by radioassay, in addition to ICAs, in 882 first-degree relatives of patients with type I diabetes, 50 of whom later developed diabetes with a median follow-up of 2.0 years (maximum 11.3 years). The cutoff for each radioassay was determined by testing >200 control subjects. When autoantibody frequencies among the relatives were analyzed according to relationship to the proband, the offspring of diabetic fathers had a higher frequency of ICA512bdcAAs (P = 0.008), IAAs (P = 0.0001) and GAAs (P = 0.0001) than the offspring of diabetic mothers. ICA512bdcAAs and IAAs both showed a significant association with HLA-DR4-DQ8 (P = 0.0005). Among relatives developing diabetes, 98% had one or more of IAAs, GAAs, or ICA512bdcAAs, and 80% had two or more of these autoantibodies, compared with none of the control subjects. Using survival analysis to allow for different lengths of follow-up, there was a significant increase in the risk of diabetes with the number of these autoantibodies present, comparing zero, one, two, and three autoantibodies (P < 0.0001, log-rank test), and by Cox regression analysis, this was independent of ICAs and age. For relatives with two or more of these autoantibodies, the risk of diabetes within 3 years was 39% (95% CI, 27–52) and the risk within 5 years was 68% (95% CI, 52–84). Relatives with all three autoantibodies had a risk within 5 years estimated to be 100%. The presence of low first-phase insulin release further increased the risk for relatives with one or two autoantibodies. We conclude that the presence of two or more autoantibodies (out of IAAs, GAAs, and ICA512bdcAAs) is highly predictive of the development of type I diabetes among relatives.
Poor Capacity for Proliferation of Pancreatic β-Cells in Otsuka-Long-Evans-Tokushima Fatty Rat: A Model of Spontaneous NIDDM
Authors: Zhu Min , Noma Yoshihiko , Shima Kenji , Mizuno Akira , Sano Toshiaki
Published: September 2013

Otsuka-Long-Evans-Tokushima Fatty (OLETF) rat, a genetic model of spontaneous development of NIDDM, exhibits hyperglycemic obesity with hyperinsulinemia and insulin resistance similar to that in humans. It is still unclear whether a defect in the β-cell proliferation per se is the primary pathogenetic event in OLETF rat. To determine whether it is, we used partially pancreatectomized rats as a model, with administration of phlorizin to control blood glucose level, to examine whether the capacity for proliferation of P-cells during hyperglycemia or normoglycemia differs between OLETF and their diabetes-resistant counterparts, Long-Evans-Tokushima-Otsuka (LETO) rats. We also examined whether such a defect, if present, could be improved by nicotinamide. Male rats, 6 weeks of age, were allocated at random'to two groups: 70% pancreatectomy (Px) and sham-pancreatectomy (sham). Each group was divided into four subgroups by date of killing after surgery: 3-day, 7-day, 28-day (treated with phlorizin, nicotinamide, or saline), and 91-day. A sustained hyperglycemia was evident in the Px OLETF rats after surgery, which was associated with insufficient proliferation of β-cells, characterized by decrease in β-cell labeling index in proportion to decrease in β-cell mass and reduction in insulin content in the remnant pancreas. This defect was unaffected by restoration of normoglycemia induced by phlorizin injection. Administration of nicotinamide, however, ameliorated the sustained hyperglycemia by increasing (J-ceU proliferation. These findings suggest that OLETF rats have poor capacity for proliferation of pancreatic β-cells and that this change may be the critical pathogenetic event before the onset of overt diabetes.
Relationship Between Insulin Sensitivity and Plasma Leptin Concentration in Lean and Obese Men
Authors: Segal Karen R , Landt Michael , Klein Samuel
Published: September 2013

Alterations in the production of or the sensitivity to leptin, the protein encoded by the ob gene, cause obesity and diabetes in rodents. We evaluated the isolated relationship between leptin and insulin sensitivity in lean and obese humans. Three groups of subjects who were carefully matched for either insulin sensitivity (determined by the modified intravenous glucose tolerance test and minimal model analysis) or adiposity (determined by hydrodensitometry) were studied: 1) lean insulin-sensitive men (percentage body fat, 15 ± 1%); 2) lean insulinresistant men (percentage body fat, 16 ± 1%), matched on percentage body fat and fat mass with the lean insulinsensitive group; and 3) obese insulin-resistant men (percentage body fat, 31 ± 3), matched on insulin sensitivity with the lean insulin-resistant group. Basal plasma leptin concentrations were significantly lower in the lean insulin-sensitive than in the lean insulin-resistant men (1.90 ± 0.4 vs. 4.35 ± 1.21 ng/ml, P < 0.05) despite identical body composition. Plasma leptin in the obese men (9.27 ± 1.4 ng/ml) was significantly higher than values in the two lean groups (P < 0.01). Marked alterations in plasma glucose and insulin concentrations induced by glucose and tolbutamide injection did not cause any change in plasma leptin levels. These results demonstrate that insulin resistance is associated with elevated plasma leptin levels independent of body fat mass. However, plasma insulin itself does not acutely regulate leptin production.
Identification of Incidental Sequence Polymorphisms and Absence of the db/db Mouse and fa/fa Rat Mutations
Authors: Considine Robert V , Caro Jose F , Considine Eileen L , Williams Charlene J , Hyde Thomas M
Published: September 2013

Leptin-receptor gene expression in hypothalamic tissue from lean and obese humans was examined. The fulllength leptin receptor, that is believed to transmit the leptin signal, is expressed in human hypothalamus. There was no difference in the amount of leptin-receptor mRNA in seven lean (BMI 23.3 ± 0.9 kg/m2) and eight obese (BMI 36.9 ± 1.5) subjects as determined by reverse transcription-polymerase chain reaction. A sequence polymorphism (A→G) was detected at position 668 of the leptin receptor cDNA. This second base substitution changed a glutamine to an arginine at position 223 of the leptin receptor protein. Of 15 subjects analyzed, 11 were heterozygous for this base change and 3 were homozygous. The occurance of the polymorphic allele(s) did not correlate with BMI in the population studied. The mutation responsible for the defect in the leptin receptor in db/db mice was not detected in any obese human, nor was the fa/fa rat mutation. These results provide evidence that the leptin resistance observed in obese humans is not due to a defect in the leptin receptor.
Hexosamines and Insulin Resistance
Authors: McClain Donald A , Crook Errol D
Published: September 2013

Glucose is an important regulator of cell growth and metabolism. Thus, it is likely that some of the adverse effects of hyperglycemia are reflections of normal regulation by abnormal concentrations of glucose. How the cell senses glucose, however, is still incompletely understood. Evidence has been presented that the hexosamine biosynthesis pathway serves this function for regulation of aspects of glucose uptake, glycogen synthesis, glycolysis, and synthesis of growth factors. Excess hexosamine flux causes insulin resistance in cultured cells, tissues, and intact animals. Further evidence for the possible role of this pathway in normal glucose homeostasis and disease is that the level of activity of the rate-limiting enzyme in hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase, is correlated with glucose disposal rates (GDRs) in normal humans and transgenic mice.
The Antioxidant α-Lipoic Acid Enhances Insulin-Stimulated Glucose Metabolism in Insulin-Resistant Rat Skeletal Muscle
Authors: Jacob Stephan , Streeper Ryan S , Fogt Donovan L , Hokama Jason Y , Henriksen Erik J , Dietze Günther J , Tritschler Hans J
Published: September 2013

Insulin resistance of muscle glucose metabolism is a hallmark of NIDDM. The obese Zucker (fa/fa) rat—an animal model of muscle insulin resistance—was used to test whether acute (100 mg/kg body wt for 1 h) and chronic (5–100 mg/kg for 10 days) parenteral treatments with a racemic mixture of the antioxidant α-lipoic acid (ALA) could improve glucose metabolism in insulin-resistant skeletal muscle. Glucose transport activity (assessed by net 2-deoxyglucose [2-DG] uptake), net glycogen synthesis, and glucose oxidation were determined in the isolated epitrochlearis muscles in the absence or presence of insulin (13.3 nmol/1). Severe insulin resistance of 2-DG uptake, glycogen synthesis, and glucose oxidation was observed in muscle from the vehicle-treated obese rats compared with muscle from vehicle-treated lean (Fa/−) rats. Acute and chronic treatments (30 mgkg−1 · day−1, a maximally effective dose) with ALA significantly (P < 0.05) improved insulin-mediated 2-DG uptake in epitrochlearis muscles from the obese rats by 62 and 64%, respectively. Chronic ALA treatment increased both insulin-stimulated glucose oxidation (33%) and glycogen synthesis (38%) and was associated with a significantly greater (21%) in vivo muscle glycogen concentration. These adaptive responses after chronic ALA administration were also associated with significantly lower (15–17%) plasma levels of insulin and free fatty acids. No significant effects on glucose transporter (GLUT4) protein level or on the activities of hexokinase and citrate synthase were observed. Collectively, these findings indicate that parenteral administration of the antioxidant ALA significantly enhances the capacity of the insulinstimulatable glucose transport system and of both oxidative and nonoxidative pathways of glucose metabolism in insulin-resistant rat skeletal muscle.
The hsp60 Peptide p277 Arrests the Autoimmune Diabetes Induced by the Toxin Streptozotocin
Authors: Elias Dana , Cohen Irun R
Published: September 2013

The development of autoimmune diabetes in the NOD strain of mice (H-2g7) is marked by the presence of T-cells reactive to the p277 peptide of the 60-kDa heat shock protein (hsp60). We have found that the p277 peptide can be used as a therapeutic vaccine to arrest NOD diabetes. Recently, we found that T-cell autoimmunity to p277 also develops spontaneously in C57BL/KsJ mice (H-2d) during the induction of autoimmune diabetes by a very low dose of the β-cell toxin streptozotocin (STZ). We now report the inhibition of STZ toxin-induced autoimmune diabetes by p277 peptide therapy. Administration of two doses each of 100 µg of peptide p277 in mineral oil given 1 week after toxin induction and 85 days later was most effective. The effect of p277 on STZ toxin-induced diabetes was marked by a shift in p277 autoimmunity from a T-cell proliferative response to the production of anti-p277 antibodies. The anti-p277 antibodies were predominantly of the IgGl and IgG2b isotypes, known to be regulated by Th2 type cytokines; IgG2a antibody, known to be dependent on interferon (IFN)-γ, was induced to a much lesser degree. Peptide p277 therapy was specific: treatment of the mice with an immunogenic peptide from the sequence of another antigen, GADp34, failed to prevent the development of diabetes. The GADp34 peptide induced lower titers of specific antibodies, and the antibodies were predominantly of the IgG2a class. Thus, p277 peptide therapy, marked by the induction of Th2-type antibodies, can be effective in toxin-induced autoimmune diabetes.
Identification of the 37-kDa Antigen in IDDM as a Tyrosine Phosphatase-Like Protein (Phogrin) Related to IA-2
Authors: Hawkes Charlotte J , Wasmeier Christina , Christie Michael R , Hutton John C
Published: September 2013

Antibodies to islet cell proteins detected as 37,000 and 40,000 Mr tryptic fragments (37- and 40-kDa antigens) are strongly associated with progression to IDDM. The 40-kDa antigen has recently been identified as the tyrosine phosphatase-like protein IA-2 (ICA512) whereas the 37-kDa antigen has been suggested to be a different protein that has structural similarity to IA-2. A protein, phogrin, that has 80% amino acid sequence identity to IA-2 in the cytoplasmic domain, has recently been cloned from an insulinoma cell cDNA library. In this study, we have investigated possible relationships between the 37-kDa antigen and phogrin. Antibodies to phogrin were detected in sera from patients with IDDM, and these antibodies were strongly correlated with the presence of antibodies to the 37-kDa antigen. Trypsin treatment of immunoprecipitated phogrin generated a 37,000 Mr fragment. Recombinant phogrin was able to block autoantibody binding to the 37-kDa antigen but not to the 40-kDa antigen, and rabbit antibodies raised to different regions of phogrin depleted insulinoma cell extracts specifically of the 37-kDa antigen. These results demonstrate that the 37-kDa antigen in IDDM is indistinguishable from phogrin and show that two distinct tyrosine phosphatase-related proteins are major targets of the autoimmune response in the disease.
Efficient Gene Transfer to Dispersed Human Pancreatic Islet Cells In Vitro Using Adenovirus-Polylysine/DNA Complexes or Polycationic Liposomes
Authors: Saldeen Johan , Curiel David T , Eizirik Décio L , Andersson Arne , Strandell Eva , Buschard Karsten , Welsh Nils
Published: September 2013

The establishment of gene delivery systems that result in efficient transfection of the pancreatic β-cells may generate an important tool for the study of IDDM and may also represent one critical step toward a clinical application of gene transfer for the prevention or early treatment of the disease. Using the reporter gene vectors pCAT and pCMV β-gal, we have investigated the efficiency of transfection mediated by calcium phosphate precipitation, the monocationic liposome Lipofectin, the polycationic liposome Lipofectamine, and adenovirus-polylysine (AdpL) DNA complexes in human, mouse, rat, and fetal porcine islet cells. In all species studied, calcium phosphate–mediated transfection resulted in lower chloramphenicol acetyl transferase (CAT) activities than the other methods. Intact human, mouse, and rat islets were poorly transfected by Lipofectin, Lipofectamine, and AdpL. When dispersed by trypsin treatment, however, human, mouse, rat, and fetal pig islet cells were efficiently transfected by Lipofectamine. Moreover, transfection of dispersed human and mouse islet cells using AdpL also resulted in high CAT activities. The percentage of cells staining positively for β-galactosidase after transfection with Lipofectamine was 49% for mouse, 56% for rat, and 57% for dispersed human islet cells. Transfection of human islet cells using AdpL, however, yielded 70% βgal–positive cells. Fluorescence-activated cell sorting-purified rat islet α- and β-cells were transfected with similar efficiency using Lipofectamine. CAT expression in human islet cells transfected with either Lipofectamine or AdpL reached a peak value after 5–7 days, followed by a gradual decline. It is concluded that transfection with AdpL or Lipofectamine are both efficient means to achieve transient expression of gene constructs in human and mouse islet cells, while for rat and fetal porcine islet cells, Lipofectamine is the most efficient of the agents investigated in this study.
Regulation of Proliferation and Differentiation of Human Fetal Pancreatic Islet Cells by Extracellular Matrix, Hepatocyte Growth Factor, and Cell-Cell Contact
Authors: Beattie Gillian M , Rubin Jeffrey S , Mally Martin I , Otonkoski Timo , Hayek Alberto
Published: September 2013

Ex vivo expansion of human fetal pancreatic endocrine cells is important for biological studies and as a potential tissue source for transplantation in insulin-deficient states. In tissue culture experiments involving the use of hepatocyte growth factor/scatter factor and selected extracellular matrices, we obtained a 30-fold increase in cell number of human fetal pancreatic epithelial cells. This proliferation in monolayer culture was associated with marked downregulation of insulin and glucagon gene expression. However, gene expression increased when the cells were combined into three-dimensional aggregates, suggesting that cell-cell contact mediated mechanisms regulate the transcription of islet-specific genes, a process enhanced by nicotinamide (NIC). After transplantation into nude mice, either as cell suspensions or aggregates, only the cell aggregates treated with NIC developed into mature functional islet-like structures. These are the first experiments to describe the interactions of specific matrices and growth factors in the ex vivo expansion of human fetal pancreatic cells, and they also show the importance of cell aggregates in the context of cellular and molecular events that might positively influence islet cell transplantation.
Place Learning and Hippocampal Synaptic Plasticity in Streptozotocin-Induced Diabetic Rats
Authors: Biessels Geert-Jan , Kamal Amer , Ramakers Geert M , Urban Ivan J , Spruijt Berrie M , Erkelens D Willem , Gispen Willem Hendrik
Published: September 2013

Moderate impairment of learning and memory has been recognized as a complication of diabetes. The present study examined behavioral and electrophysiological measures of cerebral function in streptozotocin (STZ)-induced diabetic rats. Behavioral testing consisted of a spatial learning task in a water maze. Electrophysiological testing consisted of in vitro assessment of hippocampal long-term potentiation (LTP), an activity-dependent form of synaptic plasticity, which is believed to be related to the cellular mechanisms of learning and memory. Two experiments were performed: the first with severely hyperglycemic rats and the second with moderately hyperglycemic rats. Rats were tested in the water maze 11 weeks after induction of diabetes. Next, LTP was measured in vitro in trained animals. Both spatial learning and LTP expression in the CA1 field of the hippocampus were impaired in severely hyperglycemic rats as compared with nondiabetic controls. In contrast, spatial learning and hippocampal LTP were unaffected in moderately hyperglycemic rats. The association of alterations in hippocampal LTP with specific learning impairments has previously been reported in conditions other than diabetes. Our findings suggest that changes in LTP-like forms of synaptic plasticity in the hippocampus, and possibly in other cerebral structures, are involved in learning deficits in STZ-induced diabetes. The beneficial effect of moderate glycemic control on both place learning and hippocampal LTP supports the significance of the relation between these two parameters and indicates that the development of the observed deficits may be related to the level of glycemic control.
Interactions of the Kallikrein-Kinin and Renin-Angiotensin Systems in Experimental Diabetes
Authors: Vora Jiten P , Oyama Terry T , Thompson Michele M , Anderson Sharon
Published: September 2013

The kallikrein-kinin system (KKS) has been postulated to play a role in modulation of hemodynamic function in diabetes and to contribute to the hemodynamic effects of angiotensin-converting enzyme inhibition (CEI). To further explore the KKS and its interactions with the renin-angiotensin system (RAS), studies were conducted in nondiabetic control rats and in moderately hyperglycemic diabetic rats. In protocol 1, control and diabetic rats were studied before and after administration of one of two dissimilar B2 kinin receptor antagonists (BK2As), or vehicle. At a low dose (0.5 μg · kg−1 · min−1), the first generation antagonist D-Arg, [Hyp3,Thi5,8,D-Phe7]-bradykinin significantly reduced the glomerular filtration rate (GFR) and renal plasma flow rate in diabetic rats, despite variable effectiveness in blocking the hypotensive response to injected bradykinin. However, a similar hemodynamic effect occurred in nondiabetic rats, suggesting that the observed effect was not specific to diabetes. Higher doses (20 μg bolus, then 1 μg · kg−1 · min−1 infusion) did not affect hemodynamics in either group, perhaps because of partial agonist effect. The second BK2A tested was the newer compound, icatibant (Hoe 140; D-Arg,[Hyp3,Thi5,D-Tic7,Oic8]-bradykinin). Hoe 140 consistently blocked the vasodepressor action of injected bradykinin, but had no effect on systemic or renal hemodynamics in either control or diabetic rats. In protocol 2, control and diabetic rats were pretreated with the CEI ramipril for 1–2 weeks, after which renal function was studied before and after Hoe 140 (0.1 mg s.c. and i.v.) or vehicle. CEI lowered blood pressure in both groups. Hoe 140 did not affect renal function in control rats, but in diabetic rats pretreated with ramipril, it induced a modest but significant decline in GFR. Ramipril induced the predicted changes in the systemic and intrarenal RAS, while acute BK2A had no consistent effect on RAS parameters. These studies suggest that the endogenous KKS has only a minor role in modulation of renal hemodynamics in the euvolemic diabetic rat, in the absence of KKS stimulation by CEI. However, angiotensin-converting enzyme is also kininase II, which serves to increase endogenous kinin activity. The increased kinin activity resulting from CEI treatment may participate, to a modest degree, in hemodynamic regulation of the diabetic kidney.
Potential Role of an Endothelium-Specific Growth Factor, Hepatocyte Growth Factor, on Endothelial Damage in Diabetes
Authors: Morishita Ryuichi , Nakamura Shigefumi , Nakamura Yoshio , Aoki Motokuni , Moriguchi Atsushi , Kida Iwao , Yo Yoshikage , Matsumoto Kunio , Nakamura Toshikazu , Higaki Jitsuo , Ogihara Toshio
Published: September 2013

Endothelial cells are known to secrete various antiproliferative and vasodilating factors. Although injury of endothelial cells has been postulated as an initial trigger of the progression of atherosclerosis in patients with diabetes, the mechanisms of endothelial injury in diabetes are not yet clarified. Therefore, it is important to know the effects of high glucose on the factors that may influence endothelial cell growth. A novel member of endothelium-specific growth factors, hepatocyte growth factor (HGF), is produced in vascular cells. To investigate the effects of high glucose on vascular cells, we examined 1) the effects of high glucose on endothelial cell and vascular smooth muscle cell (VSMC) growth and 2) the effects of high glucose on local HGF production in endothelial cell and VSMC. Treatment of human aortic endothelial cell with a high concentration of D-glucose, but not mannitol and L-glucose, resulted in a significant decrease in cell number. Interestingly, addition of recombinant HGF attenuated high D-glucose–induced endothelial cell death. Therefore, we measured local HGF secretion of endothelial cell. Importantly, local HGF production was significantly decreased by high D-glucose treatment. In contrast, high D-glucose treatment resulted in a significant increase in the number of human aortic VSMCs, whereas local HGF production was significantly decreased in accordance with increase in D-glucose concentration. No significant changes in numbers were observed in VSMC treated with high mannitol and L-glucose. We also studied the mechanisms of local HGF suppression by high D-glucose. High D-glucose treatment stimulated transforming growth factor-β (TGF-β) concentration in endothelial cell and VSMC. Decreased local vascular HGF production was abolished by addition of anti-TGF-β antibody. As TGF-β inhibited local HGF production in endothelial cell and VSMC, increased TGF-β induced by high D-glucose may suppress local HGF production. This study demonstrated that high D-glucose induced endothelial cell death, stimulated VSMC growth, and decreased local HGF production through the stimulation of TGF-β production both in endothelial cell and VSMC. Overall, decrease in a local endothelial stimulant, HGF, by high D-glucose may be a trigger of endothelial injury in diabetes, potentially resulting in the progression of atherosclerosis.
Induction of Hepatic Glucose-6-Phosphatase Gene Expression by Lipid Infusion
Authors: Massillon Duna , Barzilai Nir , Hawkins Meredith , Prus-Wertheimer Dina , Rossetti Luciano
Published: September 2013

The distal enzymatic step in the process of glucose output is catalyzed by the glucose-6-phosphatase (Glc-6-Pase) complex. The recently cloned catalytic unit of this complex has been shown to be regulated by insulin, dexamethasone, cAMP, and glucose. Using a combination of intralipid and/or nicotinic acid infusions and a pancreatic clamp technique, we maintained plasma free fatty acids (FFAs) at three different levels (0.26 ± 0.07, 0.56 ± 0.09, and 1.59 ± 0.12 mmol/1) in the presence of well-controlled hormonal and metabolic conditions. An increase in the plasma FFA concentration within the physiological range caused a rapid, greater than threefold increase in the mRNA and protein levels of the catalytic subunit of Glc-6-Pase in the liver. These data indicate that the in vivo gene expression of Glc-6-Pase in the liver is regulated by circulating lipids independent of insulin and thus that prolonged hyperlipidemia may contribute to the increased production of glucose via increased expression of this protein.
Transgenic Expression of Mouse Proinsulin II Prevents Diabetes in Nonobese Diabetic Mice
Authors: French Michelle B , Allison Janette , Cram David S , Thomas Helen E , Dempsey-Collier Majella , Silva Anabel , Georgiou Harry M , Kay Thomas W , Harrison Leonard C , Lew Andrew M
Published: September 2013

IDDM in humans and in nonobese diabetic (NOD) mice is a T-cell–dependent autoimmune disease in which the β-cells of the pancreatic islets are destroyed. Several putative β-cell autoantigens have been identified, but insulin and its precursor, proinsulin, are the only ones that are β-cell specific. (Pro)insulin may be a key autoantigen in IDDM. To address the role of proinsulin in the development of IDDM, we generated NOD mice transgenic for the mouse proinsulin II gene driven off a major histocompatibility complex (MHC) class II promoter to direct expression of the transgene to MHC class II bearing cells, including those in the thymus, with the aim of deleting proinsulin-reactive T-cells. The mononuclear cell infiltration of the islets (insulitis) is almost completely absent, and diabetes is prevented in these transgenic NOD mice. The mononuclear cell infiltration of the salivary glands (sialitis) and immune responses to ovalbumin (OVA) are not altered, indicating that the protective effect of the transgene is specific for islet pathology and not due to general immunosuppression. We conclude that autoimmunity to proinsulin plays a pivotal role in the development of IDDM.
A Stable Isotope Dilution Assay for the In Vivo Determination of Insulin Levels in Humans by Mass Spectrometry
Authors: Stöcklin Reto , Vu Lan , Vadas Laszlo , Cerini Fabrice , Kippen Alistair D , Offord Robin E , Rose Keith
Published: September 2013

Insulin levels in humans were measured by a new assay, the isotope dilution assay (IDA), based on stable isotope dilution mass spectrometry. A known amount of a deuterated analog of insulin was used as an internal standard and added to the serum samples before sample processing. After isolation by immunoaffinity chromatography and solid phase extraction, followed by a purification step on reversed-phase microbore high-performance liquid chromatography (HPLC), the insulincontaining fraction was analyzed by mass spectrometry. The relative intensity of the signals due to insulin and its deuterated analog in the mass spectrum was used to determine the concentration of insulin in the sample. Using serum samples of 0.5–2.0 ml, we were able to measure insulin levels in the range of 3–1700 pmol/l in several clinical samples from type II diabetic patients. The basal level of endogenous insulin was also determined in two normal subjects and found to be∼20 pmol/l. Insulin secretion was followed after the ingestion of 75 g glucose in one healthy volunteer. Finally, the determination of the insulin level of one hemolyzed post-mortem blood sample, for which immunoassays gave inconsistent results, was performed to help forensic investigations. Our results showed a good correlation with standard immunoassay data, except in six samples where much lower values were obtained by our stable isotope dilution assay, suggesting an overestimation of insulin levels by immunoassay in some cases. As it is not subject to immunological interferences by insulinrelated compounds, this new assay has a major clinical advantage in that it avoids confusions related to hyperinsulinemia.
Oscillations in Oxygen Consumption by Permeabilized Clonal Pancreatic β-Cells (HIT) Incubated in an Oscillatory Glycolyzing Muscle Extract: Roles of Free Ca2+, Substrates, and the ATP/ADP Ratio
Authors: Civelek Vildan N , Deeney Jude T , Fusonie Glenn E , Corkey Barbara , Tornheim Keith
Published: September 2013

To determine whether oscillations in glycolysis could underlie the oscillations in O2 consumption observed in intact islets, we evaluated the capacity of an islet extract to exhibit spontaneous oscillations in glycolysis. When a cell-free extract obtained from ∼1,000 islets was supplied with glucose and glycolytic cofactors, oscillations in NADH fluorescence were obtained. After this demonstration of spontaneous oscillations in islet extracts, we bathed permeabilized clonal β-cells in the more plentiful spontaneously oscillating glycolytic muscle extract that generates pulses of α-glycerophosphate and pyruvate and induces oscillations in free Ca2+ and the ATP/ADP ratio. This preparation was used to investigate whether changes in Ca2+ and possibly α-glycerophosphate or pyruvate supply could underlie observed oscillations in O2 consumption and explain coordination between cytosolic and mitochondrial metabolism. We found that oscillations of O2 consumption and Ca2+ of a similar period were induced. Removal of medium Ca2+ with EGTA did not prevent the oscillations in O2 consumption nor were they greatly affected by the substantial rise in medium Ca2+ on treatment with thapsigargin to inhibit sequestration into the endoplasmic reticulum. The O2 oscillations were also not eliminated by the addition of relatively high concentrations of pyruvate or α-glycerophosphate. However, they were lost on addition of fructose-2,6-P2 at concentrations that prevent oscillations of glycolysis and the ATP/ADP ratio. Addition of a high concentration of ADP increased O2 consumption and also prevented O2 oscillations. These results suggest that the changes in respiration reflected in the O2 oscillations occur in response to the oscillations in the ATP/ADP ratio or ADP concentration and that this parameter is a primary regulator of O2 consumption in the pancreatic β-cell.
Effect of ACE Inhibition on Spontaneous and Insulin-Stimulated Endothelin-1 Secretion: In Vitro and In Vivo Studies
Authors: Desideri Giovambattista , Ferri Claudio , Bellini Cesare , De Mattia Giancarlo , Santucci Anna
Published: September 2013

To evaluate the effect of angiotensin-converting inhibition enzyme on spontaneous and insulin-stimulated endothelin-1 (ET-1) secretion in vitro and in vivo, human endothelial cells derived from umbilical cord veins were cultured onto acellular collagen-coated permeable membrane, thus mimicking in vivo conditions with a luminal and abluminal side. Insulin (10−6,−8,−9 mol/l) significantly stimulated ET-1 secretion by cultured cells (P < 0.05 starting from 2-h incubation). Captopril (10−7,−8,−9 mol/l) significantly reduced both spontaneous and insulin-stimulated ET-1 secretion, while increasing nitric oxide production. Considering each cell side, captopril significantly inhibited the apical secretion of ET-l, while its effect on the basolateral compartment was modest. In the presence of D-Arg,[Hyp3,Thi5,8,D-Phe7]-bradykinin (10−6 mol/l), a bradykinin B2 receptor antagonist, captopril had no effects on ET-1 and nitric oxide production and also when insulin was added to the culture media. With regard to in vivo experiments, oral captopril therapy (25 mg twice daily for 1 week) was given to normotensive (n = 5) and hypertensive (n = 6) subjects and significantly decreased plasma ET-1 concentration (normotensive subjects, before: 0.98 ± 0.09 pg/ml; after: 0.55 ± 0.08 pg/ml, P < 0.0001; hypertensive subjects, before: 1.05 ± 0.03 pg/ml; after: 0.56 ± 0.05 pg/ml, P < 0.0001). Transient hyperinsulinemia was accompanied by a significant rise in plasma ET-1 concentrations in both groups (P < 0.0001 at 180 and 210 min) before but not after captopril treatment. In conclusion, captopril inhibits both spontaneous and insulinstimulated ET-1 secretion by endothelial cells, acting on angiotensin-converting enzyme bound to the luminal cell side. In vivo, captopril significantly reduces plasma ET-1 levels in both basal and insulin-stimulated conditions.