bluuebunny/crossref_metadata_2025 huggingface dataset:
Normalization by Insulin Treatment of Low Mitochondrial Glycerol Phosphate Dehydrogenase and Pyruvate Carboxylase in Pancreatic Islets of the GK Rat
Authors: MacDonald Michael J , Efendic Suad , Ostenson Claes-Goran
Published: September 2013

The enzyme activity of the mitochondrial glycerol phosphate dehydrogenase (mGPD) in the pancreatic islet has been reported to be less than one-half of normal in the Goto-Kakizaki (GK) rat, a genetic model of NIDDM. In the current study, mGPD enzyme activity and the amount of mGPD protein, as judged by Western analysis, were 35–40% of normal in the islets of these animals. With the exception of pyruvate carboxylase, the activities of other enzymes were not abnormal. The assayable activity and amount of pyruvate carboxylase protein were decreased ∼50% in the islets of the GK rats. Because mGPD, which is the key enzyme of the glycerol phosphate shuttle, and pyruvate carboxylase, which is the key component of the pyruvate malate shuttle, have been proposed to be essential for stimulus-secretion coupling in the pancreatic β-cell, an important question is whether the decreases in these enzymes have a causal role in the hyperglycemia or whether the diabetic syndrome caused the decreases. To attempt to differentiate between these two possibilities, GK rats were treated with insulin to normalize their blood sugars. The activities of both mGPD and pyruvate carboxylase were also normalized by insulin treatment. An incidental discovery of this study was the identification of a high level of propionyl-CoA carboxylase activity and a lesser amount of methylcrotonyl-CoA carboxylase activity in pancreatic islets. These enzymes were normal in the islets of the GK rats. This is the first report on the presence of these two carboxylases in the islet and of low pyruvate carboxylase activity in the islet in NIDDM. We conclude that the decreased mGPD and pyruvate carboxylase in the pancreatic islet of the GK rat result from the diabetic syndrome.
Impaired Coupling of Glucose Signal to the Exocytotic Machinery in Diabetic GK Rats
Authors: Abdel-Halim Samy M , Guenifi Amel , Khan Akhtar , Larsson Olof , Berggren Per-Olof , Ostenson Claes-Goran , Efendic Suad
Published: September 2013

The GK rat is a spontaneous model of NIDDM. The insulin response to 16.7 mmol/l glucose was markedly impaired in both isolated perfused pancreas and isolated islets from GK rats compared with control Wistar rats. Depolarization with 30 mmol/l KC1 in the presence of 3.3 mmol/l glucose and 250 μmol/l diazoxide induced similar insulin responses in perfused pancreases of GK and control rats. In contrast, the glucose-stimulated insulin release was also severely impaired in GK pancreases in the depolarized state. Forskolin (1 μmol/l) markedly enhanced insulin release at 3.3 mmol/l glucose in GK but not control pancreases (54 ± 15 vs. 3 ± 1 pmol/l0 min, P < 0.001). Dibutyryl cAMP (1 mmol/l) exerted effects similar to forskolin on insulin release in the perfused pancreas. In depolarized pancreases of GK but not control rats, forskolin also induced a marked insulin response at 3.3 mmol/l glucose (163 ± 48 vs. 16 ± 1 pmol/20 min,< P < 0.03). Similarly, in studies on isolated islets from GK rats cultured in 5.5 or 16.7 mmol/l glucose for 48 h, forskolin (5 μmol/l) restored insulin release in response to 16.7 mmol/l glucose but had no effect on islet glucose utilization at 3.3 or 16.7 mmol/l glucose. Forskolin markedly stimulated insulin release at 3.3 mmol/l glucose in GK but not control rat islets cultured for 48 h in 5.5 mmol/l glucose, whereas 20 mmol/l arginine had an almost identical effect in both islet varieties. However, in islets cultured in 16.7 mmol/l glucose, forskolin stimulated insulin release similarly both in control and GK islets at 3.3 mmol/l glucose. In conclusion, this study suggests that the insulinotropic effects of glucose are coupled to a direct regulation of the exocytotic machinery in the pancreatic 3-cell. This pathway is markedly impaired in GK rats, contributing to defective insulin response to glucose. In this model, cAMP generation restores the insulin response to 16.7 mmol/l glucose and exerts a marked insulin release even at 3.3 mmol/l glucose.
Lithium Stimulation of Rat Pancreatic β-Cell Replication Is Mediated Through Pertussis Toxin-Sensitive GTP-Binding Proteins and Occurs Independently of Ca2+ Influx, cAMP, or Protein Kinase C Activation
Authors: Sjöholm Åke
Published: September 2013

We recently demonstrated the mitogenic and secretagogic actions of lithium in the insulin-producing pancreatic β-cell (Sjöholm Å, Welsh N, Hellerström C: Lithium increases DNA replication, polyamine content and insulin secretion by rat pancreatic β-cells in vitro. Am J Physiol 262:C391-C395, 1992). In this study, the influence of lithium on β-cell signal transduction pathways was monitored and their importance for the stimulated cell replication and hormone secretion was elucidated by selective pharmacological probes. To this end, fetal rat pancreatic islets enriched in β-cells were isolated and cultured for 3 days with or without 10 mmol/l LiCl. This resulted in a marked mitogenic response by the β-cells, of similar magnitude to that obtained by pharmacological activation of cAMP-dependent protein kinases by forskolin or the Sp-diastereomer of cAMP or protein kinase C stimulation by phorbol ester. However, neither did lithium affect the islet content of cAMP (whereas forskolin did), nor was the mitogenic response to the ion impeded when islets were pretreated with the Rp-diastereomer of cAMP, a specific antagonist of cAMP-dependent protein kinases, or by the Ca2+ channel blocker D-600. The protein kinase C inhibitor l-(5′-isoquinolinesulfonyl)-2-methylpiperazine prevented the mitogenicity of phorbol ester, but not that of lithium. Conversely, addition of increasing concentrations of inositol along with lithium also did not affect the mitogenicity of the ion, providing indirect evidence against the involvement of protein kinase C in mediating the growth-promoting effect of lithium in this system. It was found that pretreatment of islets with pertussis toxin, which inactivates GTP-binding proteins by ADP-ribosylation, prevented the mitogenicity and part of the secretagogic action of lithium. It is concluded that although specific activation of the cAMP and protein kinase C signaling systems appears sufficient to trigger a mitogenic response of the β-cell, lithium seemingly does not work through any of these systems nor via Ca2+ influx in promoting β-cell mitogenesis. Our results, moreover, suggest that the actions of lithium are conveyed by pertussis toxin-sensitive GTP-binding proteins.
The Insulin-Mimetic Agent Vanadate Promotes Receptor Endocytosis and Inhibits Intracellular Ligand-Receptor Degradation by a Mechanism Distinct From the Lysosomotropic Agents
Authors: Fantus I George , George Rajan , Tang Shangguo , Chong Paul , Poznansky Mark J
Published: September 2013

Vanadate (sodium orthovanadate) is an insulin-mimetic agent and phosphotyrosine phosphatase inhibitor that has been proposed as a potential therapeutic agent for diabetes. We previously reported that vanadate decreased the number of cell-surface insulin receptors but inhibited receptor degradation in cultured lymphocytes (IM-9) (1). To determine whether vanadate affected receptors without intrinsic tyrosine kinase activity, its effects on LDL and transferrin receptors and their ligands were examined. Vanadate exposure resulted in a doseand time-dependent decrease in LDL binding to cultured human fibroblasts associated with a decrease in cell surface receptor number while total solubilized cell LDL receptors increased. Vanadate also inhibited the LDLmediated downregulation of total cellular LDL receptors in the absence and presence of cycloheximide consistent with an inhibition of LDL receptor degradation. In the case of the ligand, vanadate augmented the accumulation of intact 125I-LDL associated with an inhibition of up to 80% of the ability of LDL to decrease cholesterol synthesis. Since these actions were similar to the effects of lysosomotropic agents, we examined the effect of vanadate on intraendosomal pH using the fluorescent probe acridine orange. In contrast with chloroquine and NH4C1, vanadate did not neutralize the pH of the acidic intracellular compartment. Furthermore, after a transient insulin-like effect, chronic exposure to vanadate diminished 125I-diferric transferrin binding to rat adipocytes. In contrast with the inhibitory action of NH4C1, intracellular 59Fe uptake remained unaffected and was proportional to cell-surface binding capacity in the presence of vanadate. These data demonstrate a chronic effect of vanadate to promote the accumulation of intracellular receptors and to inhibit ligand and receptor degradation. The latter effect is not mediated by pH changes, appears to be localized to a late endosoma/Mysosomal compartment, and suggests a possible role for tyrosine dephosphorylation in the regulation of receptor-ligand degradation.
Kinetics of Intraperitoneally Infused Insulin In Rats: Functional Implications for the Bioartificial Pancreas
Authors: De Vos Paul , Vegter Djoeke , De Haan Bart J , Strubbe Jan H , Bruggink Jan E , Van Schilfgaarde Reinout
Published: September 2013

Intraperitoneal transplantation of encapsulated islets can restore normoglycemia in diabetic recipients but not normal glucose tolerance nor normal insulin responses to a physiological stimulus. This study investigates whether the intraperitoneal implantation site as such contributes to the interference with optimal transport kinetics between the islets and the bloodstream. Insulin was infused into the peritoneal cavity of conscious and freely moving rats in doses of 20, 40, and 80 pmol · I−1 · min−1 during 15 min, to mimic the gradual release of insulin from an encapsulated, i.e., a nonvascularized, islet graft. With 20 pmol · I−1 · min−1, we observed virtually no rise of insulin levels, and it took 30 min until glucose levels had dropped significantly. With 40 and 80 pmol · I−1 · min−1 insulin infusions, there was a dose-dependent rise of insulin and decrease of glucose levels. When compared with intraportal infusions with the same insulin dosages, however, they were strongly delayed and reduced as well as prolonged. Similar results were obtained when inulin instead of insulin was intraperitoneally infused, which indicates that the transport of insulin from the peritoneal cavity to the bloodstream is mainly by passive diffusion. With a view on the clinical efficacy of the bioartificial pancreas, our findings indicate that we should focus on finding or creating a transplantation site that, more than the unmodified peritoneal cavity, permits close contact between the bloodstream and the encapsulated islet tissue.
Insulin Sensitivity and Body Weight Changes in Young White Carriers of the Codon 64 Amino Acid Polymorphism of the β3-Adrenergic Receptor Gene
Authors: Urhammer Søren A , Clausen Jesper O , Hansen Torben , Pedersen Oluf
Published: September 2013

Recently, a missense mutation replacing tryptophan with arginine at codon 64 of the β3-adrenergic receptor gene was shown to be associated with insulin resistance in nondiabetic subjects and to an earlier onset of NIDDM in Pima Indians. We studied whether the codon 64 amino acid polymorphism of the β3-adrenergic receptor gene in a cohort of young healthy Danes was associated with high birth weight, accelerated weight gain during childhood and adolescence, present obesity, or impaired insulin sensitivity. The protocol included 380 unrelated white subjects in whom insulin sensitivity and secretion were measured during a combined intravenous glucose and tolbutamide tolerance test. A number of biochemical and anthropometric characteristics were determined for each subject. The subjects were genotyped for the codon 64 polymorphism by applying polymerase chain reaction restriction fragment-length polymorphism screening with the use of endonuclease BstNl. The allelic frequency of the mutated allele was 7% (95% CI: 5–10%), and it was similar in obese and nonobese subjects. The β3-adrenergic receptor gene variant was not related to birth weight or weight gain during childhood or adolescence. In its heterozygous form, the gene variant was not associated with an altered insulin sensitivity index (SI) or other features of the insulin resistance syndrome (BMI, blood pressure, fasting serum lipid levels, or fasting serum fibrinolytic variables). Three homozygous carriers of the polymorphism were identified, and each had a significantly higher BMI (27.4 ± 1.3 vs. 23.5 ± 3.7 kg/m2 [mean ± SD]; P = 0.032), lower SI [4.9 ± 2.9 vs. 15.4 ± 9.0 10−5 × (min × pmol/l)−1; P = 0.013], and higher fasting serum C-peptide (730 ± 155 vs. 471 ± 158 pmol/l; P = 0.016) than the wild-type carriers. The homozygous carriers also had significantly higher levels of fasting serum triglyceride (P = 0.042) and serum LDL cholesterol (P = 0.013). When adjustments were made for age, sex, BMI, and VO2max in a multiple regression analysis, a significantly negative association was found between homozygosity for the codon 64 variant and the SI (P = 0.009). We conclude that in young healthy Danes, the homozygous form but not the heterozygous form of the codon 64 amino acid polymorphism of the β3-adrenergic receptor may be associated with obesity and, independent of BMI, with a low SI Since only three homozygous carriers were identified among 380 subjects, the results must be interpreted with caution, and studies of larger population samples are needed.
Lymphocyte Function Associated Antigen-1, Integrin α4, and L-Selectin Mediate T-Cell Homing to the Pancreas in the Model of Adoptive Transfer of Diabetes in NOD Mice
Authors: Fabien Nicole , Bergerot Isabelle , Orgiazzi Jacques , Thivolet Charles
Published: September 2013

The involvement of adhesion molecule in the process of T-cell homing to the pancreas was investigated in the model of the T-cell transfer of type I diabetes in NOD mice. Treatment of mice using monoclonal anti-lymphocyte function associated antigen (LFA)-l, anti-integrin α4, anti-intercellular adhesion molecule (ICAM)-1, and anti-L-selectin antibodies (monoclonal antibodies [mAbs]) gave rise to a partial or complete prevention of diabetes via different mechanisms of protection. On day 20 posttransfer, diabetes was only observed in control mice (26 of 32) and in few mice treated with the anti-L-selectin mAbs (3 of 24). On day 60, the best protection was observed using the anti-LFA-1 or the anti-integrin α4 mAbs with 3 of 11 and 2 of 5 diabetic mice, respectively. On day 20, no insulitis was observed in the pancreases of mice treated with these mAbs compared with the pancreases of controls, suggesting that such treatment blocked the penetration of T-cells into the islets. In vitro adhesion assays confirmed that adhesion of T-cells to the pancreatic endothelium was blocked, except when using the anti-L-selectin mAb, which induced a modification of the traffic of the transferred T-cells; the ability of T-cells to migrate into the pancreatic lymph nodes was significantly reduced (10.4 vs. 22%). Anti-LFA-1 mAbs did not modify such T-cell trafficking. The present study, therefore, elucidates the role of LFA-1, integrin α4, and L-selectin in T-cell homing to the pancreas, first step of the cascade of events leading to type I diabetes.
Abnormalities of Retinal Metabolism in Diabetes or Experimental Galactosemia. III. Effects of Antioxidants
Authors: Kowluru Renu A , Kern Timothy S , Engerman Ronald L , Armstrong Donald
Published: September 2013

Effects of antioxidants on hyperglycemia-induced alterations of retinal metabolism were evaluated in rats diabetic or experimentally galactosemic for 2 months. Oxidative stress was estimated by measuring lipid peroxides (measured as thiobarbituric acid reactive substances [TEARS]) in retina and plasma. Erythrocyte osmotic fragility, another measure of oxidative stress, also was determined in the same groups of rats. In diabetic rats, TBARS were elevated by 74% in retina and 87% in plasma. In galactose-fed rats, TBARS were significantly elevated in retina (P < 0.05), but were normal in plasma. The administration of supplemental dietary ascorbic acid and α-tocopherol acetate for 2 months prevented the elevation of retinal TBARS and the decrease of Na+-K+- ATPase and calcium ATPase activities in retinas of diabetic animals without having any beneficial effect on plasma TBARS. In galactosemic rats, these antioxidants had a partial beneficial effect on the activity of retinal Na+-K+-ATPase, but failed to have any effect on calcium ATPase. The beneficial effects of antioxidants in diabetes and experimental galactosemia were not caused by the amelioration of hyperglycemia or retinal polyol accumulation. Erythrocyte osmotic fragility was increased by more than twofold in diabetes, but was normal in experimental galactosemia, and antioxidants prevented diabetes- induced increases in erythrocyte osmotic fragility. Diabetes-induced increased oxidative stress and subnormal ATPase activities in the retina can be inhibited by dietary supplementation with antioxidants.
Effect of Mitochondrial and/or Cytosolic Glycerol 3-Phosphate Dehydrogenase Overexpression on Glucose-Stimulated Insulin Secretion From MIN6 and HIT Cells
Authors: Ishihara Hisamitsu , Nakazaki Mitsuhiro , Kanegae Yumi , Inukai Kouichi , Asano Tomoichiro , Katagiri Hideki , Yazaki Yoshio , Kikuchi Masatoshi , Miyazaki Jun-ichi , Saito Izumu , Oka Yoshitomo
Published: September 2013

The glycerol phosphate shuttle consists of FAD-linked mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) and its cytosolic NAD-linked isoform (cGPDH). Impaired mGPDH activity has recently been suggested to be one of the primary causes of insulin secretory defects in β-cells. We found that mGPDH and cGPDH activities in MIN6 cells are comparable to those of isolated islets and higher than those in HIT cells by eightfold and threefold, respectively. Therefore, we selected the MIN6 cell line as a β-cell model with normally regulated insulin secretion and normal shuttle enzyme activities and the HIT cell line as a β-cell model with impaired insulin secretion and lower activities of these enzymes. The role of these dehydrogenases in glucose-stimulated insulin secretion was addressed by examining the effects of overexpression of mGPDH and/or cGPDH via recombinant adenoviruses in these cells. Infection with recombinant adenovirus with a cDNA encoding the Escherichia coli β-galactosidase gene resulted in expression of its gene in 90% of MIN6 and HIT cells. Infection with a recombinant adenovirus with mGPDH cDNA (AdexlCAmGPDH) caused 2.1- fold and 5.7-fold increases in dehydrogenase activity as compared with those of control MIN6 and HIT cells, respectively. Infection with a recombinant adenovirus with cGPDil cDNA (AdexlCAcGPDH) caused a more than 50-fold increase in activity in both cell lines. Glycerol phosphate shuttle flux, as estimated by [2-3H]glycerol conversion to [3H]H2O, was increased to 120–130% by infection with AdexlCAmGPDH, but not with AdexlCAcGPDH infection, in both MIN6 and HIT cells. No further increase in flux through the glycerol phosphate shuttle was detected when the cells were infected with AdexlCAmGPDH together with AdexlCAcGPDH. Furthermore, neither [U-14C]glucose oxidation nor the insulin secretory response to glucose was affected in either cell line. Thus, mGPDH abundance in MIN6 and HIT cells is not directly related to their insulin secretory capacity in response to glucose, and reduced expression of mGPDH is not the primary cause of abnormal insulin secretory responses in HIT cells. The present data indicate that the emerging hypothesis pointing to mGPDH deficiency as a possible cause of NIDDM needs to be carefully evaluated.
The Influence of Diabetes on the Vasomotor Responses of Saphenous Vein and the Development of Infra-Inguinal Vein Graft Stenosis
Authors: Hicks Robert C J , Moss Jill , Higman Dan J , Greenhalgh Roger M , Powell Janet J
Published: September 2013

There has been a prejudice that diabetes modulates the function of saphenous vein in a manner that predisposes to bypass graft failure, although most of the evidence accrues from animal studies. We have investigated the effect of diabetes on the vasodilator responses and ultrastructure of saphenous vein harvested from patients undergoing infrainguinal bypass surgery for limb salvage and the development of stenoses within the vein grafts. Of 55 consecutive patients undergoing vein bypass surgery for critical ischemia, 16 (29%) were diabetic: diabetes was not a risk factor for graft stenosis, which occurred in 17 of 56 (30%) grafts. Endotheliumdependent relaxation by nitric oxide pathways stimulated after receptor activation (bradykinin and thrombin) was not different in vein rings from diabetic (n = 12) and nondiabetic patients (n = 12). Prostanoid-mediated vasorelaxation was absent in vein rings from diabetic patients, and the production of 6-keto prostaglandin F1α (PGFlα) from diabetic vein was only 66 ± 27 pg · cm−2 · min™1 compared with 112 ± 20 pg · cm−2 · min−1 from control vein (P = 0.011). Fibrinogen-mediated vasorelaxation, normally inhibited by K+ channel blockers, was negligible in vein from diabetic patients. No ultrastructural differences were observed between the endothelium of saphenous vein harvested from diabetic and nondiabetic patients. However, diabetes was associated significantly with the presence of spiraled collagen in media. The maintenance of receptor-activated stimulation of nitric oxide pathways and the damping of the response to fibrinogen in saphenous vein endothelium may provide, in part, for the good prognosis of vein graft surgery in diabetic patients: diabetes is not a risk factor for early (12 months) infrainguinal vein graft stenosis.
Oxidative and Chemical Stress Mimic Insulin by Selectively Inhibiting the Expression of Phosphoenolpyruvate Carboxykinase in Hepatoma Cells
Authors: Sutherland Calum , Tebbey Paul W , Granner Daryl K
Published: September 2013

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the rate-limiting step in hepatic gluconeogenesis. Glucagon (via the second messenger cAMP), retinoic acid, and glucocorticoids stimulate transcription of the PEPCK gene, whereas insulin and phorbol esters have a dominant inhibitory effect. We now show that oxidative and chemical stress (hydrogen peroxide and sodium meta-arsenite, respectively) also produce a dominant inhibitory effect, both on the endogenous PEPCK gene and on a stably transfected PEPCK–chloramphenicol acetyl transferase (CAT) fusion gene. Wortmannin, an inhibitor of 1-phosphatidylinositol 3- kinase (PI 3-kinase), blocks the inhibition of glucocorticoid and cAMP-induced PEPCK gene transcription by insulin; however, it has no effect on the inhibition elicited by oxidative or chemical stress. Thus, the mechanism(s) used by hydrogen peroxide and sodium meta-arsenite to regulate PEPCK gene expression are PI 3-kinase independent. This suggests that these agents operate by a pathway distinct from that used by insulin or that the pathways converge at a point downstream of PI 3-kinase. The reactivating kinase (RK, also known as p38 mitogen activated protein kinase) is induced by insulin, hydrogen peroxide, or sodium metaarsenite in hepatoma cells, and these effects are blocked by SB203580, a selective inhibitor of RK. However, SB203580 has no effect on the ability of any of these agents to regulate PEPCK-CAT fusion gene expression. Thus, although RK has a role in the regulation of lymphokine gene expression in monocytes, it is not required for the regulation of PEPCK expression by either insulin or oxidative and chemical stress in hepatoma cells. From the Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee.
Effect of Extracellular Matrix Glycation on Endothelial Cell Adhesion and Spreading: Involvement of Vitronectin
Authors: Boobink Inge W G , de Boer Hetty C , Tekelenburg Winnie L H , Banga Jan-Drik , de Groot Philip G
Published: September 2013

Glycation of proteins of the vessel wall is thought to play an important role in the pathogenesis of vascular complications in diabetes by affecting structure and function of these proteins. Adhesive proteins in the extracellular matrix (ECM) of endothelial cells (ECs) are essential for attachment of ECs to the subintima. In this study, we investigated the effect of glycation of ECM and purified adhesive proteins on EC adhesion and spreading. ECM was incubated with the reactive sugar glucose-6-phosphate (0–500 mmol/l) for different time periods (0–14 days) at 37°C. Degree of glycation, measured in an enzyme-linked immunosorbent assay using a monoclonal antibody specific for advanced glycation end products, increased in a timeand concentration-dependent manner. Glycation of ECM with 50 mmol/l glucose-6-phosphate resulted in increased coverage by ECs as measured in a cell adhesion assay and was the result of an increase in number of adhered cells, while cell size was unaffected. Glycation of ECM with higher concentrations of glucose-6-phosphate resulted in decreased coverage by ECs caused by both a reduction in number of adhered ECs and impaired spreading. Experiments with purified glycated matrix proteins indicate that the decrease in EC adhesion and spreading on glycated ECM may result from glycation of vitronectin. Impaired EC adhesion and spreading caused by vitronectin glycation may result in impaired endothelial function and contribute to vascular disease.
Impaired β-Cell Function and Deposition of Fat Droplets in the Pancreas as a Consequence of Hypertriglyceridemia in OLETF Rat, a Model of Spontaneous NIDDM
Authors: Man Zhi-Wei , Zhu Min , Noma Yoshihiko , Toide Kiyotaka , Sato Takashi , Asahi Yoshihiko , Hirashima Tsukasa , Mori Shigehito , Kawano Kazuya , Mizuno Akira , Sano Toshiaki , Shima Kenji
Published: September 2013

Hypertriglyceridemia is known to be a feature of obesity-related NIDDM, but the patho-etiological significance of this association is obscure. The effects of triglycerides (TGs) on β-cell function and morphological changes in pancreas were examined using in vivo and in vitro approaches in male OLETF rats at ages 6, 12, and 30 weeks, with their diabetes-resistant counterpart, LETO rats, as normal controls. The results showed that, in the fasting state, plasma TGs in OLETF rats were increased 2.5-fold at age 6 weeks, 3.3-fold at age 12 weeks, and 6.2-fold at age 30 weeks, compared with age-matched LETO rats. The TG content in islets from 12-week-old OLETF rats was significantly increased when compared with those from their age-matched counterparts, but this was not the case with the 6-week-old OLETF rats. Therefore, the islets from 6-week-old rats were cultured with either free fatty acids (FFAs; 1.0 mmol/1 sodium oleate) or TG (5.0 mmoM Intralipide) for 72 h. Several abnormalities in OLETF rats were evident, in contrast to the results from control LETO rats: 1) glucose-induced insulin secretion was more inhibited by either FFAs or TGs in the presence of 27.7 mmol/l glucose, a result associated, at least in part, with reduced glucokinase activity in the islets; 2) a marked elevation in TG content was found in the islets; and 3) the deposition of fat droplets in the enlarged islets, even in the β-cells, was found by Oil Red O-insulin double staining at age 30 weeks. In conclusion, hypertriglyceridemia resulted in significant TG stores in the islets, which subsequently inhibited glucose-induced insulin secretion, at least in part, via reduced glucokinase activity in the islets. Fat droplets in islets, therefore, may play an important role in hastening the development of NIDDM in this rat model.
Insulin Signaling in Human Skeletal Muscle: Time Course and Effect of Exercise
Authors: Wojtaszewski Jørgen F P , Hansen Bo F , Kiens Bente , Richter Erik A
Published: September 2013

Activation of early steps in the insulin signaling cascade in human skeletal muscle was investigated using a one-step euglycemic-hyperinsulinemic (∼100 μU/ml) clamp in seven healthy young men 3 h after one-legged exercise. Concomitant insulin stimulation (three- to six-fold [P < 0.05]) of thigh glucose clearance, muscle insulin receptor tyrosine kinase (IRTK), insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, and IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) was observed in the rested leg. Twenty minutes after cessation of insulin infusion, the level of these parameters returned toward basal. A twofold higher insulin-stimulated glucose clearance in the exercised compared with the rested thigh was accompanied by unaltered maximal IRTK activation and IRS-1 tyrosine phosphorylation, and by a decreased (∼50%, P < 0.05) maximal IRS-1 associated PI 3-kinase activation. Prior exercise caused significantly faster insulin-stimulated tyrosine phosphorylation of IRS-1, PI 3-kinase activity, and glucose clearance compared with those in the rested thigh. In conclusion, physiological hyperin-sulinemia activates IRTK, IRS-1 tyrosine phosphorylation, and PI 3-kinase in human skeletal muscle. However, increased insulin action after exercise is not caused by potentiation of these steps in the insulin signaling cascade. In contrast, at steady state, paradoxically decreased insulin-stimulated IRS-1-associated PI 3-kinase activity was observed in exercised muscle. Thus, the activity of IRS-1-associated PI 3-kinase and glucose uptake may not always be tightly coupled during insulin stimulation in human muscle.
Evidence of an Increased Number of Type IIb Muscle Fibers in Insulin-Resistant First-Degree Relatives of Patients with NIDDM
Authors: Nyholm Birgit , Qu Zhuqing , Kaal Andreas , Pedersen Steen Bønnelykke , Gravholt Claus Højbjerg , Andersen Jesper Løvind , Saltin Bengt , Schmitz Ole
Published: September 2013

Insulin resistance is a common feature in first-degree relatives of NIDDM patients. To explore the mechanism(s) behind this condition in more detail, a percutaneous muscle biopsy (vastus lateralis) was performed in 25 first-degree relatives of NIDDM patients and 21 control subjects to examine muscle fiber composition and capillary density. Insulin-stimulated glucose disposal (Rd) was determined employing a hyperinsulinemic-(insulin infusion rate 0.6 mU · kg−1 · min−1) euglycemic clamp. Rd (5.76 ± 0.35 vs. 8.06 ± 0.36 mg · kg lean body weight [LBW]−1 · min−1 P < 0.001) and estimated Vo2max (49.3 ± 2.8 vs. 57.2 ± 3.5 mg · kg LBW−1 · min−1, 0.05 < P < 0.10) were decreased in the relatives. The number of type lib fibers (29.5 ± 2.5 vs. 21.0 ± 2.8%, P < 0.05) was increased in the relatives, whereas no significant differences were found in other fiber types or capillary density between the groups. Correlations were observed between number of type I fibers (positive), number of type lib fibers (negative), and capillary density (positive) versus Rd as well as estimated Vo3max (P < 0.05). In a multiple linear regression analysis with Rd as a dependent variable, estimated Vo2max, family history of NIDDM, and number of type lib fibers (P < 0.001, r2 = 0.64) significantly determined the level of Rd, whereas capillary density did not. In conclusion, insulin-resistant first-degree relatives of NIDDM patients are characterized by an increased number of type lib muscle fibers. Whether this finding reflects a reduced physical activity level and fitness in the relatives or is of primary genetic origin remains to be determined.
Aerobic Exercise Capacity Remains Normal Despite Impaired Endothelial Function in the Micro- and Macrocirculation of Physically Active IDDM Patients
Authors: Veves Aristidis , Saouaf Rola , Donaghue Valerie M , Mullooly Catherine A , Kistler Joy A , Giurini John M , Horton Edward S , Fielding Roger A
Published: September 2013

The aim of the present study was to examine if diabetes in the absence of neuropathy affects the exercising capacity of IDDM patients, and whether regular, intense training has a beneficial effect on endothelial function. Five groups of subjects were studied: 23 healthy control subjects who exercised regularly (age 33 ± 6 years), 23 nonneuropathic type 1 diabetic patients who exercised regularly (age 33 ± 6 years, IDDM duration 11 ± 8 years), 7 neuropathic type 1 diabetic patients who exercised regularly (age 36 ± 7 years, IDDM duration 22 ± 8 years), 18 healthy subjects who did not exercise regularly (age 34 ± 7 years), and 5 nonneuropathic type 1 diabetic patients who did not exercise regularly (age 31 ± 4 years, IDDM duration 20 ± 3 years). All groups were matched for age, sex, and body weight. No differences existed in the energy expenditure per week in physical activity among the three exercising groups or between the two nonexercising groups. The maximal oxygen uptake was similar between control and diabetic nonneuropathic exercisers, and among diabetic neuropathic exercisers, control nonexercisers, and diabetic nonexercisers; however, a significant difference existed between the first two and the last three groups (P < 0.0001). A stepwise increase was found in the resting heart rate among the groups, ranging from the lowest in control exercisers to the highest in diabetic nonexercisers, but the maximal heart rate was lower only in diabetic neuropathic exercisers compared with all other groups (P < 0.05). Assessments of endothelial function in both macro- and microcirculation were performed in 12 control exercisers, 10 diabetic nonneuropathic exercisers, 5 diabetic neuropathic exercisers, 17 control nonexercisers, and 4 diabetic nonexercisers. When all diabetic patients were considered as one group and all control subjects as another, the microcirculation endothelial function in the diabetic group was reduced compared with the control subjects (91 ± 49 vs. 122 ± 41% flux increase over baseline; P < 0.05). In contrast, no differences existed among the three diabetic groups or between the two control groups. Similarly, in macro-circulation, a reduced response during reactive hyperemia was observed in the diabetic patients compared with control subjects (7.0 ± 4.5 vs. 11.2 ± 6.6% diameter increase; P < 0.05), whereas again no difference existed among the three diabetic groups or between the two control groups. These data suggest that diabetes per se does not affect aerobic exercise capacity (Vo2max) in physically active individuals, but is reduced in the presence of neuropathy. In addition, regular exercise training involving the lower extremities does not improve the endothelial function in the micro- and macrocirculation of the nonexercised upper extremity in type 1 diabetic patients.
Association of HLA-DR, DQ Genotype With Different β-Cell Functions at IDDM Diagnosis in Japanese Children
Authors: Sugihara Shigetaka , Sakamaki Tateo , Konda Susumu , Murata Atsushi , Wataki Kunio , Kobayashi Yasuyuki , Minamitani Kanshi , Miyamoto Shigeki , Sasaki Nozomu , Niimi Hiroo
Published: September 2013

Japanese IDDM patients have been demonstrated to have unique and different HLA associations from white patients. To elucidate the effect of HLA-associated genetic factors on the clinical heterogeneity of IDDM in Japanese people, HLA-DRB1, DQA1, and DQB1 genotypes in 88 childhood-onset Japanese IDDM patients were examined by polymerase chain reaction-sequence-specific oligonucleotide (PCR-SSO) or sequence-specific primers (SSP). Of the 88 IDDM patients, 26 (29.5%) had DRB1*0405-DQA1*0302-DQB1*0401/X (DR4-DQ4/X), 38 (43.2%) had DRB1*0901-DQA1*0302-DQB1*0303/X (DR9-DQ9/X), and 9 (10.2%) were DR4/9-DQ4/9 heterozygous in the present study (X does not contain protective alleles). Clinical heterogeneity such as age distribution at onset, prevalence and serum level of anti-GAD antibodies (GADAb), and residual pancreatic β-cell function after diagnosis were compared between patients with HLA-DR4-DQ4 and DR9-DQ9. The frequency of DR9-DQ9 genotype was significantly higher in the younger (0–10 years) than in the older (11–16 years) age-group of onset, but the frequency of DR4-DQ4 was higher in the older (11–16 years) age-group. Although no association of DR-DQ genotypes with the prevalence and serum level of GADAb was found among newly diagnosed patients, long-standing DR9-DQ9 patients had significantly higher levels of GADAb than those with DR4-DQ4. While no difference in time course of serum C-peptide (CPR) levels was detected between GADAb+ and GADAb− patients, a remarkable difference was demonstrated between DR9-DQ9 and DR4-DQ4 patients. The residual pancreatic β-cell function was retained more in patients with DR4-DQ4 than in those with DR9-DQ9 at diagnosis through 12–18 months after diagnosis. These results suggest that the DR9-DQ9 genotype may induce stronger autoimmune destructive response (T-helper 1 function) against target β-cells than the DR4-DQ4 genotype does. Our findings may warrant further studies on the association of diabetogenic autoimmune response with HLA class II molecules and contribute to a clarification of interracial differences in HLA-encoded susceptibility to IDDM.
Effect of Insulin on GLUT4 Cell Surface Content and Turnover Rate in Human Skeletal Muscle as Measured by the Exofacial Bis-Mannose Photolabeling Technique
Authors: Lund Sten , Holman Geoffrey D , Zierath Juleen R , Rincon Jorge , Nolte Lorraine A , Clark Avril E , Schmitz Ole , Pedersen Oluf , Wallberg-Henriksson Harriet
Published: September 2013

Insulin-stimulated glucose transport across the skeletal muscle cell membrane is a major regulatory step in postprandial glucose disposal. To estimate the total molar concentration of GLUT4 as well as the turnover rate of GLUT4 in human vastus lateralis muscles at the cell surface in the basal state and after insulin exposure, we have applied the sensitive exofacial bis-mannose photolabeling technique on in vitro incubated human skeletal muscle strips from healthy subjects. In addition, we have measured 3-O-methylglucose transport in other muscle strips prepared from the same surgically removed human skeletal muscle biopsies to compare glucose transport with cell surface level of GLUT4. Maximal in vitro insulin stimulation (2,400 pmol/l) resulted in a twofold increase compared with basal in both surface GLUT4 content (0.38 ± 0.05 vs. 0.19 ± 0.03 pmol/g wet muscle wt, P < 0.005) and 3-O-methylglucose transport (1.24 ± 0.13 vs. 0.63 ± 0.08 umol · ml−1 · h−1 P < 0.005). The insulin-induced increment in 3-O-methylglucose transport was strongly correlated with the insulin-induced increase in cell surface GLUT4 content (r2 = 0.91; P < 0.005). The calculated turnover rate of human skeletal muscle GLUT4 amounted to ∼8 × 104 min−1 at 35δC and was unaffected by insulin. In conclusion, maximal in vitro insulin stimulation of vastus lateralis muscle strips from healthy subjects resulted in a twofold rise in glucose transport as well as in cell surface content, whereas the turnover rate of GLUT4 was unaffected by insulin under the chosen experimental conditions.
Retardation or Acceleration of Diabetes in NOD/Lt Mice Mediated by Intrathymic Administration of Candidate β-Cell Antigens
Authors: Cetkovic-Cvrlje Marina , Gerling Ivan C , Muir Andrew , Atkinson Mark A , Elliott John F , Leiter Edward H
Published: September 2013

A single injection of syngeneic islet cells into the thymus of 4-week-old NOD/Lt female mice strongly retards diabetogenesis. The present study used the intrathymic route of antigen administration to compare the relative efficacy of peptides/proteins derived from two major candidate pancreatic β-cell autoantigens, insulin and GAD65, to modulate diabetogenesis. Intrathymic administration of insulin B chain or recombinant human GAD65 significantly suppressed diabetogenesis during a 20-week follow-up period, whereas no protection was mediated by either insulin A chain or a synthetic peptide (A2) derived from it. Quite unexpectedly, two GAD65-derived peptides near the COOH-terminus (p34 and p35) accelerated diabetes onset. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed on cDNAs from isolated islets or whole pancreases of NOD/Lt females 4 weeks after intrathymic injections. Protection mediated by intrathymic administration with either intact islet cells or GAD65 were correlated with an upregulation of mRNA for T-helper 2 (Th2)-associated cytokines (interleukin [IL]-4, IL-10), concomitant with downregulation of Th1-associated interferon (IFN) transcripts (all normalized to T-cell receptor Cβ transcripts) in islet-infiltrating lymphocytes. Protection mediated by the intrathymic administration of insulin B chain, however, correlated only with a modest upregulation of IL-4 and IL-10 transcript levels, and no diminution in IFN-γ transcripts. In contrast, the diabetes-accelerating GAD65 p34 and p35 peptides were not associated with an immune deviation, expressing levels of IFN-γ characteristic of islet-infiltrating lymphocytes in vehicleinjected NOD controls. Hence, Th1-to-Th2 immune deviation provides only a partial explanation for peptide immunotherapy of diabetes in NOD mice. The finding that certain peptides can accelerate rather than retard diabetogenesis as a function of route and age of administration adds a cautionary note to this type of therapy.
Leptin Gene Expression Increases With Age Independent of Increasing Adiposity in Rats
Authors: Li Hua , Matheny Michael , Nicolson Margery , Türner Nihal , Scarpace Philip J
Published: September 2013

Humans and rats tend to gain weight as they age. Leptin is one regulator of food intake and energy expenditure. To determine if the increase in adiposity with age is related to altered leptin gene expression, we assessed adiposity levels, leptin mRNA levels in epididymal and inguinal white adipose tissue (EWAT and IWAT), and uncoupling protein (UCP1) mRNA levels in interscapular brown adipose tissue (IBAT) from F344 × BN rats ages 3, 12, 18, 24, and 30 months (n = 8/age). Levels of adiposity determined by the adiposity index and the Lee index increased between ages 3 and 24 months, with a decrease at age 30 months. There were parallel increases with age in body weight, EWAT, and IWAT depot size up to age 24 months, followed by a nonsignificant decrease at age 30 months. Daily food intake was unchanged with age. In EWAT, leptin mRNA per microgram of RNA was unchanged with age, whereas in IWAT, it increased up to 24 months, then declined at 30 months. Total leptin mRNA levels in both IWAT and EWAT depots increased with age, peaking at age 24 months, and were correlated with adiposity. Serum leptin levels increased with age, also peaking at age 24 months, and were correlated with total leptin mRNA in WAT pads and adiposity. The rate of increase in serum leptin was greater than the increase in adiposity with age, suggesting contributions from both the increase in leptin expression per unit of WAT and the increase in WAT depot size. In addition, UCP1 mRNA levels in IBAT did not change with age. These data suggest that adiposity increases with age and cannot be attributed to increased food intake, impaired leptin gene expression, or decreased UCP1 mRNA level in IBAT. Furthermore, leptin gene expression in IWAT increases with age independent of increasing adiposity.
Method of Insulin Administration Has No Effect on Insulin Sensitivity Estimates From the Insulin-Modified Minimal Model Protocol
Authors: Saad Mohammed F , Steil Garry M , Riad-Gabriel Maggy , Khan Arshad , Sharma Alok , Boyadjian Rima , Jinagouda Sujata D , Bergman Richard N
Published: September 2013

The effect of the method of insulin administration on insulin sensitivity estimates from the insulin modified minimal model (MINMOD) protocol was evaluated using the tolbutamide-boosted protocol as a reference. The study included 21 nondiabetic men ages 40 ± 2 years (mean ± SE) with a BMI of 26.6 ± 1.1 kg/m2. Each subject underwent four frequently sampled intravenous glucose tolerance tests (FSIGTT), one with tolbutamide and three with the same insulin dosage (0.03 U/kg) given as a bolus or infusion over 5 or 10 min. The insulin sensitivity index (SI) of each subject was calculated from each FSIGTT with MINMOD. Insulin sensitivity indexes from the four FSIGTTs were highly correlated (r > 0.85, P < 0.001). SI(insulin) from the bolus and the 5- and 10-min infusion protocols were similar, but were 21 ± 5, 29 ± 5, and 23 ± 4% lower than SI(tolbntamde), respectively. SG(tolbutamide) and SG(insulin) were not different among the four protocols and were significantly correlated (r > 0.55, P < 0.01). Thus the tolbutamide and insulin protocols must not be used interchangeably in any single cross-sectional or longitudinal study. When the same insulin dosage is used, the method of its administration has no bearing on insulin sensitivity estimates from the insulin-modified FSIGTT. The same method of insulin administration should be used, however, in any single study for purpose of standardization.
Altered Platelet Membrane Dynamic Properties in Type 1 Diabetes
Authors: Mazzanti Laura , Rabini Rosa A , Fumelli Paolo , Martarelli Daniela , Staffolani Roberto , Salvolini Eleonora , Curatola Giovanna
Published: September 2013

A modified platelet response to aggregating stimuli is supposed to play a role in the pathogenesis of diabetic macroangiopathy. We studied the fluidity and microheterogeneity of the external surface of the platelet membrane and the activities of the plasma membrane Na+-K+-ATPase and Ca2+-ATPase in 21 men with type 1 diabetes and in 20 control subjects before and after in vitro thrombin addition. In the resting state, platelets from type 1 diabetic patients showed an increased fluidity and microheterogeneity of the platelet membrane, a higher Ca2+-ATPase activity, and a reduced Na+-K+-ATPase activity in comparison with platelets from healthy subjects. The fatty acid composition was also modified, with increased C 16:1 and decreased C 18:0 content. Control cells incubated with thrombin showed a modification of the membrane parameters opposite to the response observed in type 1 cells after the stimulation. The incubation of control platelets in the resting state with high concentrations of glucose modified the fluidity of the plasma membrane Na+-K+-ATPase and Ca2+-ATPase activities in an opposite way in comparison with the alterations observed in type 1 platelets. This study suggests that in type 1 diabetic patients, the platelet membrane responds to activation with a molecular remodeling different from the response of healthy subjects. The abnormal organization of the membrane might contribute to the altered platelet functions in type 1 diabetic patients, but acute exposure to high glucose levels does not seem able to modify the platelet membrane in the way observed in type 1 diabetes.