bluuebunny/crossref_metadata_2025 huggingface dataset:
Dietary Fiber in the Management of Diabetes
Authors: Nuttall Frank Q
Published: September 2013

It generally is accepted that a diet high in fiber, particularly soluble fiber, is useful in the management of the plasma glucose concentration in individuals with diabetes. This is one of the reasons several national diabetes associations have recommended that diabetic individuals ingest a diet high in fiber-containing foods. However, more recent data obtained in carefully controlled studies with more definitive end points, indicate this may not be the case. It has been shown clearly that addition of water-soluble, gel-forming fiber in the form of guar gum and perhaps gum tragacanth to an ingested glucose solution or to a mixed meal will reduce the expected rise in glucose concentration. This has been demonstrated in both normal subjects and subjects with IDDM and NIDDM. However, it is only observed when large amounts of fiber are added. The fiber also must be mixed with the administered glucose or food. Other less viscous soluble fiber sources such as the pectins and psyllium powder are not effective. In long-term, well-controlled trials, guar gum, pectin, beet fiber, or cereal bran fiber ingested with meals has been of little or no value in controlling the plasma glucose concentration in individuals with NIDDM. Several studies have been conducted in which a high-carbohydrate diet has been reported to reduce the plasma glucose concentration. In these diets, foods with a high fiber content have been emphasized. In general, they were not well controlled, and several confounding variables such as weight loss, decreased food energy intake, different food sources with potential for differences in starch digestibility, and decreased dietary fat content were present. Thus, it has not been possible to determine if dietary fiber was playing a significant role. The results of studies in which specific, defined fibers have been added to the diet would suggest that the naturally occurring fiber in foods is likely to play only a minor role.
Loss of Potentiating Effect of Hypoglycemia on the Glucagon Response to Hyperaminoacidemia in IDDM
Authors: Caprio Sonia , Tamborlane William V , Zych Kathleen , Gerow Karen , Sherwin Robert S
Published: September 2013

IDDM subjects lose the ability to release glucagon during hypoglycemia. Because replacement of basal levels of amino acids enhances the glucagon response to hypoglycemia in healthy subjects, we tested whether raising amino acid levels during hypoglycemia could reverse the defective α-cell response in IDDM patients. For this purpose, 11 IDDM patients (HbA1 9.4 ± 0.6%) and 8 healthy, nondiabetic subjects received two hypoglycemic insulin clamp studies (0.8 mU.kg−1 · min−1) in which plasma glucose was clamped at 55 mg/dl (3.08 mM) for 180 min. During one of the studies, an infusion of amino acids was superimposed between 120 and 180 min (0.3 g.kg−1 · h−1). This dose of amino acids had a small effect on plasma glucagon levels during euglycemic hyperinsulinemia that was comparable in normal and IDDM subjects. In healthy control subjects, plasma glucagon rose by 80% during the initial hypoglycemic phase of the study. The addition of amino acids produced a further sharp (200–250 ng/L, P < 0.02) rise in plasma glucagon, such a change did not occur in the absence of amino acids. In contrast, plasma glucagon in IDDM patients failed to increase during hypoglycemia alone and rose by only 40–50 ng/L (P < 0.05 vs. controls) when amino acid infusion was superimposed, even though plasma amino acid levels rose to the same extent in IDDM and control subjects. More importantly, the rise in glucagon produced by amino acids was comparable during hypoglycemic and euglycemic hyperinsulinemia in the IDDM patients, results strikingly different from those observed in nondiabetic control subjects. We conclude that hypoglycemia greatly amplifies the α-cell response to hyperaminoacidemia in healthy subjects but not in IDDM.
Benfluorex Normalizes Hyperglycemia and Reverses Hepatic Insulin Resistance in STZ-Induced Diabetic Rats
Authors: Serradas Patricia , Blondel Olivier , Bailbe Danielle , Portha Bernard
Published: September 2013

We have examined the effect of chronic (20 days) oral administration of benfluorex (35 mg/kg) in a rat model of NIDDM, induced by injection of STZ 5 days after birth and characterized by frank hyperglycemia, hypoinsulinemia, and hepatic and peripheral insulin resistance. We assessed the following: 1) basal blood glucose and insulin levels, 2) glucose tolerance and glucose-induced insulin release in vivo and in vitro, and 3) basal and insulin-stimulated in vivo glucose production and glucose utilization, using the insulin-clamp technique in conjunction with isotopic measurement of glucose turnover. The in vivo insulin response of several individual tissues also was evaluated under the steady-state conditions of the clamp, using the uptake of the glucose analogue 2-deoxy-D-glucose as a relative index of glucose metabolism. In the benfluorex-treated diabetic rats, postabsorptive basal plasma glucose levels were decreased (8.1 ± 0.2 mM compared with 10.5 ± 0.5 mM in the pair-fed untreated diabetic rats and 6.1 ± 0.2 mM in the benfluorex-treated nondiabetic rats), whereas the basal and glucose-stimulated intravenous glucose tolerance test plasma insulin levels were not improved. Such a lack of improvement in the glucose-induced insulin release after benfluorex treatment was confirmed under in vitro conditions (perfused pancreas). In the pair-fed untreated diabetic rats, the basal glucose production and overall glucose utilization were significantly increased, and during hyperinsulinemia both liver and peripheral tissues revealed insulin resistance. In the benfluorex-treated diabetic rats, the basal glucose production and basal overall glucose utilization were normalized. After hyperinsulinemia, glucose production was normally suppressed, whereas overall glucose utilization was not significantly improved. Among the range of peripheral tissues investigated, only brown adipose tissue showed a significant increase in glucose utilization after benfluorex treatment. Because benfluorex exerts a predominant action on the liver in the this rat model of diabetes, and because increased basal hepatic glucose output is a major metabolic abnormality and is responsible for much of the elevated fasting blood glucose levels in NIDDM, the use of such a compound in NIDDM may be potentially relevant.
IDDM in Rats Induced by Thymectomy and Irradiation
Authors: Stumbles Philip A , Penhale W John
Published: September 2013

A diabetic syndrome closely resembling human IDDM has been induced in rats of specific pathogen-free origin by a combination of thymectomy and irradiation, with an overall incidence (10 wk postirradiation) in female rats of 34% for acute disease and 47% for islet lesions. Males were slightly more susceptible than females. Clinical features of the syndrome included hyperglycemia, insulinopenia, ketosis, and lipidemia, and corresponding islet pathology ranged from diffuse atrophy to focal atrophy and insulitis. Onset was usually acute, and the disease fatal unless early insulin therapy was initiated. Lymphocytic thyroiditis also was observed in a proportion of thymectomized and irradiated rats (49% in females) over the same period but with no apparent correlation to the occurrence of diabetes. A significant decrease in the incidence of disease was found in thymectomized and irradiated rats of conventional origin when compared with genetically identical specific pathogen-free rats, implicating a role for environmental factors in disease susceptibility. Diabetes inducement also was found to be strain related but not RT1u dependent. Both clinical signs and islet lesions were inhibited by early reconstitution of thymectomized and irradiated animals with syngeneic lymphoid cells from normal donors. Islet lesions and glucose intolerance could be transferred to syngeneic recipients by concanavalin A–activated lymphoid cells from acute diabetic donors. The close similarities between this experimental syndrome induced by immunological manipulation and the clinical condition in humans provide further evidence for an immune-mediated pathogenesis for IDDM.
Glucokinase Gene Variants in the Common Form of NIDDM
Authors: Chiu Ken C , Tanizawa Yukio , Permutt M Alan
Published: September 2013

To determine whether a structural defect in glucokinase could be a primary cause of glucose intolerance in the common form of NIDDM, the prevalence of mutations in the gene in 60 American black NIDDM patients was investigated. First, by Southern blot analysis of DNA from a subset of randomly selected subjects (n = 20), no gross deletions, insertions, or rearrangements of the gene were detected. Next, the 5′-untranslated and coding regions of the gene were amplified directly from genomic DNA by the polymerase chain reaction. PCR products were screened for mutations by using single-strand conformational polymorphism analysis. A total of nine variants were identified, with two in the 5′-UT regions of islet exon 1, two in the 5′-UT region of liver exon 1, and five in the coding regions. For islet exon 1, 5 of 60 NIDDM patients had both variants in the 5′-UT region; and for liver exon 1, two variants each occurred in 1 of 60 NIDDM patients. The coding region variants included a missense mutation in islet exon 1, substitution of Ala11 (GCC) with Thr11 (ACC), found in 2 patients. The biological consequences of this mutation and the mutations in the 5′-UT portion of the gene have yet to be determined. The rest of the variants were third base pair changes of codons, i.e., silent. A common polymorphism, which was in linkage equilibrium with microsatellite repeats GCK1 and GCK2, was found in intron 9, and a variant in intron 2 in both alleles of 1 patient. From these studies it was concluded that, although genetic variations in the coding sequence of the glucokinase gene are common, the amino acid sequence is highly conserved. The previous association of glucokinase alleles with NIDDM in American blacks has not been clarified by this analysis. Primary structural abnormalities of glucokinase do not play a significant role in the common form of NIDDM in this group.
Compromised Hormonal Counterregulation, Symptom Awareness, and Neurophysiological Function After Recurrent Short-Term Episodes of Insulin-Induced Hypoglycemia in IDDM Patients
Authors: Lingenfelser Thomas , Renn Walter , Sommerwerck Ute , Jung Matthias F , Buettner Ulrich W , Zaiser-Kaschel Heike , Kaschel Rainer , Eggstein Manfred , Jakober Bernhard
Published: September 2013

To test the hypothesis that recurrent short-term hypoglycemic episodes may impair hormonal counterregulation, symptom awareness, and neurophysiological function during subsequent hypoglycemia, we examined two groups of IDDM patients (n = 18), neither of whom exhibited signs of autonomic neuropathy. Two sequential euglycemic-hypoglycemic clamp studies were performed three days apart with stable glycemic plateaus of 5.6, 3.3, 2.2, and 1.7 mM, at which the patients' awareness of and response to hypoglycemia was evaluated. In the intervention group (n = 11), three short-term hypoglycemic episodes preceded the second clamp study. Counterregulatory hormones increased significantly during hypoglycemia, but adrenaline (P < 0.03), cortisol (P < 0.01), and ACTH (albeit not significant) showed a blunted response after repetitive hypoglycemic events. In this group, the perception of hypoglycemic symptoms was significantly reduced and was most evident for the autonomic symptoms of sweating (P < 0.05), heart pounding (P < 0.01), and warmness (P < 0.03). The deterioration of neurophysiological function, as assessed from the middle latency auditory evoked potentials, was more pronounced in the intervention group (latency shift of the Pa component, P < 0.05). These data suggest that alterations of neuroendocrine counterregulation, symptom perception, and certain aspects of cerebral function may occur as a consequence of recurrent short-term hypoglycemic episodes. These adaptation phenomena may contribute to the increased incidence of severe hypoglycemia in IDDM patients on intensive insulin therapy.
Chemical Cross-Linking Detects Association of Insulin Receptors With Four Different Class I Human Leukocyte Antigen Molecules on Cell Surfaces
Authors: Reiland Jane , Edidin Michael
Published: September 2013

Class I major histocompatibility complex molecules have been shown to be physically associated with insulin receptor molecules on the surfaces of a number of different cell types. The class I human leukocyte antigen (human MHC) phenotype of B lymphoblasts correlates with the affinity of IR for insulin. This correlation could be the result of association of some but not other HLA molecules with IR, or could reflect differences in the way in which association with an HLA molecule affects the function of IR. To distinguish between these possibilities, this study isolated complexes of four different class I HLA molecules with IR. Expression of some of these molecules correlates with high-affinity IR, whereas expression of others correlates with 5- to 10-fold lower affinity IR. This study found that chemical cross-linking was necessary to stabilize HLA-IR complexes, and all four of the class I HLA molecules studied, HLA-A1, HLA-A2, HLA-B5, and HLA-B8, can form complexes with IR.
Autoantibodies in IDDM Primarily Recognize the 65,000-Mr Rather Than 67,000-Mr Isoform of Glutamic Acid Decarboxylase
Authors: Hagopian William A , Michelsen Birgitte , Karlsen Allan E , Larsen Fleming , Moody Alistar , Grubin Catherine E , Rowe Rachel , Petersen Jacob , McEvoy Robert , Lernmark Åke
Published: September 2013

Glutamic acid decarboxylase autoantibodies may aid in rapid screening strategies predicting IDDM before clinical onset. Rat islets contain GAD65 and GAD67 autoantibody targets, but human islets express only GAD65, now confirmed by direct immunoprecipitation from radiolabeled rat and human islets. Because human IDDM involves β-cell-specific autoimmunity, we tested 190 new IDDM patients and 51 healthy control subjects for antibodies to recombinant human islet GAD65, rat islet GAD67, or human insulinoma/cerebellum GAD67, each expressed separately in hamster fibroblasts. By using immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometric fluorogram scanning, 132 of 190 (70%) of new IDDM patients had GAD65 autoantibodies, whereas only 17 of 190 (9%) had antibodies to rat GAD67 (P < 0.001). Of healthy control subjects, 2 of 51 (3.9%) and 1 of 51 (1.9%) had antibodies to GAD65 and GAD67, respectively. All 17 GAD67 antibody-positive patients also had GAD65 antibodies; 14 of 17 with greater GAD65 than GAD67 index. Control studies showed comparable reactivity between recombinant rat and human GAD67 and between different subcellular preparations of recombinant GAD67 of either species. In conclusion, only GAD65 is expressed in human islets, the autoantibody response is primarily to this isoform, and GAD67 antibodies add little to IDDM detection.
The New Elements of Insulin Signaling: Insulin Receptor Substrate-1 and Proteins With SH2 Domains
Authors: Myers Martin G , White Morris F
Published: September 2013

Since the discovery of insulin and its receptor, the downstream elements responsible for the pleiotropic insulin signal have been difficult to define. The recently discovered insulin receptor substrate, IRS-1, provides an innovative and simple way to think about this problem: IRS-1 may mediate the control of various cellular processes by insulin. Overexpression of IRS-1 enhances insulin-stimulated DNA synthesis in Chinese hamster ovary cells, and microinjection of IRS-1 protein potentiates the maturation of Xenopus oocytes. We suspect that insulin signals are enabled when the activated insulin receptor kinase phosphorylates specific tyrosine residues in IRS-1. These phosphorylated sites associate with high affinity to cellular proteins that contain SH2 (src homology-2) domains. This association is specific and depends on the amino acid sequence surrounding the phosphotyrosine residue and the isoform of the SH2 domain. A growing number of SH2 domain–containing proteins have been identified, and we suspect that IRS-1 has the potential to simultaneously regulate many of them. We have only begun to identify the specific proteins that associate with phosphorylated IRS-1. One of them, the phosphatidylinositol 3′-kinase, is activated when the SH2 domains in its 85,000-Mr regulatory subunit bind to phosphorylated IRS-1. IRS-1 also interacts with other proteins such as SHPTP2, a novel SH2 domain–containing Tyr phosphatase, and GRB-2/sem-5, a protein that is implicated in p21ras signaling. The interaction between phosphorylated IRS-1 and multiple SH2 domain–containing proteins may ultimately explain the pleiotropic effects of insulin.
An RT6a Gene Is Transcribed and Translated in Lymphopenic Diabetes-Prone BB Rats
Authors: Crisá Laura , Sarkar Probir , Waite Debra J , Friedrich Friedrich Haag , Rajan T V , Mordes John P , Handler Eugene S , Thiele Heinz-Günter , Rossini Aldo A , Greiner Dale L
Published: September 2013

T-cells expressing the RT6 surface alloantigen appear to perform important immunoregulatory functions in the rat. Diabetes-prone BB rats lack circulating RT6+ T-cells and spontaneously develop autoimmune diabetes mellitus and thyroiditis. The coisogenic diabetes-resistant BB rat does circulate RT6+ T-cells and is free of disease. Transfusions leading to engraftment of RT6+ T-cells prevent both diabetes and thyroiditis in the diabetes-prone rat. To investigate the absence of this subset in the lymphopenic BB rat, we used both molecular and biochemical procedures and made the following observations: 1) an mRNA encoding RT6 protein is present in diabetes-prone spleen cells; 2) nucleotide sequencing of this transcript reveals an intact coding sequence for the RT6.1 alloantigen; 3) sensitive chemiluminescent assay of diabetes-prone lymph node cell detergent extracts shows that diabetes-prone RT6 mRNA is translated in vivo; 4) quantitatively, diabetes-prone lymph node cells express ≤ 10% of the RT6.1 protein found on similar numbers of diabetes-resistant BB cells; and 5) finally, we obtained evidence of an intact phosphatidylinositol linkage of the molecule to the cell surface and successfully immunoprecipitated the phosphatidylinositol-linked protein with DS4.23 monoclonal antibody, indicating that the RT6.1 antigen is correctly processed and folded in diabetes-prone lymph node cells. We conclude that the near total absence of RT6+ T-cells in the diabetes-prone BB rat is unlikely to be because of a defect in RT6 gene expression per se. Defects in RT6 gene regulation or other cellular defects leading to premature cell death in the T-cell lineage, alone or in combination, may instead be responsible.
Fasting Does Not Impaird Insulin-Stimulated Glucose Uptake But Alters Intracellular Glucose Metabolism in Conscious Rats
Authors: Youn Jang H , Buchanan Thomas A
Published: September 2013

Effects of 24-h and 48-h fasting on maximal insulin-stimulated whole-body and muscle glucose uptake, glycogen synthesis, and glycolysis were studied in conscious rats by combining the glucose clamp technique with tracer methods. Fasting decreased body weight and basal plasma glucose, plasma insulin, hepatic glucose output, and glucose clearance (P < 0.05 for all). However, maximal insulin-stimulated whole-body glucose uptake, normalized to body weight, was almost identical in fed, 24-h fasted, and 48-h fasted rats (191 ± 8, 185 ± 14, and 182 ± 5 μmol · kg−1 · min−1, respectively; P > 0.7). Similarly, rates of insulin-stimulated glucose uptake by four different skeletal muscles, estimated by the 2-deoxyglucose injection technique, were not different among the three groups. In contrast to glucose uptake, insulin-stimulated whole-body glycolysis was decreased significantly after fasting (36% after 48 h fasting; P < 0.05), whereas insulin-stimulated whole-body glycogen synthesis was increased (44% after 48 h fasting; P < 0.05). In fed rats, glycolysis was the major pathway for glucose metabolism during hyperinsulinemia, accounting for 60 ± 5% of glucose uptake. This fraction was decreased significantly by fasting (P < 0.01), so that after a 48-h fast, glycolysis accounted for only 40 ± 3% of insulin-stimulated glucose uptake and glycogen synthesis became predominant pathway, accounting for 60 ± 3% of whole-body glucose utilization. Whole-body patterns of glucose metabolism during hyperinsulinemia were paralleled by glucose metabolism in individual muscles. These data indicate that fasting for up to 48 h in rats had no effect on insulin-stimulated glucose disposal (whole body or muscle) but resulted in a change of the primary metabolic path for insulin-stimulated glucose utilization from glycolysis to glycogen synthesis.
Organization of the Human GLUT2 (Pancreatic β-Cell and Hepatocyte) Glucose Transporter Gene
Authors: Takeda Jun , Kayano Toshiaki , Fukomoto Hirofumi , Bell Graeme I
Published: September 2013

The gene encoding the predominant facilitative glucose transporter expressed in pancreatic β-cells and hepatocytes, termed GLUT2, has been cloned and characterized. The human GLUT2 gene is composed of 11 exons spanning ∼30 kilobases. The sequence of the promoter region and all exons and adjacent intron regions has been determined and deposited in the GenBank database. Two highly polymorphic simple tandem repeat DNA polymorphisms useful for linkage studies were localized in introns 1 and 4a. In addition, a 168-base pair insertion/deletion polymorphism was identified in intron 3. The characterization of the human GLUT2 gene will facilitate studies of its role in the development of diabetes mellitus.
Hyperglycemic Pseudohypoxia and Diabetic Complications
Authors: Williamson Joseph R , Chang Katherine , Frangos Myrto , Hasan Khalid S , Ido Yasuo , Kawamura Takahiko , Nyengaard Jens R , Den Enden Maria van , Kilo Charles , Tilton Ronald G
Published: September 2013

Vasodilation and increased blood flow are characteristic early vascular responses to acute hyperglycemia and tissue hypoxia. In hypoxic tissues these vascular changes are linked to metabolic imbalances associated with impaired oxidation of NADH to NAD+ and the resulting increased ratio of NADH/NAD+. In hyperglycemic tissues these vascular changes also are linked to an increased ratio of NADH/NAD+, in this case because of an increased rate of reduction of NAD+ to NADH. Several lines of evidence support the likelihood that the increased cytosolic ratio of free NADH/NAD+ caused by hyperglycemia, referred to as pseudohypoxia because tissue partial pressure oxygen is normal, is a characteristic feature of poorly controlled diabetes that mimics the effects of true hypoxia on vascular and neural function and plays an important role in the pathogenesis of diabetic complications. These effects of hypoxia and hyperglycemia-induced pseudohypoxia on vascular and neural function are mediated by a branching cascade of imbalances in lipid metabolism, increased production of superoxide anion, and possibly increased nitric oxide formation.
Effects of Acute Hyperinsulinemia on VLDL Triglyceride and VLDL ApoB Production in Normal Weight and Obese Individuals
Authors: Lewis Gary F , Uffelman Kristine D , Szeto Linda W , Steiner George
Published: September 2013

The effects of short-term hyperinsulinemia on the production of both VLDL triglyceride and VLDL apoB were determined semiquantitatively before and during a 6-h euglycemic hyperinsulinemic clamp (40 mU · m−2 · min−1) in 17 women (8 chronically hyperinsulinemic obese, BMI = 35.7 kg/m2; 9 normal weight, BMI = 22.5 kg/m2). During acute hyperinsulinemia, plasma FFA decreased by ∼ 95% within 1 h in both groups. VLDL triglyceride production decreased 66% in the control subjects (P = 0.0003) and 67% in obese subjects (P = 0.0003). ApoB production decreased 53% in control subjects (P = 0.03) but only 8% in obese (NS). Plasma triglycerides decreased by 40% from baseline in control subjects (P < 0.0001) but only by 10% in obese subjects (P = NS). Despite the similar decrease in triglyceride and apoB production in control subjects, VLDL particle size (triglyceride-to-apoB ratio) decreased with hyperinsulinemia (P = 0.003). In obese subjects, despite a decrease in triglyceride production similar to that in control subjects but no change in apoB production, VLDL size did not change appreciably. Acute hyperinsulinemia in humans: 1) suppresses plasma FFA equally in control and obese subjects at this high dose of insulin; 2) inhibits VLDL triglyceride production equally in control and obese subjects, perhaps secondary to the decrease in FFA; 3) inhibits VLDL apoB production in control but less so in obese subjects, suggesting that obese subjects may be resistant to this effect of insulin; 4) decreases plasma triglyceride and VLDL particle size in control subjects, reflecting either stimulation of LPL activity or a greater relative decrease in triglyceride to apoB production; and 5) does not decrease plasma triglyceride or VLDL size in obese subjects to the same extent as it does in control subjects. Thus, the insulin resistance of obesity affects some but not all aspects of VLDL metabolism.
Decreased Catecholamine Secretion From the Adrenal Medullae of Chronically Diabetic BB-Wistar Rats
Authors: Wilke Russell A , Riley Danny A , Lelkes Peter I , Hillard Cecilia J
Published: September 2013

Many humans with IDDM eventually lose the capacity to secrete epinephrine from their adrenal medullae. The mechanism for this pathological change is unknown. We hypothesized that this abnormality is attributable to neuropathic changes in the greater splanchnic nerves or in the chromaffin cells that they innervate. To study this hypothesis, we isolated rat adrenal glands, perfused them ex vivo, and measured the epinephrine content of the perfusate under various conditions of stimulation. We used transmural electrical stimulation (20–80 V, at 10 Hz) to induce epinephrine secretion indirectly by selectively activating residual splanchnic nerve terminals within the isolated glands. Under these conditions, epinephrine secretion was severely attenuated in glands from female BB-Wistar rats with diabetes of 4 mo duration compared with their age-matched, nondiabetic controls. These perfused diabetic adrenal medullae also demonstrated decreased catecholamine release in response to direct chromaffin cell depolarization with 20 mM K+, evidence that a functional alteration exists within the chromaffin cells themselves. Nonetheless, total catecholamine content of adrenal medullae from these diabetic rats was not significantly different from controls, indicating that the secretory defect was not simply attributable to a difference in the amount of catecholamines stored and available for release. Herein, we also provide histological evidence of degenerative changes within the cholinergic nerve terminals that innervate these glands.
Aberrant Distribution of CD4 and CD8 Lymphocyte Subsets in Recent-Onset IDDM Patients: Effect of Cyclosporin
Authors: Chatenoud Lucienne , Volpini Walkyria , Timsit José , Boitard Christian , Bach Jean-François
Published: September 2013

The monoclonal antibodies 2H4 and 4B4 are specifically directed against CD45RA and CD29 antigens, respectively, which are expressed on both CD4+ and CD8+ lymphocytes. We used them to monitor the distribution of these T-cell subsets in 19 recent-onset IDDM patients (26 ± 7 yr of age [mean ± SD throughout]) receiving cyclosporin, and who had been treated with insulin for < 1 mo. Blood samples were examined before starting cyclosporin and after 3 and 9 – 12 mo of therapy. CD45RA and CD29 are expressed on distinct subsets of CD4+ T-cells, whereas in both healthy subjects and IDDM patients, 2H4 and 4B4 labeling does not always differentiate two distinct CD8+ populations. Various proportions of double-labeled CD8+ CD45RA+ CD29+ cells were detected in 59% of IDDM patients and 70% of healthy control subjects. Before cyclosporin treatment, recent-onset IDDM patients had a significantly lower proportion of CD4+CD29+ cells than the healthy control subjects (42 ± 11 vs. 52 ± 9%, P < 0.004). This imbalance corresponded to a significant increase in the CD4+ CD45RA+/CD4+ CD29+ ratio in the IDDM patients (1.37 ± 0.93 vs. 0.78 ± 0.36, P < 0.014). Independent of the presence of double-labeled cells, the proportion of CD8+ lymphocytes expressing CD45RA was significantly higher in the patients than in control subjects (72 ± 15 vs. 57 ± 15%, P < 0.004). The mean density of the CD45RA antigen (but not that of CD29) on both CD4+ and CD8+ cells was significantly higher in the IDDM patients before treatment than in the healthy control subjects. Cyclosporin induced complete or partial remission of the disease in 12 of 19 patients at 4–6 mo of treatment, but did not correct the imbalance between the regulatory T-cell subsets, regardless of clinical effectiveness. Cyclosporin use was associated with a significant decrease in CD45RA antigen density on both CD4+ and CD8+ T-cells.
The Effects of Hyperinsulinemia and Hyperglycemia on GLUT4 and Hexokinase II mRNA and Protein in Rat Skeletal Muscle and Adipose Tissue
Authors: Postic Catherine , Leturque Armelle , Rencurel Frank , Printz Richard L , Forest Claude , Granner Daryl K , Girard Jean
Published: September 2013

The GLUT4 glucose transporter and type II hexokinase are predominantly expressed in skeletal muscle and adipose tissue. The effects of insulin and glucose on the expression of GLUT4 and HKII were studied in vivo by using the euglycemic-hyperinsulinemic and hyperglycemic-hyperinsulinemic clamp methods. The clamps were maintained in conscious rats for 6 or 24 h after a 1-day starvation period. Adipose tissue GLUT4 mRNA was increased 4-fold after 6 h and 23-fold after 24 h of hyperinsulinemia; HKII mRNA was increased by four- and eightfold after 6 and 24 h, respectively. In contrast, GLUT4 mRNA was not significantly changed in skeletal muscle by either the euglycemic- or hyperglycemic-hyperinsulinemic clamps. Each of these treatments resulted in a fourfold induction of HKII mRNA. No changes of GLUT4 protein and hexokinase activity were detected after 6 h of hyperinsulinemia in either skeletal muscle or adipose tissue. After 24 h of hyperinsulinemia, adipose tissue GLUT4 protein had doubled, whereas skeletal muscle GLUT4 was unchanged. In contrast, hexokinase activity increased by two- to eightfold in skeletal muscle and adipose tissue. Hyperinsulinemia alone was sufficient to mediate the effects observed, because no additional effects were seen when hyperglycemia accompanied hyperinsulinemia. These results reveal the lack of coordinate regulation of GLUT4 and HKII in adipose tissue and skeletal muscle. Whereas hyperinsulinemia increases both GLUT4 and HKII mRNA and protein levels in adipose tissue, this treatment increases HKII mRNA and protein in skeletal muscle, but has no effect on GLUT4 in this tissue.
Human Skeletal Muscle Insulin Receptor Substrate-1: Characterization of the cDNA, Gene, and Chromosomal Localization
Authors: Araki Eiichi , Sun Xiao-Jian , Haag Burritt L , Chuang Lee-Ming , Zhang Yitao , Yang-Feng Teresa L , White Morris F , Kahn C Ronald
Published: September 2013

Insulin receptor substrate-1 is a major substrate of insulin receptor Tyr kinase. We have now cloned the IRS-1 cDNA from human skeletal muscle, one of the most important target tissues of insulin action, localized and cloned the human IRS-1 gene, and studied the expression of the protein in Chinese hamster ovary cells. Human IRS-1 cDNA encodes a 1242 amino acid sequence that is 88% identical with rat liver IRS-1. The 14 potential Tyr phosphorylation sites include 6 Tyr-Met-X-Met motifs and 3 Tyr-X-X-Met motifs that are completely conserved in human IRS-1. Human IRS-1 has >50 possible Ser/Thr phosphorylation sites and one potential ATP-binding site close to the NH2-terminal. The human IRS-1 gene contains the entire 5ʹ-untranslated region and protein coding region in a single exon and was localized on chromosome 2 q36–37 by in situ hybridization. By Northern blot analysis, IRS-1 mRNA is rare and consists of two species of 6.9 and 6 kilobase. By using quantitative polymerase chain reaction after reverse transcription of total RNA from human fetal tissues, IRS-1 mRNA could be identified in all tissues. When human IRS-1 cDNA was expressed in Chinese hamster ovary cells, the protein migrated between 170,000–180,000 Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was rapidly Tyr phosphorylated upon insulin stimulation. Thus, IRS-1 is widely expressed and highly conserved across species and tissues. Compared with rat protein, human IRS-1 contains more potential Ser/Thr phosphorylation sites and only one nucleotide binding site. The entire protein coding sequence is contained within a single exon.
Effect of Glucocorticoid and Growth Hormone Treatment on Proinsulin Levels in Humans
Authors: Kahn Steven E , Horber Fritz F , Prigeon Ronald L , Haymond Morey W , Porte Daniel
Published: September 2013

Treatment with glucocorticoids is associated with a disproportionate elevation in the PI/IRI ratio. To determine whether growth hormone—another agent capable of producing insulin resistance and changing B-cell function—also alters the PI/IRI ratio and whether growth hormone and glucocorticoids have a synergistic effect on PI and IRI levels, we examined these variables in four groups of young healthy subjects (n = 8/group) after 7 days of treatment with placebo, prednisone (0.8 mg.kg−1 · day−1), rhGH (0.1 mg.kg−1 · day−1), and the combination of prednisone and rhGH. Fasting plasma glucose levels increased significantly above those of the control group in subjects receiving prednisone or prednisone and rhGH but not in subjects receiving rhGH alone. The basal concentration of IRI increased in response to prednisone, rhGH, and the combination of prednisone and rhGH. However, this increase in IRI was largely due to an increase in PI, so that the PI/IRI ratio increased from 14.7 ± 2.4% in control subjects to 33.9 ± 5.3% in subjects on prednisone (P < 0.005), 40.9 ± 4.3% in individuals receiving rhGH (P < 0.001 vs. control subjects), and 58.1 ± 9.2% in subjects receiving both prednisone and rhGH (P < 0.001 vs. control subjects). We suggest that this change in PI/IRI with glucocorticoid and growth hormone treatment may be due to an alteration in B-cell synthesis or release of PI. This change in the PI/IRI ratio is not dependent on fasting hyperglycemia but may contribute to the hyperglycemia often observed with these agents. Furthermore, these data show that IRI is not a reliable indicator of true insulin levels or insulin sensitivity in either growth hormone- or glucocorticoid-treated subjects.
The Design of Trials for Prevention of IDDM
Authors: Eisenbarth George S , Verge Charles F , Allen Holley , Rewers Marian J
Published: September 2013

Several large clinical trials for the prevention of IDDM in islet cell antibody positive first-degree relatives are planned or underway. The design of these trials rests in part on assumptions about the natural history of autoimmunity during the prediabetic period and on the likely effectiveness of the intervention being tested. At this time, most of the factors that influence the required sample size can only be roughly estimated.
Glycosylation of Asn397 or Asn418 is Required for Normal Insulin Receptor Biosynthesis and Processing
Authors: Bastian William , Zhu Jian , Way Barbara , Lockwood Dean , Livingston James
Published: September 2013

Two N-linked sites of glycosylation in the insulin receptor were examined for their contribution to insulin binding, tyrosine kinase activity, and receptor biosynthesis. Asn397 and Asn418 were replaced by Gln using site-directed mutagenesis either as single mutations, i.e., Q-397 and Q-418, or as a double mutation in which both sites were removed (Q-D). The mutations were transiently expressed in COS cells and the findings compared with cells that transiently expressed the wild-type human insulin receptor. Q-397 and Q-418 mutant insulin receptors had insulin-binding characteristics similar to the wild-type human insulin receptor, whereas no insulin-binding activity could be detected above the control level in cells transfected with Q-D. Flow cytometry with antibodies against the human insulin receptor indicated the presence of Q-397, Q-418, and wild-type human insulin receptors in the surface of COS cells and failed to demonstrate a Q-D receptor. Insulin-induced autophosphorylation was similar in Q-397, Q-418, and wild-type human insulin receptors as was their ability to phosphorylate an artificial substrate, poly Glu-Tyr (4:1). Our inability to detect Q-D receptors was not caused by a lack of Q-D mRNA. COS cells transfected with Q-D cDNA generated as much Q-D mRNA as the amount of wild-type human insulin receptor mRNA present in cells transfected with wild-type receptor cDNA. Finally, pulse-chase experiments with [35S]Met were able to detect 190,000-Mr) proreceptors and the alpha-subunits for Q-397, Q-418, and wild-type human insulin receptors. In these experiments, Q-D-transfected cells produced a small amount of 170,000-Mr protein (presumably the Q-D proreceptor) that disappeared after 2 h and was not processed into α- and β-subunits. The marked discrepancy in the synthesis and/or processing of the single and double mutants suggests a functional redundancy that requires the presence of carbohydrates in a certain region of the receptor, but the precise location of the carbohydrates within the region can vary.
Physiological Increments in Plasma Insulin Concentrations Have Selective and Different Effects on Synthesis of Hepatic Proteins in Normal Humans
Authors: De Feo Pierpaolo , Volpi Elena , Lucidi Paola , Cruciani Guido , Reboldi Gianpaolo , Siepi Donatella , Mannarino Elmo , Santeusanio Fausto , Brunetti Paolo , Bolli Geremia B
Published: September 2013

These studies tested the hypothesis that physiological increments in plasma insulin concentrations have selective effects on the synthesis of hepatic proteins in humans. Leucine kinetics and fractional synthetic rates of albumin, fibrinogen, antithrombin III, and apoB-100 were determined in 6 normal subjects, on two different occasions during either the infusion of saline (control study) or a euglycemic-hyperinsulinemic (0.4 mU.kg−1 · min−1 for 240 min) clamp, by a primed-constant infusion of [1-14C]Leu. The insulin infusion significantly decreased the rates of nonoxidative Leu disposal (1.70 ± 0.10 vs. control 2.06 ± 0.09 mol.kg−1 · min−1), increased the albumin (7.2 ± 0.4 vs. 6.2 ± 0.6%/day), decreased the fibrinogen (18 ± 1 vs. 23 ± 2%/day), and antithrombin III (28 ± 3 vs. 40 ± 4%/day) fractional synthetic rate, whereas it did not affect the total apoB-100 (49 ± 5 vs. 52 ± 6%/day) fractional synthetic rate. Thus, the insulin-induced decrement in the estimates of whole-body protein synthesis (nonoxidative Leu disposal) represents the mean result of opposite effects of hyperinsulinemia on the synthesis of proteins with different functions. The positive effect of insulin on albumin synthesis may play an important anabolic role during nutrient absorption by promoting the capture of a relevant amount of dietary essential amino acids into the protein, whereas the negative effect of insulin on fibrinogen synthesis might, at least partially, account for the increased plasma fibrinogen concentrations previously reported in poorly controlled diabetic patients.
Antihyperglycemic Effect of Dehydroepiandrosterone Analogue 16α-Fluoro-5-androsten-17-one in Diabetic Mice
Authors: Pashko Laura L , Schwartz Arthur G
Published: September 2013

The adrenocortical steroid, dehydroepiandrosterone, has been shown previously to produce an antidiabetic effect in C57BL/KsJ db/db mice. Preliminary clinical data suggest that this steroid may enhance insulin sensitivity in humans. The therapeutic use of dehydroepiandrosterone may be limited by its androgenic action. In a previous study, high-dose dehydroepiandrosterone therapy to postmenopausal women produced marked elevations in plasma testosterone (9-fold) and dihydrotestosterone (20-fold) levels. We previously developed the synthetic steroid, 16α-fluoro-5-androsten-17-one, which lacks the androgenic action of dehydroepiandrosterone yet has retained other biological activities of the native steroid. In this study, administration of 16α-fluoro-5-androsten-17-one in the diet (0.2 and 0.3%) to male C57BL/KsJ db/db mice markedly reduced plasma glucose levels. In contrast, treatment with dehydroepiandrosterone was effective in reducing plasma glucose levels at the 0.2% dose but had no effect at the 0.3% dose, possibly as a result of the androgenic state induced at the higher dose. Dehydroepiandrosterone treatment also produced a 25-fold elevation in plasma testosterone levels and a significant increase in seminal vesicle weights, whereas treatment with 16α-fluoro-5-androsten-17-one had no apparent effect on the weight of the seminal vesicle glands.
Insulin Response to an Intravenous Glucose Load During Development of Obesity in Gold Thioglucose-Injected Mice
Authors: Blair Stephen C , Caterson Ian D , Cooney Gregory J
Published: September 2013

The insulin secretory response to an intravenous glucose load was examined in chronically catheterized, conscious mice 2, 5, and 10 wk after induction of obesity by a single injection of gold thioglucose. At 2 wk after administration of gold thioglucose, a significant increase in both the insulinemia and incremental area under the curve of insulin release after intravenous glucose were observed (incremental area under the curve for 2-wk control mice, 852 ± 54 min/pM; incremental area under the curve for 2-wk GTG-injected mice, 1140 ± 114 min/pM; P < 0.05). At this stage, no significant difference existed in the glucose tolerance or body weight of control and gold thioglucose-injected mice. By 5 wk, the gold thioglucose-injected mice were ∼33% heavier than their lean controls and showed a marked glucose intolerance. This was accompanied by overt hyperinsulinemia in both the basal state and also in response to an intravenous glucose bolus as indicated by the increase in the incremental area under the curve of insulin (5-wk control mice, 816 ± 114 min/pM; 5-wk gold thioglucose-injected mice, 1374 ± 156 min/pM; P < 0.05). At 10 wk after gold thioglucose administration, body weight and the degree of glucose intolerance were increased. Although 10-wk gold thioglucose-injected mice showed basal hyperinsulinemia, an intravenous glucose bolus elicited a smaller insulin secretory response than that observed in the age-matched lean control animals (10-wk control mice, 672 ± 54 min/pM; 10-wk gold-thioglucose-injected mice 186 ± 42 min/pM; P < 0.05). Thus, during the early stages of gold thioglucose-induced obesity, a marked increase in insulin secretion in response to an intravenous glucose load occurs, but as the animals become chronically hyperinsulinemic and hyperglycemic, the insulin secretory response to intravenous glucose decreases, possibly indicating glucose toxicity of the pancreas. The increase in insulin secretion observed in 2-wk gold thioglucose-injected animals may be an important abnormality in the initiation of obesity responsible for the onset of the hyperlipogenesis and insulin resistance in this animal model.
Metformin Blocks Downregulation of Cell Surface GLUT4 Caused by Chronic Insulin Treatment of Rat Adipocytes
Authors: Kozka Izabela J , Holman Geoffrey D
Published: September 2013

Large decreases in insulin-responsive glucose transport occur in rat adipocytes maintained in culture for 24 h in the continuous presence of insulin. After 24 h in culture, an acute treatment with insulin increased 3-O-methyl-D-glucose transport by only approximately fivefold. In chronically insulin-treated cells, the transport activity was more severely reduced. The transport activity was only approximately twofold higher than in basal cells. To attribute changes in transport to alterations in cell surface transporters, we labeled the cell surface GLUT4 and GLUT1 transporters with the impermeant photoaffinity label 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos -4-yloxy)-2-propylamine. Cell surface labeling was compared with the labeling obtained in digitonin-permeabilized cells where the normally impermeant reagent had access to the total cellular pool of transporters. Labeling showed that in basal cells the proportions of GLUT4 and GLUT1 at the cell surface were 20 and 22% of the total. After an acute treatment with insulin, the proportions of GLUT4 and GLUT1 at the cell surface were increased to 49 and 37% of the total, respectively. The chronic insulin treatment was associated with a very low proportion of GLUT4 (25% of the total) at the cell surface. The downregulation of GLUT4 observed after chronic insulin treatment was alleviated by metformin, and the proportion of GLUT4 at the cell surface was maintained at 60% of the total. Furthermore, cells that were chronically treated with insulin showed severe resistance to subsequent acute insulin restimulation of transport and GLUT4 recruitment to the cell surface. This effect was also alleviated by inclusion of metformin during the chronic insulin treatment.
HLA-DQA1 and DQB1 Gene Polymorphisms in Type I Diabetic Patients from Central Italy and Their Use for Risk Prediction
Authors: Buzzetti Raffaella , Nisticò Lorenza , Osborn John F , Giovannini Claudio , Chersi Alberto , Sorrentino Rosa
Published: September 2013

Susceptibility to type I diabetes has been shown to be highly correlated with the presence of an amino acid other than Asp at position 57 of the DQ β-chain (non-Asp57) and also with the presence of an Arg at position 52 of the DQ α-chain (Arg52). In this study we analyzed the DQA1 and DQB1 gene polymorphisms in 65 patients from central Italy and 93 randomly selected control subjects. Polymerase chain reaction amplification of DNA encoding the first polymorphic domain of the DQB1 and DQA1 chains was performed, and DQB1 gene polymorphism was evaluated by dot blot analysis using 11 sequence-specific oligonucleotide probes. For DQA1 typing, a new simple procedure based on allele-specific amplification and analysis of heteroduplex DNA molecules formed by the annealing of mismatched allelic strands was used. This technique allows the discrimination of Arg52 and non-Arg52 DQA1 alleles. We then calculated by logistic regression the contribution of these genetic markers to the development of diabetes. Frequencies and odds ratios relative to the amino acid in position 57 of the DQ β-chain and the amino acid in position 52 of the DQ α-chain showed that the highest odds ratio (odds ratio = 161; 95% confidence interval 19–1386) was that of the homozygous combination of the two susceptibility markers (non-Asp57 and Arg52). Based on the incidence estimates of type I diabetes in the continental Italian population, the annual incidence rate of the disease was estimated for the different genotypes grouped according to the number of potentially formed susceptible heterodimers as 212.53, 12.60, 3.24, and 1.33/100,000 individuals per yr for the 4, 2 , 1 , and 0 susceptible heterodimers groups, respectively.