bluuebunny/crossref_metadata_2025 huggingface dataset:
Repeat Treatment of Obese Mice With BRL 49653, a New Potent Insulin Sensitizer, Enhances Insulin Action in White Adipocytes: Association With Increased Insulin Binding and Cell-Surface GLUT4 as Measured by Photoaffinity Labeling
Authors: Young Paul W , Cawthorne Michael A , Coyle Paul J , Holder Julie C , Holman Geoffrey D , Kozka Izabela J , Kirkham David M , Lister Carolyn A , Smith Stephen A
Published: September 2013

(±)-5-([4-[2-Methyl-2(pyridylamino)ethoxy]phenyl]methyl) 2,4-thiazolidinedione (BRL 49653) is a new potent antidiabetic agent that improves insulin sensitivity in animal models of NIDDM. In C57BL/6 obese (ob/ob) mice, BRL 49653, included in the diet for 8 days, improved glucose tolerance. The half-maximal effective dose was 3 μmol/kg diet, which is equivalent to ∼0.1 mg/kg body wt. Improvements in glucose tolerance were accompanied by significant reductions in circulating triacylglycerol, nonesterified fatty acids, and insulin. The insulin receptor number of epididymal white adipocytes prepared from obese mice treated with BRL 49653 (30 μmol/kg diet) for 14 days was increasedtwofold. The affinity of the receptor for insulin was unchanged. In the absence of added insulin, the rates of glucose transport in adipocytes from untreated and BRL 49653-treated obese mice were similar. Insulin (73 nmol/l) produced only a 1.5-fold increase in glucose transport in adipocytes from control obese mice, whereas after BRL 49653 treatment, insulin stimulated glucose transport 2.8-fold. BRL 49653 did not alter the sensitivity of glucose transport to insulin. The increase in insulin responsiveness was accompanied by a 2.5-fold increase in the total tissue content of the glucose transporter GLUT4. Glucose transport in adipocytes from lean littermates was not altered by BRL 49653. To establish the contribution of changes in glucose transporter trafficking to the BRL 49653-mediated increase in insulin action, the cell-impermeant bis-mannose photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-yloxy)-2-[2-3H]-propylamine was used to measure adipocyte cell-surface–associated glucose transporters. In these experiments, the increase in maximal insulin-stimulated glucose transport (4.2-fold) produced after BRL 49653 treatment was correlated with a 2.6-fold increase in cell-surface–associated GLUT4. Photolabeled cell-sur-face GLUT1 was not detectable in any adipocyte preparation. These results suggest that the improvement in glycemic control produced by repeated administration of BRL 49653 to obese mice is mediated by increased insulin responsiveness of target tissues. BRL 49653 potentiates insulin-stimulated glucose transport in adipocytes from insulin-resistant obese mice, both by increasing insulin receptor number and by facilitating translocation of GLUT4, from an expanded intracellular pool, to the cell surface. In addition, the increased intrinsic activity of cell-surface glucose transporters may also contribute to an increased insulin responsiveness of adipose tissue.
Isolation and Characterization of the Human Muscle Glycogen Synthase Gene
Authors: Orho Marju , Nikula-Ijäs Pirjo , Schalin-Jäntti Camilla , Permutt M Alan , Groop Leif C
Published: September 2013

Impaired glycogen synthase (GS) activity in skeletal muscle has been considered to be an inherited trait in patients with non-insulin-dependent diabetes mellitus (NIDDM). We therefore isolated the human muscle GS gene from genomic libraries and determined the genomic structure. The entire coding region, the 5′-flanking region, and the exon-intron boundaries were sequenced. The gene consists of 16 exons spanning ∼27 kb of DNA and exists as a single copy in the human genome. The negatively charged parts with all known phosphorylation sites were coded by the first andthe last exon. A single transcription initiation site was located 167 nucleotides upstream of the initiation codon. All of the exons and the putative promoter region were analyzed by single-strandconformation polymorphism in 30 insulin-resistant Finnish NIDDM patients, and three polymorphic sites were found. A missense mutation Gly464/Ser in exon 11 was found in 2 of 228 NIDDM patients screened but in 0 of 154 control subjects. These two patients were characterized further by severe insulin resistance and premature arteriosclerosis. The characterization of the genomic structure of the human muscle GS gene will facilitate studies of its role in the development of insulin resistance and NIDDM.
Treatment of Autoimmune Diabetes and Insulitis in NOD Mice With Heat Shock Protein 60 Peptide p277
Authors: Elias Dana , Cohen Irun R
Published: September 2013

We recently showed that a peptide of the Mr 60,000 heat shock protein molecule, designated peptide p277, is a target of T-cells in autoimmune diabetes in NOD mice. Indeed, the p277 peptide could be used as a therapeutic agent to arrest the autoimmune process even after it was faradvanced. The present study was done to document the effects of p277 therapy on inflammation of the islets and on T-cell responsiveness to p277. Groups of female NOD mice of various ages up to 17 weeks were treated with a single inoculation of p277 given before or after the onset of overt hyperglycemia. We now report that fragments of p277 can affect diabetes but that optimal therapy requires the whole peptide. The positive response to p277 was dependent on administration of a threshold dose of peptide. Therapy was accompanied by the regression of intra-islet inflammation and the reappearance of histologically normal islets. Successful peptide therapy was associated with downregulation of T-cell immunity to p277. Adoptive transfer experiments demonstrated that the spleen cells of p277-treated mice were no longer diabetogenic and also could suppress the diabetogenic potential of cotransferred spleen cells of untreated female NOD mice. These results indicate that specific treatment of diabetes with a defined peptide can reprogram the autoimmune response.
Enhanced Insulin Action Due to Targeted GLUT4 Overexpression Exclusively in Muscle
Authors: Tsao Tsu-Shuen , Burcelin Rémy , Katz Ellen B , Huang Lily , Charron Maureen J
Published: September 2013

Dysregulation of GLUT4, the insulin-responsive glucose transporter, is associated with insulin resistance in skeletal muscle. Although skeletal muscle is the major target of insulin action, muscle GLUT4 has not been linked causally to whole-body insulin sensitivity and regulation of glucose homeostasis. To address this, we generated a line of transgenic mice that overexpresses GLUT4 in skeletal muscle. We demonstrate that restricted overexpression of GLUT4 in fast-twitch skeletal muscles of myosin light chain (MLC)–GLUT4 transgenic mice induces a 2.5-fold increase in insulin-stimulated 2-deoxyglucose uptake in transgene-overexpressing cells. Consequently, glycogen content is increased in the fast-twitch skeletal muscles under insulin action (5.75 ± 1.02 vs. 3.24 ± 0.26 mg/g). This indicates that insulin-stimulated glucose transport is partly rate-limiting for glycogen synthesis. At the whole-body level, insulin-stimulated glucose turnover is increased 2.5-fold in unconscious MLC-GLUT4 mice. Plasma glucose and insulin levels in MLC-GLUT4 mice are altered as a result of increased insulin action. In 2- to 3-month-old MLC-GLUT4 mice, fasting insulin levels are decreased (0.43 ± 0.05 vs. 0.74 ± 0.10 microgram/l), whereas normal fasting glycemia is maintained. Conversely, 7- to 9-month-old MLC-GLUT4 mice exhibit decreased fasting glycemia (5.75 ± 0.73 vs. 8.11 ± 0.57 mmol/l) with normal insulin levels. Fasting plasma lactate levels are elevated in both age groups (50–100%). Additionally lipid metabolism is affected by skeletal muscle GLUT4 overexpression. This is indicated by changes in plasma free fatty acid and β-hydroxybutyrate levels. These studies underscore the importance of GLUT4 in the regulation of glucose homeostasis and its interaction with lipid metabolism.
Glucose-Regulated Translational Control of Proinsulin Biosynthesis With That of the Proinsulin Endopeptidases PG2 and PC3 in the Insulin-Producing MIN6 Cell Line
Authors: Skelly Robert H , Schuppin George T , Ishihara Hisamitsu , Oka Yoshitomo , Rhodes Christopher J
Published: September 2013

In the short term (< 2 h), proinsulin biosynthesis is predominately glucose regulated at the translational level; however, the details at the molecular level behind this mechanism are not well defined. One of the major hindrances for gaining a better understanding of the proinsulin biosynthetic mechanism has been a lack of an abundant source of β-cells that express a phenotype of regulated proinsulin biosynthesis in the appropriate 2.8–16.7 mmol/l glucose range as defined in normal pancreatic islets. In this study, we demonstrate that in the MIN6 cell line, specific glucose-regulated translational control of proinsulin biosynthesis is present in the appropriate glucose concentration range. In addition to that of proinsulin, the biosynthesis of the two proinsulin conversion endopeptidases, PC2 and PC3, was coordinately glucose regulated in MIN6 cells, whereas that of the exopeptidase, carboxypeptidase H, was unaffected by glucose. Proinsulin, PC2 and PC3 biosynthesis was specifically stimulated over that of total MIN6 cell protein synthesis above a threshold of 4 mmol/l glucose that reached a maximum rate between 8 and 10 mmol/l glucose. Glucose-induced proinsulin, PC2, and PC3 biosynthesis was rapid (occurring after a 20-min lag period but reaching a maximum by 60 min), unaffected by the presence of actinomycin D; and in parallel experiments, stimulatory glucose concentrations did not alter MIN6 cell total preproinsulin, PC2, or PC3 mRNA levels. Thus, short-term (< 2 h) glucose stimulation of proinsulin, PC2 and PC3 biosynthesis in MIN6 cells, like that in isolated islets, was mediated at the translational level. Intracellular signals for mediating glucose-stimulated proinsulin PC2 and PC3 biosynthesis translation in MIN6 cells also appeared to be similar to those in pancreatic islets, requiring glucose metabolism and a supporting role for protein kinase A. However, protein kinase C or a Ca2+-dependent protein kinase did not appear to be required for glucose-regulated proinsulin biosynthesis in MIN6 cells, as in islets. MIN6 cells are the first β-cell line that indicate glucose-regulated proinsulin biosynthesis translation essentially identical to that in differentiated islet β-cells and will be an important experimental model to better define the mechanism of proinsulin biosynthesis in detail.
Pathogenic and Protective Roles of CD45RBlow CD4+ Cells Correlate With Cytokine Profiles in the Spontaneously Autoimmune Diabetic Mouse
Authors: Shimada Akira , Rohane Patricia , Fathman C Garrison , Charlton Brett
Published: September 2013

The adoptive transfer of splenocytes from diabetic NOD mice to NOD-scid/scid (NOD-scid) recipients results in diabetes. This model was used to test the effect of cotransfer of splenocyte subsets from young nondiabetic NOD mice. As shown previously in other NOD models, the CD4+ subset from young nondiabetic mice significantly delayed the onset of diabetes in splenocyte cotransfers (P < 0.001). The data presented here showed that the development of diabetes in NOD-scid recipients correlated with a rapid increase in peripheral CD45RBlow CD4+ cells. However, the CD45RBlow subset of CD4+ cells from young nondiabetic mice protected from diabetes transfer in this model. We therefore examined whether CD45RBlow CD4+ cells from diabetic mice were pathogenic rather than protective. CD45RBlow CD4+ splenocytes from diabetic NOD mice were transferred along with CD8+ splenocytes from diabetic mice into NOD-scid recipients, and all of the recipients became diabetic within 5 weeks posttransfer. In contrast, no recipients (0 of 10) of CD45RBhigh CD4+ cells along with CD8+ splenocytes from diabetic mice became diabetic within 5 weeks posttransfer (P < 0.001). A correlate for the difference between CD45RBlow CD4+ cells from diabetic NOD mice and CD45RBlow CD4+ cells from nondiabetic mice, which showed protective effect in splenocyte cotransfers, was found in cytokine production after stimulation with anti-CD3 antibodies in vitro. CD45RBlow CD4+ cells from diabetic mice showed a significantly higher ratio (approx-imately fivefold) of γ-interferon (IFN-γ) to interleukin (IL)-4 when compared with CD45RBlow CD4+ cells from nondiabetic mice (P < 0.001). In conclusion, the function of the CD45RBlow population of CD4+ cells changes from a protective to a pathogenic one during the development of disease in the NOD mouse. This change in function correlates with cytokine production in vitro; increased IFN-γ-to-IL-4 ratio is associated with pathogenic potential and occurs coincident with (or after) the onset of diabetes.
Insulin Transport From Plasma Into the Central Nervous System Is Inhibited by Dexamethasone in Dogs
Authors: Baura Gail D , Foster David M. , Kaiyala Karl , Porte Daniel , Kahn Steven E , Schwartz Michael W
Published: September 2013

We have previously shown that transport of plasma insulin into the central nervous system (CNS) is mediated by a saturable mechanism consistent with insulin binding to blood-brain barrier insulin receptors and subsequent transcytosis through microvessel endothelial cells. Since glucocorticoids antagonize insulin receptor-mediated actions both peripherally and in the CNS, we hypothesized that glucocorticoids also impair CNS insulin transport. Nine dogs were studied both in the control condition and after 7 days of high-dose oral dexamethasone (DEX) administration (12 mg/day) by obtaining plasma and cerebrospinal fluid (CSF) samples over 8 h for determination of immunoreactive insulin levels during a 90-min euglycemic intravenous insulin infusion (plasma insulin ∼700 pmol/l). From these data, the kinetics of CNS insulin uptake and removal were determined using a mathematical model with three components (plasma → intermediate compartment, hypothesized to be brain interstitial fluid → CSF). DEX increased basal insulin levels 75% from 24 ± 6 to 42 ± 30 pmol/l (P < 0.005) and slightly increased basal glucose levels from 5.0 ± 0.7 to 5.3 ± 1.0 mmol/l (P < 0.05). DEX also lowered the model rate constant characterizing CNS insulin transport by 49% from 5.3 × 10−6 ± 4.0 × 10−6 to 2.7 × 10−6 ± 1.2 × 10−6 min−2 (P ≤ 0.001). As glucocorticoids are known to reduce CSF turnover, we also hypothesized that the model rate constant associated with CSF insulin removal would be decreased by DEX. As expected, the model rate constant for CSF insulin removal decreased 47% from 0.038 ± 0.013 to 0.020 ± 0.088 min−1 (P ≤ 0.0005) during DEX treatment. We conclude that DEX impairs CNS insulin transport. This finding supports our hypothesis that insulin receptors participate in the CNS insulin transport process and that this process may be subject to regulation. Moreover, since increasing brain insulin transport reduces food intake and body adiposity, this observation provides a potential mechanism by which glucocorticoid excess leads to increased body adiposity.
Potential Role of Bradykinin in Forearm Muscle Metabolism in Humans
Authors: Dietze Günther J , Wicklmayr Matthias , Rett Kristian , Jacob Stephan , Henriksen Erik J
Published: September 2013

Using the euglycemic-hyperinsulinemic glucose clamp and the human forearm technique, we have demonstrated that the improved glucose disposal rate observed after the administration of an angiotensin-converting enzyme (ACE) inhibitor such as captopril may be primarily due to increased muscle glucose uptake (MGU). These results are not surprising because ACE, which is identical to the bradykinin (BK)-degrading kininase II, is abundantly present in muscle tissue, and its inhibition has been observed to elicit the observed metabolic actions via elevated tissue concentrations of BK and through a BK B2 receptor site in muscle and/or endothelial tissue. These findings are supported by several previous studies. Exogenous BK applied into the brachial artery of the human forearm not only augmented muscle blood flow (MBF) but also enhanced the rate of MGU. In another investigation, during rhythmic voluntary contraction, both MBF and MGU increased in response to the higher energy expenditure, and the release of BK rose in the blood vessel, draining the working muscle tissue. Inhibition of the activity of the BK-generating protease in muscle tissue (kallikrein) with aprotinin significantly diminished these functional responses during contraction. Applying the same kallikrein inhibitor during the infusion of insulin into the brachial artery significantly reduced the effect of insulin on glucose uptake into forearm muscle. This is of interest, because in recent studies insulin has been suggested to elicit its actions on MBF and MGU via the accelerated release of endothelium-derived nitric oxide, the generation of which is also stimulated by BK in a concentration-dependent manner. This new evidence obtained from in vitro and in vivo studies sheds new light on the discussion of whether BK may play a role in energy metabolism of skeletal muscle tissue.
Bradykinin B2 Receptors on Skeletal Muscle are Coupled to Inositol 1,4,5-Trisphosphate Formation
Authors: Rabito Sara F , Minshall Richard D , Nakamura Fumiaki , Wang Li-Xiu
Published: September 2013

To determine the presence of bradykinin receptors in skeletal muscle, we examined in both displacement and saturation studies the binding of [125I-Tyr8]bradykimn or [3H]bradykinin in three types of skeletal muscle preparations: membrane fractions from guinea pig hindlimb quadriceps, dog semimembranosus and semitendinosus muscles, and L8 rat skeletal muscle myoblasts. Scatchard analysis of [125I-Tyr8]bradykinin × bradykinin competition binding demonstrated specific bradykinin binding of 4.9 and 3.2 fmol/mg protein in dog and guinea pig skeletal muscle preparations, respectively. Unlabeled bradykinin specifically displaced [125I-Tyr8] bradykinin with IC50 values of 36.5 ± 6 and 118.0 ± 16.0 pmol/1 from dog and guinea pig muscle membranes, respectively. The B2 bradykinin receptor antagonist HOE 140 and the B1 bradykinin receptor antagonist des-Arg9[Leu8]bradykinin displaced the binding of [3H]bradykinin from dog membranes with IC50 values of 0.38 and 217.3 nmol/l, respectively, suggesting that bradykinin binds to a B2-type receptor. In addition, unlabeled bradykinin competed with [3H]bradykinin for binding to dog skeletal muscle membrane preparations in a biphasic manner. To assess whether this represents multiple bradykinin receptor subtypes present in skeletal muscle homogenates or several affinity states of a single binding site, we examined bradykinin receptors on a pure skeletal muscle system, the L8 neonatal rat skeletal muscle myoblast cell line. These myoblasts also contain specific [3H ]bradykinin-binding sites with a Bmax of 271 fmol/mg protein and a Kd of 0.83 nmol/1. Competitive agonist binding curves were biphasic (high-affinity IC50 = 3.9 poml/l, low-affinity IC50 = 22.6 nmol/1) in the absence of guanosine 5′-O-(3-thio-trisphosphate) (GTPγS); they shifted to a model of one affinity (8.1 nmol/1) in the presence of GTPγS. Because the enzyme neutral endopeptidase 24.11 is an important kininase in skeletal muscle, we examined the effect of the neutral endopeptidase inhibitor phosphoramidon on the binding of bradykinin to dog skeletal muscle membranes. We found that phosphoramidon decreased the apparent Bmax from 7.3 to 5.8 fmol/mg protein. In addition, in this cell line we investigated the action of bradykinin on phosphoinositide hydrolysis. Inositol 1,4,5-trisphosphate (IP3) was measured with a radioreceptor assay. Bradykinin (0.1 nmol/1 to 1 μmol/l) induced IP3 formation in a dose-dependent manner (EC50 = 1.42 nmol/1) from a basal level of 72.8 ± 16 pmol/mg protein to 433 ± 35.5 at the highest (1 μmol/l) concentration. We conclude that bradykinin B2receptors are expressed in skeletal muscle. Phosphoinositide hydrolysis upon stimulation of this receptor is an indicator of intracellular signal transduction. Part of the bradykinin binding in skeletal muscle is due to interaction with the enzyme neutral endopeptidase.
Role of Kinins in the Pathophysiology of Myocardial Ischemia: In Vitro and In Vivo Studies
Authors: Linz Wolfgang , Wiemer Gabriele , Schölkens Bernward A
Published: September 2013

In ischemia, the heart generates and releases kinins as mediators that seem to have cardioprotective actions. Kinin-generating pathways are present in the heart. Kininogen, kininogenases, kinins, and B2 kinin receptors can be measured in cardiac tissue. Kinins are released under conditions of ischemia. In anesthetized rats and dogs with coronary artery ligation and in human patients with myocardial infarction, kinin plasma levels are increased. In isolated rat hearts, the outflow of kinins is enhanced during ischemia but markedly attenuated after deendothelialization, pointing to the coronary vascular endothelium as the main possible source. Kinins administered locally exert beneficial cardiac effects. In isolated rat hearts with ischemia-reperfusion injuries, perfusion with bradykinin (BK) reduces the duration and incidence of ventricular fibrillation, improves cardiodynamics, reduces release of cytosolic enzymes, and preserves energy-rich phosphates and glycogen stores. In anesthetized animals, intracoronary BK is followed by comparable beneficial changes and limits infarct size. Inhibition of breakdown of BK and related peptides induces beneficial cardiac effects. Treatment with ACE inhibitors such as ramipril increases cardiac kinin levels and reduces postischemic reperfusion injuries in isolated rat hearts and infarct size in anesthetized animals. The importance of an intact endothelium that continuously generates kinins is supported by observations that basal and ramipril-in-duced release of kinins and PGI2 is markedly reduced after deendothelialization of isolated hearts. Blockade of B2 kinin receptors increases ischemia-induced effects. Endothelial formation of NO and P6I2 by ACE inhibition is prevented by the specific B2 kinin receptor antagonist icatibant. In isolated hearts, ischemia-reperfusion injuries deteriorate with icatibant, which also abolishes the cardioprotective effects of ACE inhibitors and of exogenous BK. Infarct size reduction by ACE inhibitors and by BK in anesthetized animals is reversed by icatibant. Kinins contribute to the cardioprotective effects associated with ischemie preconditioning because preconditioning or BK-induced antiarrhythmic and infarct sizelimiting effects are attenuated by icatibant. In conclusion, kinins may act as mediators of endogenous cardioprotective mechanisms. Kinins are generated and released during ischemia, with subsequent formation of PGI2 and NO probably derived mainly from the coronary vascular endothelium. Their cardioprotective profile resembles that of ACE inhibitors.
Insulin-Induced Redistribution of GLUT4 Glucose Carriers in the Muscle Fiber: In Search of GLUT4 Trafficking Pathways
Authors: Zorzano Antonio , Muñoz Purificación , Camps Marta , Mora Conchi , Testar Xavier , Palacín Manuel
Published: September 2013

Insulin rapidly stimulates glucose transport in muscle fiber. This process controls the utilization of glucose in skeletal muscle, and it is deficient in various insulinresistant states, such as non-insulin-dependent diabetes mellitus. The effect of insulin on muscle glucose transport is mainly due to the recruitment of GLTJT4 glucose carriers to the cell surface of the muscle fiber. There is increasing evidence that the recruitment of GLUT4 carriers triggered by insulin affects selective domains of sarco-lemma and transverse tubules. In contrast, GLUT1 is located mainly in sarcolemma and is absent in transverse tubules, and insulin does not alter its cellular distribution in muscle fiber. The differential distribution of GLUT1 and GLUT4 in the cell surface raises new questions regarding the precise endocytic and exocytic pathways that are functional in the muscle fiber. The current view of insulin-induced GLUT4 translocation is based mainly on studies performed in adipocytes. These studies have proposed the existence of intracellular compartments of GLUT4 that respond to insulin in a highly homogeneous manner. However, studies performed in skeletal muscle have identified insulin-sensitive as well as insulin-insensitive intracellular GLUT4-containing membranes. These data open a new perspective on the dynamics of intracellular GLUT4 compartments in insulin-sensitive cells.
Molecular Responses of Endothelial Tissue to Kinins
Authors: Busse Rudi , Fleming Ingrid
Published: September 2013

The endothelial response to kinin stimulation is the result of a series of complex intracellular reactions involving changes in the intracellular concentration of free calcium ([Ca2+]i) and intracellular pH, enhanced phosphorylation of several proteins via the activation of at least four distinct families of protein kinases, and activation of membrane ion transport systems. Some of the more recent developments in this field suggest that endothelial tyrosine kinases and tyrosine phosphatases as well as serine/threonine phosphatases are also activated in response to bradykinin. In addition, the finding that the mitogen-activated protein kinase (MAP kinase) pathway was tyrosine phosphorylated, and presumably activated, in endothelial cells after an increase in [Ca2+]i has wide-ranging implications for these cells. Indeed, MAP kinase recognizes many different substrates in the cell, including growth factor receptors, microtubule-associated proteins, specific serine-threonine protein kinases, phospholipase A2, and transcription factors. Further recent studies of interest have underscored the role of endothelium-derived hyperpolarizing factor in addition to nitric oxide and prostacyclin in the coronary vasculature. Indeed, this mediator, which seems to be an endothelium-derived, cytochrome P450-derived metabolite of arachidonic acid, would now appear to represent a substantial constitutive component of the vasodilator response to bradykinin.
Nuclear Magnetic Resonance Studies of Muscle and Applications to Exercise and Diabetes
Authors: Shulman R G , Rothman D L , Price T B
Published: September 2013

Natural-abundance 13C nuclear magnetic resonance (NMR) spectroscopy is a noninvasive technique that enables in vivo assessments of muscle and/or liver glycogen concentrations. When directly compared with the traditional needle biopsy technique, NMR was found to be more precise. Over the last several years, we have developed and used 13C-NMR to obtain information about human glycogen metabolism both under conditions of altered blood glucose and/or insulin and with exercise. Because NMR is noninvasive, we have been able to obtain more data points over a specified time course, thereby dramatically improving the time resolution. This improved time resolution has enabled us to document subtleties of the resynthesis of muscle glycogen after severe exercise that have not been observed previously. An added advantage of NMR is that we are able to obtain information simultaneously about other nuclei, such as 31P. With interleaved 13C- and 31P-NMR techniques, we have been able to follow simultaneous changes in muscle glucose-6-phosphate and muscle glycogen. In this article, we review some of the work that has been reported by our laboratory and discuss the relevance of our findings for the management of diabetes.
Autoantibodies to Protein Tyrosine Phosphatase-Like Proteins In Type I Diabetes: Overlapping Specificities To Phogrin And ICA512/IA-2
Authors: Kawasaki Eiji , Eisenbarth George S , Wasmeier Christina , Hutton John C
Published: September 2013

An insulin granule membrane protein, phogrin (phosphatase homologue of granules from rat insulinoma), with homology to islet cell antigen (ICA) 512/IA-2 has recently been cloned from an insulinoma cDNA expression library with antigranule membrane sera. We have developed a radioimmunoassay for detecting antiphogrin autoantibodies using in vitro transcribed and translated phogrin and have established the sensitivity and specificity of this assay. Thirty-two of 57 (56%) new-onset patients with type I diabetes and 26 of 44 (59%) first-degree relatives followed to diabetes had anti-phogrin antibody levels exceeding the 99th percentile of 108 normal control subjects. Levels of antiphogrin autoantibodies correlated with ICA512/IA-2 autoantibodies (r = 0.82, P < 0.0001), but minimally with insulin autoantibodies (r = 0.20, P = 0.05) and not with GAD65 autoantibodies (r = 0.16, P = 0.12). Ninety-eight percent (57 of 58) of patients positive for anti-phogrin autoantibodies were also positive for autoantibodies against ICA512/IA-2. Nine percent (9 of 101) of new-onset patients and relatives followed to diabetes were ICA512/IA-2 autoantibody-positive but anti-phogrin autoantibody-negative. Preincubation of sera with recombinant ICA512/IA-2 protein completely for the majority and partially for a minority inhibited binding to in vitro translated phogrin. In three relatives in which ICA512/IA-2 autoantibodies converted to positivity with sequential follow-up, anti-phogrin autoantibodies developed at the same time. These results suggest that anti-phogrin and ICA512/IA-2 autoantibodies are related subsets of anti-islet autoantibodies.
Both CD4+ and CD8+ T-Cells in Syngeneic Islet Grafts in NOD Mice Produce Interferon-γ During β-Cell Destruction
Authors: Suarez-Pinzon Wilma , Rajotte Ray V , Mosmann Tim R , Rabinovitch Alex
Published: September 2013

Syngeneic pancreatic islet grafts in diabetic NOD mice are infiltrated by mononuclear leukocytes, β-cells are selectively destroyed, and autoimmune diabetes recurs. This model was used to identify islet graft-infiltrating mononuclear leukocytes associated with β-cell destruction and diabetes recurrence. We compared cell surface antigen and cytokine-producing phenotypes of mononuclear leukocytes in islet grafts from NOD mice that were protected from diabetes recurrence by complete Freund's adjuvant (CFA) administration (β-cell nondestructive insulitis) and in islet grafts from control phosphate-buffered saline (PBS)-injected NOD mice (β-cell destructive insulitis). Islet grafts from CFA-injected mice contained fewer CD4+ and CD8+ cells and more B cells; also fewer interferon gamma (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α)-positive cells and more IL-4 and IL-10 positive cells. By performing two-color immunostaining of cell surface antigens and intracellular IFN-γ, we found that IFN-γ positive cells in islet grafts from CFA- and PBS-injected mice were ∼ equally divided between CD4+ and CD8+ T-cell subsets. Also, the frequencies of both CD4+ IFN-γ+ and CD8+ IFN-γ+ cells were decreased in islet grafts from CFA-injected mice. These findings suggest that destruction of β-cells in syngeneic islets transplanted into NOD mice is promoted by cells producing Th1-type cytokines (IFN-γ, IL-2, and TNF-α) and prevented by cells producing TH2-type cytokines (IL-4 and IL-10). Furthermore, both CD4+ and CD8+ IFN-γ-producing T-cells in the islet grafts appear to be involved in β-cell destruction and diabetes recurrence.
Insulin Levels and the Natural History of Glucose Intolerance in Nauruans
Authors: Dowse Gary K , Zimmet Paul Z , Collins Veronica R
Published: September 2013

Longitudinal changes in serum insulin concentrations in relation to the natural history of glucose intolerance and factors associated with the incidence of NIDDM were studied in 838 nondiabetic Micronesian Nauruans over the 5.1-year period from 1982 to 1987. In 13 individuals who had data at three time-points and who developed NIDDM only at the final test, 2-h insulin levels followed an inverted V-shaped pattern as glucose tolerance declined to NIDDM. Subjects who were normal (n = 651) or had impaired glucose tolerance (IGT) (n = 187) at the 1982 baseline survey were divided into six natural history categories depending on glucose tolerance in 1987. Changes in glucose tolerance were accompanied by changes in mean 2-h insulin concentration that paralleled the inverted V pattern seen in the 13 individuals. Longitudinal changes in fasting insulin were less consistent, but mean levels increased as subjects developed NIDDM. The 5.1-year incidence of NIDDM was strongly related to baseline fasting and 2-h glucose concentrations, but associations with insulin levels were weak and inconsistent. Neither fasting nor 2-h insulin concentrations contributed to logistic regression models predicting deterioration in glucose tolerance, whereas fasting and 2-h glucose levels were included in all models and BMI also predicted deterioration from normal. These data showing sequential changes in insulin concentrations support the β-cell exhaustion theory of NIDDM pathogenesis. However, in contrast to glucose concentrations and obesity, insulin levels are poor predictors of NIDDM risk in Nauruans. This reflects the complexity of interactions with other metabolic markers and the inability of a single examination to characterize the point along the inverted V curve of insulin secretion that an individual has reached.
Epinephrine Exerts Opposite Effects on Peripheral Glucose Disposal and Glucose-Stimulated Insulin Secretion: A Stable Label Intravenous Glucose Tolerance Test Minimal Model Study
Authors: Avogaro Angelo , Toffolo Gianna , Valerio Anna , Cobelli Claudio
Published: September 2013

Epinephrine (EPI) plays a pivotal role in regulating glucose metabolism both in splanchnic and peripheral tissues. Nevertheless, previous studies did not clarify the mechanisms by which EPI affect both glucose disposal processes in peripheral tissues and β-cell secretion. The aim of this study was to investigate, in six normal volunteers, the effects of elevated EPI concentration on peripheral glucose disposal and insulin secretion by using the stable labeled (either [6,6-2H2] or [2-2H1]glucose) intravenous glucose tolerance test (IVGTT) in conjunction with the minimal models of labeled glucose disappearance and C-peptide secretion. Elevated plasma EPI concentration significantly decreased glucose effectiveness (SG*) by 29% (0.0059 ± 0.0013 vs. 0.0083 ± 0.0011 min−1, P < 0.05), and even more, 61%, insulin sensitivity (SI*); (22 ± 6 · 10(4) vs. 54 ± 20 · 10(4) min−1.pmol.l−1; P < 0.01). These findings are not due to an isotopic effect induced by an enhanced glycogen breakdown, because the [2-2H1]glucose tracer, which is not incorporated into glycogen, gave results similar to those of [6,6-2H2]glucose tracer. No differences were observed in first phase cell sensitivity, phi 1, in the EPI study (199 ± 91 vs. 245 ± 144 10(9), NS), but there was a significant increase in the second-phase cell sensitivity to glucose phi 2, (15.2 ± 1.7 vs. 17.7 ± 4.4 10(9).min−1, P < 0.05). In conclusion, EPI selectively impairs peripheral glucose metabolism because of its unique ability to simultaneously and independently decrease glucose effectiveness and insulin sensitivity. Furthermore, EPI enhances phi 2, the ratio between the C-peptide amount secreted during the second phase and the area under the curve of the glucose signal, indicating that the observed increase of C-peptide concentration is due not only to the augmented glucose signal but also to a specific EPI-mediated enhancement of β-cell responsivity to glucose.
The Rat T-Cell Surface Protein RT6 Is Associated With src Family Tyrosine Kinases and Generates an Activation Signal
Authors: Rigby Mark R , Bortell Rita , Greiner Dale L , Czech Michael P , Klarlund Jes K , Mordes John P , Rossini Aldo A
Published: September 2013

RT6 is a glycosyl-phosphatidylinositol-linked surface molecule present on most mature rat T-cells. RT6+ T-cells can prevent the expression of autoimmune diabetes in the BB rat, but the mechanism is unknown. Because cross-linking of other glycosyl-phosphatidylinositol-linked T-cell proteins is known to activate T-cells, we investigated the signaling properties of RT6. Antibody cross-linking of RT6 enhanced expression of the α subunit of the interleukin-2 (IL-2) receptor and potentiated the proliferation of rat T-cells cultured in the presence of phorbol ester plus recombinant IL-2 (rIL-2) and/or rIL-4. RT6 was found to coimmunoprecipitate with five tyrosine phosphorylated proteins including p60fyn and p56lck, members of the src tyrosine kinase family. Pretreatment of T-cells with phorbol ester increased the phosphorylation of proteins that coimmunoprecipitated with RT6, altered the electrophoretic mobility of several of these coimmunoprecipitated phosphoproteins, and increased the amount of p60fyn and p56lck coimmunoprecipitated with RT6. These data indicate that RT6-mediated signaling events may prime T-cells to respond to exogenous cytokines, suggesting a possible mechanism by which surface RT6 may influence T-cell function.
Insulin and Cortisol Promote Leptin Production in Cultured Human Fat Cells
Authors: Wabitsch Martin , Bo Jensen Per , Blum Werner F , Christoffersen Claus T , Englaro Piera , Heinze Eberhard , Rascher Wolfgang , Teller Walter , Tornqvist Hans , Hauner Hans
Published: September 2013

The aim of this study was to investigate the regulation of leptin expression and production in cultured human adipocytes using the model of in vitro differentiated human adipocytes. Freshly isolated human preadipocytes did not exhibit significant leptin mRNA and protein levels as assessed by reverse transcriptase (RT)-polymerase chain reaction (PCR) and radioimmunoassay (RIA). However, during differentiation induced by a defined adipogenic serum-free medium, cellular leptin mRNA and leptin protein released into the medium increased considerably in accordance with the cellular lipid accumulation. In fully differentiated human fat cells, insulin provoked a dose-dependent rise in leptin protein. Cortisol at a near physiological concentration of 10−8 mol/l was found to potentiate this insulin effect by almost threefold. Removal of insulin and cortisol, respectively, was followed by a rapid decrease in leptin expression, which was reversible after readdition of the hormones. These results clearly indicate that both insulin and cortisol are potent and possibly physiological regulators of leptin expression in human adipose tissue.
Cloning, Tissue Expression, and Chromosomal Localization of SUR2, the Putative Drug-Binding Subunit of Cardiac, Skeletal Muscle, and Vascular KATP Channels
Authors: Chutkow William A , Simon M Celeste , Le Beau M Michelle , Burant Charles F
Published: September 2013

ATP-sensitive inwardly rectifying potassium channels are expressed in a variety of tissues, including heart, skeletal, and smooth muscle, and pancreatic β-cells. Physiological and pharmacological studies suggest the presence of distinct KATP channels in these tissues. Recently, the KATP channel of β-cells has been reconstituted in functional form by coexpression of SUR, the sulfonylurea-binding protein, and the inwardly rectifying K+ channel subunit, KIR6.2. In this article, we describe the isolation of cDNAs encoding SUR-like proteins from mouse, SUR2A and SUR2B. Northern blotting showed that the highest expression of the SUR2 isoforms is in the heart and skeletal muscle, with lower levels in all other tissues. By reverse transcription-polymerase chain reaction, SUR2B is ubiquitously expressed, while the apparently alternatively spliced variant, SUR2A, is expressed exclusively in heart. In situ hybridization shows that the SUR2 isoforms are expressed in the parenchyma of the heart and skeletal muscle and in the vascular structures of other tissues. Human SUR2 was localized to chromosome 12, p12.1 by fluorescent in situ hybridization. The structure of the predicted protein and expression pattern of SUR2 suggests that it is the drug-binding channel-modulating subunit of the extrapancreatic KATP channel. Differences in sequence between SUR and between SUR2 isoforms may underlie the tissue-specific pharmacology of the KATP channel.
Disturbed Development of the Preimplantation Embryo in the Insulin-Dependent Diabetic BB/E Rat
Authors: Lea Richard G , McCracken Jane E , McIntyre Susan S , Smith William , Baird Joyce D
Published: September 2013

Although improved regulation of maternal IDDM during pregnancy has resulted in a major fall in the stillbirth rate, the rates for other problems, such as spontaneous preterm labor, fetuses small for gestational age, congenital malformation, and the incidence of large placentas, remain raised. This has suggested the possibility that the damaging effect of conventionally treated but poorly regulated IDDM may operate primarily at the earliest stages of gestation, even before the diagnosis of pregnancy has been made. This study shows that spontaneous autoimmune IDDM in the Bio Breeding/Edinburgh (BB/E) rat is associated with severe disturbance in the development of the preimplantation embryo in a majority of pregnancies, as indicated by a fivefold increase in the incidence of degenerate fragmented embryos and a 33% reduction in the number of expanded blastocysts and in those blastocysts that reach the expanded stage a 20% cellular deficit in the inner-cell mass without any change in trophectoderm cell number. In addition, we find that blastocysts removed from diabetic rats and cultured in vitro for 24 h show no sign of “catch-up” growth of the inner-cell mass, although under these conditions, the trophectoderm exhibits a 25% cellular accretion. It is tempting to speculate that these phenomena are a presage of the characteristic combination of fetal growth retardation and large placentas, which are a feature of both BB/E rat and human IDDM pregnancy.
Glycation of Insulin in the Islets of Langerhans of Normal and Diabetic Animals
Authors: Abdel-Wahab Yasser H A , O'Harte Finbarr P , Ratcliff Helen , McClenaghan Neville H , Barnett Christopher R , Flatt Peter R
Published: September 2013

The glycation of immunoreactive insulin (IRI) was assessed in extracts of pancreas and islets from control and hyperglycemic animal models. Glycated and nonglycated IRI were separated by affinity chromatography and quantified by radioimmunoassay. Hydrocortisone-treated Wistar rats (80 mg · kg−1 · day−1 and obese hyperglycemic (ob/ob) mice showed significant increases in plasma glucose (P < 0.001), percentage glycated hemoglobin (P < 0.001), plasma IRI (P < 0.01), and total pancreatic IRI content (P < 0.01), compared with their respective controls. These diabetic groups also demonstrated significant increases (P < 0.05) in the percentage of glycated pancreatic IRI above the controls. Streptozotocin-treated (200 mg/kg) Swiss TO mice exhibited significant increases in plasma glucose (P < 0.001), glycated hemoglobin (P < 0.001), and percentage glycated pancreatic IRI (P < 0.05), compared with untreated controls, despite a marked decrease in both plasma IRI (P < 0.001) and total pancreatic IRI content (P < 0.001). Significant elevations in the percentage of glycated IRI were also observed in islets isolated from obese hyperglycemic (ob/ob) mice (P < 0.001), compared with islets from lean controls, and when lean mouse islets were cultured in hyperglycemic media for 24 h (33.3 vs. 5.6 mmol/l D-glucose; P < 0.001). The contribution of glycated plus nonglycated insulin and proinsulin to the total IRI was estimated in lean and obese mouse pancreatic extracts following high-performance liquid chromatography separation. The contribution of proinsulin to the total IRI was ∼ 10%. Proinsulin represented 27–28% of the total glycated IRI. These data indicate that the glycation of insulin and proinsulin occurs within the pancreatic islets and is elevated in both insulin-deficient and insulin-resistant diabetic animal models.
A Comparison of the Effects of Selective Increases in Peripheral or Portal insulin on Hepatic Glucose Production in the Conscious Dog
Authors: Sindelar Dana K , Balcom James H , Chu Chang A , Neal Doss W , Cherrington Alan D
Published: September 2013

We investigated the mechanisms by which peripheral or portal insulin can independently alter liver glucose production. Isotopic ([3-3H]glucose) and arteriovenous difference methods were used in conscious overnight-fasted dogs. A pancreatic clamp (somatostatin plus basal insulin and basal glucagon infusions) was used to control the endocrine pancreas. After a 40-min basal period, a 180-min experimental period followed in which selective increases in peripheral (PERI group, n = 5) or portal-vein (PORT group, n = 5) insulin were induced. In control dogs (CONT group, n = 10), insulin was not increased. Glucagon levels were fixed in all studies, and basal euglycemia was maintained by peripheral glucose infusion in the two experimental groups. In the PERI group, arterial insulin rose from 36 ± 12 to 120 ± 12 pmol/l, while portal insulin was unaltered. In the PORT group, portal insulin rose from 108 ± 42 to 192 ± 42 pmol/l, while arterial insulin was unaltered. Neither arterial nor portal insulin changed from basal in the CONT group. With a selective rise in peripheral insulin, the net hepatic glucose output (NHGO; basal, 11.8 ± 0.7 µmol · kg−1 · min−1) did not change initially (11.8 ± 2.1 µmol · kg−1 · min−1, 30 min after the insulin increase), but eventually fell (P < 0.05) to 6.1 ± 0.9 µmol · kg−1 · min−1 (last 30 min). With a selective rise in portal insulin, NHGO dropped quickly (P < 0.05) from 10.0 ± 0.9 to 5.6 ± 0.6 µmol · kg−1 · min−1 (30 min after the insulin increase) and eventually reached 3.1 ± 1.1 µmol x kg−1 · min−1 (last 30 min). When insulin levels were not increased (CONT group), NHGO dropped progressively from 10.1 ± 0.6 to 8.3 ± 0.6 µmol · kg−1 · min−1 (last 30 min). Conclusions drawn from the net hepatic glucose balance data were confirmed by the tracer data. Net hepatic gluconeogenic substrate uptake (three carbon precursors) fell 2.0 µmol · kg−1 · min−1 in the PERI group, but rose 1.2 µmol · kg−1 · min−1 in the PORT group and 1.2 µmol · kg−1 · min−1 in the CONT group. A selective 84 pmol/l rise in arterial insulin was thus associated with a fall in NHGO of ∼ 50%, which took 1 h to manifest. Conversely, a selective 84 pmol/l rise in portal insulin was associated with a 50% fall in NHGO, which occurred quickly (15 min). From the control data, it is evident that in either case ∼ 30% of the fall in NHGO was due to a drift down in baseline and that 70% was due to the rise in insulin. In conclusion, an increment in portal insulin had a rapid inhibitory effect on NHGO, caused by the suppression of glycogenolysis, while an equal increment in arterial insulin produced an equally potent but slower effect that resulted from a small increase in hepatic sinusoidal insulin, from a suppression of gluconeogenic precursor uptake by the liver, and from a redirection of glycogenolytic carbon to lactate rather than glucose.