bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Exercise Training on Insulin-Regulatable Glucose-Transporter Protein Levels in Rat Skeletal Muscle
Authors: Rodnick Kenneth J , Holloszy John O , Mondon Carl E , James David E
Published: September 2013

Exercise training has been shown to enhance the ability of insulin to stimulate glucose uptake in responsive tissues. The purpose of this study was to determine the effects of exercise training on the levels of the insulin-regulatable glucose transporter (IRGT) in rat skeletal muscle. After 6 wk of voluntary running in exercise-wheel cages, male Sprague-Dawley rats were rested for ∼27 h and fasted overnight before removal of plantaris and soleus muscles. The concentration of glucose transporters per unit of muscle protein or DNA was quantitated by immunoblotting with an anti-IRGT polyclonal antibody raised against a synthetic peptide. The IRGT protein was increased by 60% (141 ± 14 vs. 229 ± 24 counts/min [cpm]/25 μg protein, P < 0.01) in plantaris muscle from exercise-trained rats compared with controls. Total protein yield, DNA content, and 5-nucleotidase activity were not different in plantaris muscle from control and exercise-trained rats. In contrast, there was no significant increase in the IRGT protein in soleus muscle after training when data were expressed per unit of muscle protein (292 ± 22 vs. 346 ± 16 cpm/25 μg protein). These data indicate that the increase in the IRGT in plantaris muscle is a selective response to exercise training that does not reflect an overall increase in muscle protein. The changes in IRGT for these muscles with exercise training parallel changes observed in insulin-mediated glucose uptake. We propose that this increase in the total number of glucose transporters may be a major component of the increase in insulin-mediated glucose uptake that is observed with exercise training.
Wisconsin Epidemiologic Study of Diabetic Retinopathy: XII. Relationship of C-Peptide and Diabetic Retinopathy
Authors: Klein Ronald , Moss Scot E , Klein Barbara E K , Davis Matthew D , Demets David L
Published: September 2013

The relationship between plasma C-peptide and the frequency and severity of diabetic retinopathy was examined in a population-based study in Wisconsin in 1984–1986. Individuals with younger- (n = 835) and older- (n = 940) onset diabetes were included. C-peptide was measured by radioimmunoassay with Heding's M1230 antiserum. Retinopathy was determined from stereoscopic fundus photographs. The highest frequencies and most severe retinopathy were found in insulin-using individuals with undetectable or low plasma C-peptide (<0.3 nM), whereas the lowest frequencies of retinopathy were found in older-onset overweight individuals not using insulin. In olderonset individuals using insulin, having no detectable C-peptide was significantly associated with the presence of proliferative retinopathy. Otherwise, within each group (younger onset using insulin, older onset using insulin, and older onset not using insulin), after controlling for other characteristics associated with retinopathy, there was no relationship between higher levels of C-peptide and lower frequency of or less severe retinopathy.
Elusive Proximal Signals of β-Cells for Insulin Secretion
Authors: MacDonald Michael J
Published: September 2013

The β-cell is unique because its major agonists, i.e., insulin secretagogues, undergo metabolism instead of interacting with a receptor. This perspectives presents the hypothesis that the first part of a metabolic signal of a secretagogue is specific to the secretagogue and the β-cell and can be envisioned as proximal. The second part, which occurs after transduction to more universal signaling mechanisms, is viewed as distal. Distal signaling and exocytosis in the β-cell operate the same as in other cells. Aerobic glycolysis is required for glucose-induced insulin release. Because glyceraldehyde, which enters metabolism at the triose phosphates in the glycolytic pathway, is a potent insulin secretagogue but pyruvate, which is metabolized in the mitochondrion, is not an insulin secretagogue, the proximal signal for glucose-induced insulin release originates with an interaction between the central part of the glycolytic pathway and mitochondrial metabolism. The proximal message in leucine-induced insulin release originates with leucine allosterically activating glutamate dehydrogenase, which activates endogenous glutamate metabolism, and by the metabolism of leucine itself. The methyl ester of succinate is a potent experimental insulin secretogogue. It is puzzling why the glucose signal requires the interplay of glycolysis and mitochondrial metabolism, whereas the signals from leucine and succinate originate entirely from within the mitochondrion. Leucine-induced insulin release is suppressed and glucose-induced insulin release is activated in islets cultured at a high concentration of glucose. Conversely, leucine-induced insulin release is activated and glucose-induced insulin release is suppressed in islets cultured at low glucose. We have correlated suppression of the insulinotropism of leucine and glucose with decreased expression of the genes that encode the catalytic subunit of the first component of the branched-chain ketoacid dehydrogenase complex and the pyruvate dehydrogenase complex, respectively. This indicates that the proximal signal is specific to the secretagogue, whereas distal signals are more universal and are shared by many secretagogues. The proximal signaling mechanisms have yet to be elucidated, but many distal mechanisms are known.
Insulin-Mimicking Anti-Idiotypic Antibodies in Development of Spontaneous Autoimmune Diabetes in BB/E Rats
Authors: Elias Dana , Bone Adrian J , Baird Joyce D , Cooke Anne , Cohen Irun R
Published: September 2013

BB/E rats spontaneously develop a form of autoimmune diabetes resembling insulin-dependent diabetes mellitus (IDDM) in humans. IDDM results from central destruction of the insulin-producing β-cells of the pancreatic islets. Herein, we report that the outbreak of IDDM in BB/E rats is preceded by the spontaneous development of an anti-idiotypic antibody to a particular antibody to insulin made by the rats. This anti-idiotype, designated anti-DM-id, behaves as an antibody to the insulin-hormone receptor. Thus, a spontaneous anti-idiotypic antibody network whose products can affect the peripheral utilization of insulin seems to accompany the central destruction of β-cells in developing IDDM.
Increased Susceptibility to Hypoxia of Prolonged Action Potential Duration in Ventricular Papillary Muscles From Diabetic Rats
Authors: Aomine Masahiro , Nobe Seiki , Arita Makoto
Published: September 2013

The action potential duration (APD) of ventricular muscles obtained from diabetic animals is reported to be prolonged. We studied the effect of varied periods of diabetes on APD prolongation using isolated ventricular papillary muscles from streptozocininjected rats. We found that a diabetic period >30 wk was necessary for the evolution of significant prolongation of APD. We then studied the effect of repeated hypoxia (Po2 40 mmHg) and normoxia (Po2 300 mmHg) on prolonged APD of diabetic muscles and compared the findings with those from control rats. Transmembrane potentials were recorded with conventional glass microelectrodes. Under normoxic conditions, the APDs of diabetic muscles were significantly prolonged, the maximum upstroke velocity of action potentials tended to be decreased, and the resting membrane potential was not changed significantly compared with controls. The first hypoxia (20 min) shortened the APD in both diabetic and control rats but more so in diabetic rats, thereby making the APD of diabetic rats virtually identical to the control rat APD during the hypoxia. On subsequent reoxygenation (30 min), these hypoxia-induced changes were almost recovered. The second hypoxia (20 min) produced more severe shortening of the APD in both muscle types, and the effect was again far greater in diabetic rats than controls, ending with a reversed sequence of APDs and the APD of diabetic muscles much shorter than the controls. Excessive shortening of APD in diabetic muscles during hypoxic conditions was speculated to be due to greater increases in the outward K+ current through ATPregulated K+ channels that may be secondary to the more severe reduction of intracellular ATP concentrations in diabetic versus control hearts.
Distribution of Elements and Water in Peripheral Nerve of Streptozocin-Induced Diabetic Rats
Authors: Lowery Joan M , Eichberg Joseph , Saubermann Albert J , LoPachin Richard M
Published: September 2013

Accumulating evidence suggests that alterations in Na, Ca, K, and other biologically relevant elements play a role in the mechanism of cell injury. The pathogenesis of experimental diabetic neuropathy is unknown but might include changes in the distribution of these elements in morphological compartments. In this study, this possibility was examined via electron-probe X-ray microanalysis to measure both concentrations of elements (millimoles of element per kilogram dry or wet weight) and cell water content (percent water) in frozen, unfixed, unstained sections of peripheral nerve from control and streptozocin-induced diabetic rats. Our results indicate that after 20 wk of experimental diabetes, mitochondria and axoplasm from myelinated axons of proximal sciatic nerve displayed diminished K and CI content, whereas in tibial nerve, the intraaxonal levels of these elements increased. In distal sciatic nerve, mitochondrial and axoplasmic levels of Ca were increased, whereas other elemental alterations were not observed. These regional changes resulted in a reversal of the decreasing proximodistal concentration gradients for K and CI, which existin nondiabetic rat sciatic nerve. Our results cannot be explained on the basis of altered water. Highly distinctive changes in elemental distribution observed might be a critical component of the neurotoxic mechanism underlying diabetic neuropathy.
Polymorphisms of GLUT2 and GLUT4 Genes: Use in Evaluation of Genetic Susceptibility to NIDDM in Blacks
Authors: Matsutani Akira , Koranyi Laszlo , Cox Nancy , Permutt M Alan
Published: September 2013

The liver/islet (GLUT2) and muscle/adipose tissue (GLUT4) glucose-transporter gene products, membrane proteins that facilitate glucose uptake into cells, are important molecules for normal carbohydrate metabolism. Recent isolation of the genes encoding these proteins provides a means to assess the role of possible defects that might contribute to impaired glucose-stimulated insulin secretion or impaired insulin-mediated glucose uptake, both prominent phenotypic features of non-insulin-dependent diabetes (NIDDM). A GLUT2 cDNA clone was isolated from a human liver cDNA library to search for polymorphisms at this locus in American Blacks. Three highly polymorphic sites were identified, one of which (EcoRI–Hae III) appears to be due to an insertion and/or deletion of 200 base pairs of DNA. Significant linkage disequilibrium between these sites over ∼30 kilobases of genomic DNA suggested that these polymorphisms could be in linkage disequilibrium with mutations at this locus if they exist. A GLUT4 cDNA clone was also utilized to search for polymorphisms at this locus, but only one previously described polymorphism was observed. GLUT2 and GLUT4 cDNA probes were used to evaluate DNA polymorphisms in genomic DNA from American Blacks with NIDDM. The allelic, genotypic, and haplotypic frequencies of the DNA polymorphisms at these loci did not differ from the frequencies in nondiabetic subjects. Because no associations with NIDDM were found, it appears unlikely that mutations at these loci contribute in a major way to the genetic susceptibility to NIDDM observed in American Blacks.
Role of Two Types of Glucose Transporters in Enlarged Adipocytes From Aged Obese Rats
Authors: Ezaki Osamu , Fukuda Naoko , Itakura Hiroshige
Published: September 2013

The mechanism of insulin-resistant glucose-transport activity in enlarged aged adipocytes was examined. Glucose-transport activity was assessed by measuring 3-O-methylglucose transport and the concentration of HepG2 erythrocyte/glucose transporter (GLUT1), and the muscle/adipose tissue transporter (GLUT4) was estimated by immunoblotting. Basal glucose-transport activity increased 6.3-fold/cell but remained constant per unit cellular surface area due to cell enlargement. Maximal insulin-stimulated transport activity remained constant per cell but decreased per unit cellular surface area. On a per protein basis, GLUT1 and GLUT4 from aged rats decreased to ∼60 and 10% of those from young rats, respectively. However, when the protein content of each fraction and the recoveries of marker enzymes were used for estimating the amount of transporters in intact adipocytes, the amount of GLUT1 per cell remained relatively constant, whereas that of GLUT4 decreased. In basal cells from young rats, 31% of the total GLUT1 per cell was located in the plasma membrane, whereas in those from aged rats, 63% was located in the plasma membrane. Thus, in comparing basal adipocytes from aged rats with those from young rats, GLUT1 per cell in the plasma membrane increased 2.8-fold, but this increase was less than that of transport activity (6.3-fold). In basal cells from young rats, 8% of the total GLUT4 was located in the plasma membrane, and a 4.5-fold increase was observed with insulin treatment, but the amount of GLUT4 in each fraction from aged rats markedly decreased. These findings suggest that the dramatic decrease of GLUT4 is a major cause of decreased insulin-stimulated glucose uptake and that GLUT1 located in the plasma membrane plays a part in maintaining basal glucose-transport activity.
Changes in Phosphoinositide Turnover, Ca2+ Mobilization, and Protein Phosphorylation in Platelets From NIDDM Patients
Authors: Ishii Hidehiro , Umeda Fumio , Ashimoto Toshihiko , Nawata Hajime
Published: September 2013

Enhanced platelet functions have been demonstrated in patients with non-insulin-dependent diabetes mellitus (NIDDM). This study evaluated abnormalities in platelet signal transduction in diabetic patients, including turnover of phosphoinositides, mobilization of intracellular Ca2+, and phosphorylation of 20,000- and 47,000-Mr proteins (P20 and P47). Washed platelets were obtained from 6 patients with NIDDM whose platelet aggregation rates were abnormally elevated (DM-A group), 11 NIDDM patients with normal platelet aggregation rates (DM-B group), and 8 age-matched healthy control subjects. The mass and specific radioactivity of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), and phosphatidic acid (PA) in 32P-labeled platelets were not different among the three groups. Hydrolysis of PIP2, PIP, and PI; accumulation of PA; and phosphorylation of P20 in platelets stimulated by 0.05 U/ml thrombin were significantly increased in the DM-A group compared with the control or DM-B group. There was no difference in P47 phosphorylation among the three groups. On the contrary, P20 and P47 phosphorylation induced by 50 nM of 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, was significantly decreased in the DM-A group. Additionally, the intracellular free Ca2+ concentration ([Ca2+]1) was measured with the fluorescent Ca2+ indicator fura 2. Although the basal [Ca2+]1 value was similar in the three groups, the rise in [Ca2+]1 induced by 0.05 U/ml thrombin in the presence and the absence of extracellular Ca2+ was significantly higher in the DM-A group than the other groups. These data suggest that the enhanced platelet aggregation in NIDDM may be closely related to 7) increases in phosphoinositide hydrolysis, intracellular Ca2+ mobilization, and myosin light-chain kinase-mediated P20 phosphorylation and 2) decreases in P20 and P47 phosphorylation by protein kinase C.
Tumor Necrosis Factor–Increased Hepatic Very-Low-Density Lipoprotein Production and Increased Serum Triglyceride Levels in Diabetic Rats
Authors: Feingold Kenneth R , Soued Mounzer , Adi Saleh , Staprans Ilona , Shigenaga Judy , Doerrler William , Moser Arthur , Carl Grunfeld
Published: September 2013

Previous studies demonstrated that administration of tumor necrosis factor (TNF) to diabetic rats rapidly increases serum triglyceride levels and stimulates hepatic lipogenesis without affecting the activity of adipose tissue lipoprotein lipase or serum insulin levels. The purpose of this study was to determine the mechanism by which TNF increases serum triglyceride levels and stimulates hepatic fatty acid synthesis in diabetic animals. The maximal increase (∼2-fold) in serum triglyceride levels in diabetic rats is seen with a dose of 10 μg TNF/200 g body wt, and the halfmaximal effect is observed with 5 μg TNF/200 g body wt. The clearance of labeled triglyceride-rich lipoproteins from the circulation is not affected by TNF administration (triglyceride t½: diabetic vs. TNFadministered diabetic, 3.5 ± 0.7 vs. 4.0 ± 0.6 min, respectively; NS). The production of triglyceride, measured by the Triton WR-1339 technique, is increased twofold in diabetic animals after TNF administration. These results indicate that the rapid increase in serum triglyceride levels after TNF treatment is accounted for by increased hepatic lipoprotein secretion. TNF administration did not alter either the amount or activation state of hepatic acetyl-CoA carboxylase, a key regulatory enzyme in fatty acid synthesis. There was also no change in the hepatic levels of fatty acyl-CoA, an allosteric inhibitor of acetyl-CoA carboxylase. However, there was a 71% increase in hepatic citrate concentrations. Citrate is an allosteric activator of acetyl-CoA carboxylase, and changes in hepatic citrate concentrations have been shown to mediate changes in the rates of fatty acid synthesis. These results suggest that the TNF-induced stimulation of hepatic lipogenesis is mediated by citrate activation of acetyl-CoA carboxylase. At 2 h after TNF administration, neither serum glucose nor p-hydroxybutyrate levels were adversely altered in the TNF group, indicating that the disturbances in lipid metabolism are not dependent on alterations in glycemic control. The increases in serum triglyceride levels that occur during infections or stress in diabetes may be secondary to TNF.
Nephropathy in Model Combining Genetic Hypertension With Experimental Diabetes: Enalapril Versus Hydralazine and Metoprolol Therapy
Authors: Cooper Mark E , Allen Terri J , O'Brien Richard C , Papazoglou Dimitria , Clarke Belinda E , Jerums George , Doyle Austin E
Published: September 2013

We compared the effects of the angiotensin-converting enzyme inhibitor enalapril and a conventional antihypertensive regimen (hydralazine and metoprolol) on kidney function, albuminuria, and glomerular ultrastructure in hypertensive diabetic and nondiabetic rats. Diabetes was induced with streptozocin at 8 wk of age in spontaneously hypertensive (SHR) rats. Antihypertensive drugs were administered in drinking water from the time of induction of diabetes in all groups. Blood pressure reduction was equal in the diabetic and nondiabetic SHR rats receiving either enalapril or hydralazine plus metoprolol. In diabetic SHR rats, there was a rise in serum creatinine after 32 wk, which did not occur in diabetic rats treated with either antihypertensive regimen or in nondiabetic rats. Both drug regimens reduced albuminuria in diabetic and nondiabetic SHR rats to a similar degree. Enalapril and the combination of hydralazine and metoprolol were associated with decreased glomerular basement membrane thickness and glomerular volume in diabetic and nondiabetic SHR rats without significant effect on fractional mesangial volume. Thus, antihypertensive therapy retards the development of albuminuria, glomerular basement membrane thickening, and glomerular hypertrophy in the rat in the presence or absence of diabetes. No specific benefit of angiotensin-converting enzyme inhibition was observed in these hypertensive models of nephropathy. Human studies comparing the effects of different classes of antihypertensive drugs on kidney function, proteinuria, and glomerular morphology are warranted
Coupling of β-Cell Desensitization by Hyperglycemia to Excessive Stimulation and Circulating Insulin in Glucose-Infused Rats
Authors: Sako Yasuhiro , Grill Valdemar E
Published: September 2013

Nondiabetic rats were infused with glucose for 48 h to maintain moderate or marked hyperglycemia (mean blood glucose 13.2 ± 0.7 or 22.8 ± 0.3 mM, respectively). The two levels of hyperglycemia increased plasma insulin levels severalfold but decreased the insulin response to 27 mM glucose by 19 and 95%, respectively, versus saline infusion. Diazoxide (5 mg ± kg−1 · h−1), when continuously infused during the hyperglycemia protocols, completely inhibited the glucose-induced rise in plasma insulin levels. Diazoxide transformed β-cell insensitivity to stimulation: glucose-induced insulin release was thus increased 318% after moderate hyperglycemia and 707% after marked hyperglycemia. These stimulatory effects of diazoxide were reversed by exogenous insulin infusion (8 or 2 U/24 h) in a dose-dependent manner. It is concluded that excessive β-cell stimulation rather than glucotoxicity underlies hyperglycemia-induced β-cell insensitivity. Effects of hyperinsulinemia can form part of the mechanisms whereby excessive stimulation affects β-cell secretion.
Localization of Rate-Limiting Defect for Glucose Disposal in Skeletal Muscle of Insulin-Resistant Type I Diabetic Patients
Authors: Yki-Järvinen Hannele , Sahlin Kent , Ren Jian M , Koivisto Veikko A
Published: September 2013

We searched for metabolic crossover points in muscle glucose metabolite profiles during maintenance of matched glucose fluxes across forearm muscle in insulin-resistant type I (insulin-dependent) diabetic patients and nondiabetic subjects. To classify subjects as insulin sensitive or insulin resistant, whole-body and forearm glucose disposal, oxidative and nonoxidative glucose disposal (indirect calorimetry), and glycogen synthesis (muscle glycogen content in needle biopsies) were measured under euglycemic conditions at two insulin concentrations. Wholebody and forearm muscle glucose disposal were significantly reduced in diabetic patients compared with control subjects. The reduction in total glucose disposal was due to similar relative reductions in oxidative and nonoxidative glucose disposal, pointing toward rate limitation early in glucose metabolism. The defect in nonoxidative glucose disposal was at least partly due to a defect in muscle glycogen synthesis, because muscle glycogen content failed to increase in response to an increase in the plasma insulin concentration in the diabetic patients. The mostinsulin-resistant type 1 diabetic patients were restudied under conditions where, by glucose mass action, whole-body glucose disposal was forced to be similar to that in the control subjects. Matching glucose fluxes in the two groups resulted in similar rates of forearm and whole-body oxidative and nonoxidative glucose disposal and muscle glycogen synthesis, but it did not result in accumulation of free intracellular glucose, glucose-6-phosphate, glucose-1-phosphate, fructose-6-phosphate, or lactate in muscle. These data imply that the rate-limiting defect for glucose disposal in skeletal muscle of type I diabetic patients is at the level of glucose transport.
Effects of Insulin on Diacylglycerol–Protein Kinase C Signaling in Rat Diaphragm and Soleus Muscles and Relationship to Glucose Transport
Authors: Ishizuka Tatsuo , Cooper Denise R , Hernandez Herman , Buckley Donna , Standaert Mary , Farese Robert V
Published: September 2013

Insulin was found to provoke rapid increases in diacylglycerol (DAG) content and [3H]glycerol incorporation into DAG and other lipids during incubations of rat hemidiaphragms and soleus muscles. Insulin also rapidly increased phosphatidic acid and total glycerolipid labeling by [3H]glycerol, suggesting that insulin increases DAG production at least partly through stimulation of the de novo pathway. Increased DAG production may activate protein kinase C (PKC) as reported previously in the rat diaphragm. We also observed apparent insulininduced translocation of PKC from cytosol to membrane in the rat soleus muscle. The importance of insulin-induced increases in DAG-PKC signaling in the stimulation of glucose transport in rat diaphragm and soleus muscles was suggested by 1) PKC activators phorbol esters and phospholipase C stimulation of [3H]-2-deoxyglucose (DOG) uptake and 2) PKC inhibitors staurosporine and polymixin B inhibition of insulin effects on [3H]-2-DOG uptake. Although phorbol ester was much less effective than insulin in the diaphragm, phospholipase C provoked increases in [3H]-2-DOG uptake that equaled or exceeded those of insulin. In the soleus muscle, phorbol ester, like phospholipase C, was only slightly but not significantly less effective than insulin. Similar variability in effectiveness of phorbol ester has also been noted previously in rat adipocytes (weak) and BC3HI myocytes (strong), whereas DAG, added exogenously or generated by phospholipase C treatment, stimulates glucose transport to a degree that is quantitatively more comparable to that of insulin in each of the four tissues. Differences in effectiveness of phorbol ester and DAG could not be readily explained by postulating that the latter acts independently of PKC, because DAG provoked the apparent translocation of the enzyme from cytosol to membranes in rat adipocytes, and effects of DAG on [3H]-2-DOG uptake were blocked by inhibitors of PKC in both rat adipocytes and BC3H1 myocytes. Collectively, our findings provide further support for the hypothesis that insulin increases DAG production and PKC activity, and these processes are important in the stimulation of glucose transport in rat skeletal muscle and other tissues.
Lack of Predictive Value of Islet Cell Antibodies, Insulin Antibodies, and HLA-DR Phenotype for Remission in Cyclosporin-Treated IDDM Patients
Authors: Mandrup-Poulsen Thomas , Mølvig Jens , Andersen Henrik U , Helqvist Steffen , Spinas Giatgen A , Munck Marianne ,   Canadian-European Randomized Control Trial Group
Published: September 2013

The effect of immunosuppression on the humoral immune response to islet autoantigens and exogenously administered insulin and the predictive value of islet cell cytoplasmic antibodies (ICAs), insulin antibodies (IAs), and HLA-DR phenotype for remission during immunosuppression were studied in a prospective randomized double-blind trial of cyclosporin administration in 98 newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients. HLA-DR phenotype and glycosylated hemoglobin were determined at study entry, and insulin requirement, glucagon-stimulated C-peptide, ICAs, and IAs were measured at entry and after 1, 3, 6, 9, and 12 mo of follow-up. Cyclosporin therapy caused significant suppression of the prevalence and serum concentrations of ICAs and lAs. Cyclosporin-treated IDDM patients ICA+ at study entry had higher levels of stimulated C-peptide after 1 mo of study, but the increased (β-cell function was not associated with a higher frequency of insulin-free remission at 1 mo. ICA and IA status at entry did not predict cyclosporininduced remission as assessed by the prevalence of insulin-free remission or β-cell function at 3–12 mo of study, and significant decrements in the titers or total disappearance of ICAs were not associated with an increased prevalence or duration of non-insulinrequiring remission or higher stimulated C-peptide values. There was no correlation between the serum levels of ICAs and lAs at entry and β-cell function at 12 mo of follow-up. Although patients who were HLADR3/X seemed to respond better to cyclosporin therapy at 6 mo than patients who were HLA-DR3/4 or -DR4/X when analyzed cross-sectionally, a split-plot analysis of variance with time as the split-plot factor showed that the HLA-DR phenotype failed to identify patients with an overall better response to cyclosporin therapy. These data indicate that, although cyclosporin therapy inhibits the humoral immune response to islet components and exogenous insulin, significant decrements in ICA titers are not useful in monitoring efficacy of immunosuppression with cyclosporin, and the determination of ICA and IA status and immune response phenotype at diagnosis is of no predictive value for remission in selecting recent-onset IDDM patients for cyclosporin immunointervention.
Sensitivity of Pyruvate Dehydrogenase to Insulin in Activated T Lymphocytes: Lack of Responsiveness to Insulin in Patients With Polycystic Ovarian Disease and Diabetes
Authors: Buffington Cynthia K , Givens James R , Kitabchi Abbas E
Published: September 2013

Using phytohemagglutinin-activated T lymphocytes, we studied possible mechanisms responsible for insulin resistance in patients with polycystic ovarian disease (PCO) and acanthosis nigricans (AN) by examining insulin binding to erythrocytes and activated T lymphocytes and T-lymphocyte pyruvate dehydrogenase (PDH) responsiveness to insulin in three groups. These groups of subjects consisted of six PCO-AN patients with normal glucose tolerance, six PCO-AN patients with mild non-insulin-dependent diabetes mellitus (NIDDM), and six weight-matched control subjects. We found that insulin binding to both erythrocytes and activated T lymphocytes was significantly lower in PCO and PCO-NIDDM patients than control subjects but did not differ between the PCO groups. Insulin binding to erythrocytes and T lymphocytes varied inversely with basal insulin. In activated T lymphocytes of PCO-NIDDM patients, PDH responsiveness to both submaximal and maximal insulin concentrations was impaired, the extent of which varied in proportion to their degree of carbohydrate intolerance. In contrast, PDH responsiveness to maximal amounts of insulin in T lymphocytes of PCO patients without NIDDM was similar to the weight-matched control subjects. These data may suggest that lesions at the level of the receptor are primarily responsible for insulin resistance in patients with PCO but that both receptor and postreceptor defects (i.e., PDH responsiveness to insulin) contribute to the insulin-resistant state of PCO patients with NIDDM.
Effect of Plasma Amino Acid Replacement on Glucagon and Substrate Responses to Insulin-Induced Hypoglycemia in Humans
Authors: Nair K Sreekumaran , Welle Stephen L , Tito Joseph
Published: September 2013

A defective glucagon response impairs glucose recovery from insulin-induced hypoglycemia in some insulin-dependent (type I) diabetic patients. Our objective was to determine whether the glucagon reponse to insulin-induced hypoglycemia could be stimulated in nondiabetic humans. Because insulin reduces plasma amino acid concentrations, and amino acids are known to stimulate glucagon secretion, we investigated the effect of amino acid replacement during insulin infusion on the glucagon response to hypoglycemia in six healthy nondiabetic subjects. In two separate studies, blood glucose was clamped at the postabsorptive level during a constant infusion of insulin (0.05 U · kg−1 · h−1) for 3 h. Blood glucose was then reduced to 45 mg/dl over 20 min. Hormone and substrate concentrations during recovery from hypoglycemia were monitored for 2 h. In the control study, normal saline was infused, and in the other study, an amino acid mixture was infused to prevent the insulin-induced fall in plasma amino acids. Amino acid replacement did not change the basal glucagon levels but resulted in a more robust glucagon response to hypoglycemia (from 184 ± 24 to 292 ± 36 pg/ml) than in the control study (from 176 ± 30 to an average 229 ± 32 pg/ml, P < 0.01). Plasma concentrations of epinephrine, norepinephrine, cortisol, and growth hormones increased during hypoglycemia, but the magnitude of the response was not different between the control and amino acid-replacement studies. Amino acid replacement did not affect glucose recovery but inhibited the recovery of plasma concentrations of free fatty acids, glycerol, and β-hydroxybutyrate. We conclude that the glucagon response to insulin-induced hypoglycemia is enhanced by amino acid replacement in humans and that changes in plasma amino acid concentrations modulate the effect of stress hormones on lipid metabolism during hypoglycemia.
Acetone and Acetol Inhibition of Insulin-Stimulated Glucose Oxidation in Adipose Tissue and Isolated Adipocytes
Authors: Skutches Charles L , Owen Oliver E , Reichard George A
Published: September 2013

The response of peripheral tissues to insulin is reduced in fasting and diabetes mellitus. The experiments described herein were designed to determine whether insulin-stimulated glucose oxidation is affected by the free–fatty acid–derived plasma metabolites acetone, acetol, and propylene glycol (1,2-propanediol [1,2-PD]), concentrations of which are elevated in both starvation and diabetic ketosis. In epididymal adipose tissue from fed and 48-h–fasted rats given 3% acetone drinking water for 7 days, insulin-stimulated glucose oxidation was reduced by ∼30–40%. After ingestion of 2% acetol for 7 days, basal and insulin-stimulated glucose oxidation was lowered ∼30%, whereas the consumption of 1,2-PD had no influence on either basal or insulin-stimulated glucose oxidation. Similar effects on glucose oxidation were observed in isolated adipocytes from fed rats after ingestion of 3% acetone and 2% acetol for 7 days. The reduction in insulin-stimulated glucose oxidation in adipose tissue in vitro required the consumption of 3% acetone water for >3 days. In 48-h–fasted rats that ingested 3% acetone for 5 days, insulin-stimulated glucose oxidation remained depressed 4 days after withdrawal of acetone from the drinking water. These studies imply that at least part of the insulin resistance indigenous to fasting and diabetic ketosis may be attributed to the metabolic influence of acetone and/or acetol in body fluids. In rats consuming drinking water containing 0.5–5.0% (vol/vol) acetone for 7 days, both acetol and 1,2-PD were found in plasma at concentrations related to the acetone content of the drinking water and to the degree of acetonemia, which ranged from 0.520 ± 0.076 to 15.90 ± 1.43 mM. The concentrations of acetol and 1,2-PD but not acetone were elevated in the plasma of rats given acetol in the drinking water, and only the concentration of 1,2-PD was elevated in the plasma of rats that consumed 1,2-PD. These results indicate that acetone is converted sequentially and irreversibly to acetol and 1,2-PD.
Effect of Cyclosporin on Pancreatic Events and Development of Diabetes in BB/Edinburgh Rats
Authors: Bone Adrian J , Walker Robert , Varey Anne-Marie , Cooke Anne , Baird Joyce D
Published: September 2013

The effect of cyclosporin administered from 30 to 100 days of age on pancreatic events and the development of insulin-dependent diabetes has been studied by serial pancreatic biopsy of individual diabetes-prone BB/Edinburgh rats. Cyclosporin completely prevented the development of diabetes up to 150 days of age and reduced the incidence to 50% of controls at 452 days of age. Islet cell surface antibodies paralleled the development of diabetes. Insulin autoantibodies were unrelated to diabetes and not affected by cyclosporin. Immunohistochemical analysis of pancreatic biopsies from untreated control diabetes-prone rats with monoclonal antibodies specific for rat MHC molecules and T- and B-lymphocyte and macrophage subsets showed that the first abnormality seen in rats that subsequently developed diabetes was hyperexpression of MHC class I molecules on vascular endothelium and islet cells. This was followed by accumulation of ED1 + macrophages at perivascular and periductal sites adjacent to noninfiltrated islets. Increased expression of MHC class II molecules on vascular endothelial cells was also noted. Most cells infiltrating the islets initially were also ED1+ macrophages, followed by increasing numbers of other activated effector cells including helper and cytotoxic-suppressor T lymphocytes and natural killer cells. Obliteration of insulin-containing cells was associated with regression of the infiltrate. Treatment with cyclosporin had no effect on pancreatic hyperexpression of MHC class I molecules but markedly inhibited accumulation of ED1 + cells at extraislet sites, the subsequent recruitment of immune effector cells, and islet infiltration. This resulted in a delay of the onset of diabetes in some rats and prevention of diabetes in others.
Immunological Detection of Phosphotyrosine-Containing Proteins in Rat Livers After Insulin Injection
Authors: Tobe Kazuyuki , Koshio Osamu , Tashiro-Hashimoto Yuko , Takaku Fumimaro , Akanuma Yasuo , Kasuga Masato
Published: September 2013

Tyrosine-phosphorylated proteins in Triton X-100–solubilized fractions of rat livers were examined by immunoblotting with anti-phosphotyrosine antibodies. After 2 min of insulin injection via the portal vein into livers, three major bands of 170,000, 140,000, and 95,000 Mr were stimulated. Because the incubation of nitrocellulose membrane with anti-phosphotyrosine antibodies in the presence of 40 mM phosphotyrosine completely abolished these bands, the anti-phosphotyrosine antibodies appear to recognize the phosphotyrosine residues of these proteins. Insulin injection (2–2000 μg) very quickly stimulated the tyrosine phosphorylation of these proteins in a dose-dependent fashion. In contrast, insulinlike growth factor I or epidermal growth factor injection had little effect in stimulating the tyrosine phosphorylation of these proteins. Because anti-insulin-receptor antibodies immunoprecipitated a tyrosine-phosphorylated 95,000-Mr protein, this protein must be the β-subunit of the insulin receptor; i.e., the β-subunit of the insulin receptor and two other proteins were phosphorylated at tyrosine residues in vivo by insulin injection. These data suggest that the tyrosine phosphorylation and tyrosine kinase activity of the insulin receptor may have important roles in in vivo insulin action.
Cholesterol Metabolism in Alloxan-Induced Diabetic Rabbits
Authors: O'Meara Niall M G , Devery Rosaleen A M , Owens Daphne , Collins Patrick B , Johnson Alan H , Tomkin Gerald H
Published: September 2013

The effect of diabetes control on the activities of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase), cholesterol acyltransferase (ACAT), and phenol 2-monooxygenase, the major enzymes regulating cholesterol metabolism, was determined in alloxan-induced diabetic rabbits, and the results obtained were correlated with lipid and lipoprotein levels. Although intestinal HMG-CoA reductase activity was significantly increased (P < 0.001) in poorly controlled compared with moderately controlled diabetic rabbits, there was a significant reduction in the activities of intestinal ACAT (P < 0.01), hepatic HMG-CoA reductase (P < 0.05) and ACAT (P < 0.001), and phenol 2-monooxygenase (P < 0.01). The poorly controlled animals were hypercholesterolemic (P < 0.01), and this was reflected in the very-low-density and high-density lipoprotein fractions. Serum cholesterol levels in the nondiabetic and moderately controlled diabetic groups were similar. This increase in intestinal HMG-CoA reductase activity in the poorly controlled diabetic animals occurred in the absence of hyperphagia. Although abnormalities in cellular cholesterol metabolism could be partly responsible for the alterations in serum cholesterol levels in diabetes, the precise mechanisms underlying these enzymatic changes have yet to be elucidated.
Immunodeficiency as Primary Phenotype of Diabetes Mutation db: Studies With Coxsackievirus B4
Authors: Montgomery Louise B , Loria Roger M , Chinchilli Vernon M
Published: September 2013

Restriction of food intake (R) in the C57BL/KsJ db/db diabetic mutant mouse prevents phenotypic expression of diabetes, whereas ad libitum feeding (AL) results in spontaneous diabetes. Previous results showed that coxsackievirus B4 (CB4)-infected genetically identical db/db mice with and without diabetes could be distinguished by the levels of CB4-neutralizing antibody and virus-specific antibodies as determined by enzyme-linked immunosorbent assay and the numbers of splenic antibody-forming cells. Our results show that the diabetic genotype db/db R was deficient in total spleen lymphocytes and lymphocyte subsets and was unable to produce agglutinating antibody to sheep erythrocytes (SRBCs) or specific antibody to noninfectious CB4. The db/db AL mutant expressing the diabetic phenotype was not as deficient in spleen cell parameters. The response to noninfectious CB4 was delayed but substantial. The db/db AL mouse was also unique with its higher agglutinating antibody levels after virus infection than its uninfected control or the infected or uninfected db/db R mouse. In vitro SRBC immunization of spleen lymphocytes determined that this enhanced response was largely dependent on the diabetic milieu and was not a property of the cells. Genetic predisposition to diabetes is characterized by immunodeficiency as evident from inadequate levels of antibodies to infectious or noninfectious antigens and absolute and relative deficiency in spleen lymphocyte subsets and total numbers of spleen cells. Phenotypic expression of diabetes results in partial amelioration of the immunodeficiency evident in diabetic genotype db/db R without disease.
Partial Characterization of Mechanism of Insulin Accumulation in H35 Hepatoma Cell Nuclei
Authors: Smith Robert M , Jarett Leonard
Published: September 2013

The mechanism controlling insulin accumulation in nuclei of H35 hepatoma cells was investigated by incubating intact cells with 125|-labeled insulin in the presence or absence of agents that perturb different intracellular sites involved in the processing of ligand-receptor complexes. Purified nuclei were isolated, and nuclear-associated 125|-insulin was determined. Insulin accumulation in the nuclei was time and temperature dependent. Nuclear accumulation was linear and insulin-concentration dependent between 5 and 50 ng insulin/ml. However, pharmacological concentrations of insulin increased the amount of insulin translocated to the nucleus to a far greater extent than it increased total cell-associated insulin. Chloroquine, an acidotrophic agent, increased total cell-associated and intracellular insulin but had no effect on nuclear accumulation. The monovalent ionophores monensin and nigericin inhibited nuclear accumulation of insulin at low concentrations (0.5–5.0 μM) without affecting total insulin binding or intracellular accumulation. At 10 or 25 μM, monensin and nigericin also acted as acidotrophic agents and increased total insulin binding and intracellular accumulation but inhibited nuclear accumulation by a maximum of 50%. Low concentrations of monensin and nigericin were additive; maximal concentrations were not. A 23187 and valinomycin did not affect insulin binding or intracellular and nuclear accumulation of insulin. Neither depletion of ATP by sodium azide, 2, 4-dinitrophenol, sodium cyanide, or oligomycin nor disruption of cytoskeletal elements by cytochalasin D or colchicine had any effect on nuclear accumulation of insulin. The results of this study suggest that 7) lysosomal processing of insulin does not precede translocation of the hormone to nuclei, 2) some of the insulin translocated to the nucleus may be routed through the Golgi, 3) there may be more than one intracellular pathway resulting in the accumulation of insulin in nuclei (but none are energy dependent or require cytoskeletal elements), and 4)pharmacological concentrations of insulin may affect the intracellular processing of the hormone-receptor complex. Nuclear accumulation of insulin may allow insulin to serve as its own second messenger in nuclear events involved in regulating cell growth and gene transcription.
Differential Regulation of Two Glucose Transporters in Rat Liver by Fasting and Refeeding and by Diabetes and Insulin Treatment
Authors: Thorens Bernard , Flier Jeffrey S , Lodish Harvey F , Kahn Barbara B
Published: September 2013

Two species of glucose transporter (GT) are present in the liver: the erythroid/brain GT and the newly characterized liver GT. No information is available regarding the functional role of these two species or whether their expression is regulated concordantly in states in which hepatic glucose uptake or output are markedly altered. In this study, we analyzed the effect of fasting and refeeding and streptozocin-induced diabetes and subsequent insulin treatment on the expression of the erythroid/brain and liver GT polypeptides and their mRNAs in rat liver. The erythroid/brain GT mRNA in livers of control rats corresponded to 1–3% of the amount of liver GT mRNA. After a 4-day fast, its level increased ∼3-fold and represented 8–10% of the liver GT mRNA, whereas the corresponding protein increased 4-fold. In livers from diabetic rats, levels of the erythroid/brain GT mRNA increased up to 2.4-fold and gradually returned to normal with chronic insulin treatment. Levels of the corresponding protein were minimally altered. Levels of immunoreactive liver GTs were not significantly changed by 2 days of fasting, 7 or 14 days of diabetes, or subsequent insulin treatment for 3, 5, or 7 days but increased up to 75% with refeeding for 3–48 h. Liver GT mRNA levels minimally decreased in diabetic or insulin-treated rats, decreased 45% after a 2-day fast, and increased up to 5-fold on refeeding for 24 h. These results indicate that 1) the liver GT is the main transporter species in the liver, 2) its expression is not affected by the altered metabolic states studied, with the exception of a slight elevation with refeeding, and 3) erythroid/brain GT expression in liver is increased by fasting. Because regulation of the erythroid/brain GT in liver is unlike its regulation in adipocytes under the same metabolic conditions, this indicates that the control of its expression is tissue specific. Furthermore, the two species of GTs in the liver are differentially regulated, and posttranscriptional events play a major role in determining their level of expression.
Lack of Systematically Found Insulin Autoantibodies in Spontaneously Diabetic BB Rats
Authors: Markholst Helle , Klaff Leslie J , Klöppel Günter , Lernmark ÅKe , Mordes John P , Palmer Jerry
Published: September 2013

Insulin autoantibodies (IAAs) occur in newly diagnosed human insulin-dependent diabetes mellitus (IDDM) patients, but their presence in BB rats is controversial, possibly due to assay differences or variability in the animals studied. To resolve this controversy, lAAs were measured in well-characterized inbred BB rats both in radioligand assays with 125I-labeled rat insulin I or II, respectively, and in an enzyme-linked immunosorbent assay (ELISA) with rat insulin as antigen. In prospective studies, a total of 57 serums from 16 diabetes-prone (DP) BB rats were obtained during an interval ranging from 15 wk to the last week before onset and at onset of diabetes. At comparable ages, 21 serums were obtained from 8 DP BB rats not developing diabetes, and 70 matched serums were obtained from 19 diabetes-resistant (DR) BB rats. Levels of antibody binding increased slightly with increasing age in DP and matched DR rats. Two rats were positive at onset of IDDM in all assays but not in earlier samples. Otherwise, only few isolated serums from both types of rats regardless of diabetes had increased binding in one of the assays. In a cross-sectional study, the insulin-binding levels in 150-day-old DP rats (n = 20) that had not yet developed diabetes did not correlate with insulitis present in 3 of 20 rats and did not differ from 150-day-old DR BB rats (n = 20). Diabetic BB rats (n = 20) killed at 150 days of age after treatment with protamine zinc insulin for 1–3 mo showed increased binding levels (P < 0.01–0.001) in all assays compared with the DR and DP rats. We conclude that 1) IAAs may be present at low frequency at onset of IDDM in BB rats, 2) different patterns of reactivity are found in the two radioligand assays and in the ELISA, and 3)IAAs are not a marker for later development of IDDM or insulitis in BB rats.