bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin-Induced Glucose Transporter (GLUT1 and GLUT4) Translocation in Cardiac Muscle Tissue Is Mimicked by Bradykinin
Authors: Rett K , Wicklmayr M , Dietze G J , Häring H U
Published: September 2013

The effect of bradykinin on glucose transporter translocation in isolated rat heart was compared with the effect of insulin. Hearts from male obese (fa/fa) Zucker rats were perfused under normoxic conditions and constant pressure in a classic Langendorff preparation with 12 mmol/l glucose as substrate, and a set of functional parameters was measured simultaneously. Bradykinin was administered at a concentration (10−11 mmol/l) that did not increase coronary flow. Insulin was used at a concentration (8 × 10−8 mmol/1) known to maximally stimulate glucose transport in this model. After 15 min of perfusion with insulin or bradykinin, subcellular membrane fractions of the heart were prepared, and distribution of glucose transporter protein (GLUT1 and GLUT4) in fractions enriched with surface membranes (transverse tubules [TTs] and sarcolemmal membranes [PMs]) and with low-density microsomal membranes (LDMs) were determined by immunoblotting with the respective antibodies. Both glucose transporter isoforms were translocated after stimulation with insulin (increased transporter protein content in the PM+TT-enriched fraction with a concomitant decrease in the LDM-enriched fraction) and, to a smaller extent, also with bradykinin. These data suggest that in hearts of insulin-resistant obese (fa/fa) Zucker rats, bradykinin interacts with or facilitates the translocation process of both GLUT1 and GLUT4.
Skeletal Muscle Content of Membrane Glycoprotein PC-1 in Obesity: Relationship to Muscle Glucose Transport
Authors: Youngren Jack F , Maddux Betty A , Sasson Shlomo , Sbraccia Paolo , Tapscott Edward B , Swanson Melvin S , Dohm G Lynis , Goldfine Ira D
Published: September 2013

Membrane glycoprotein PC-1, an inhibitor of insulin signaling, produces insulin resistance when overexpressed in cells transfected with PC-1 cDNA. In the present study, we determined whether PC-1 plays a role in the insulin resistance of skeletal muscle in obesity. Rectus abdominus muscle biopsies were taken from patients undergoing elective surgery. Subjects included both NIDDM patients (n = 14) and nondiabetic patients (n = 34) across a wide range of BMI values (19.5–90.1). Insulin-stimulated glucose transport was measured in incubated muscle strips, and PC-1 content, enzymatic activity, and insulin receptor content were measured in solubilized muscle extracts. Increasing BMI correlated with both an increase in the content of PC-1 in muscle (r = 0.55, P < 0.001) and a decrease in insulin stimulation of muscle glucose transport (r = −0.58, P = 0.008). NIDDM had no effect on either PC-1 content or glucose transport for any given level of obesity. Insulin stimulation of muscle glucose transport was negatively related to muscle PC-1 content (r = −0.68, P = 0.001) and positively related to insulin receptor content (r = 0.60, P = 0.005). Multivariate analysis indicated that both skeletal muscle PC-1 content and insulin receptor content, but not BMI, were independent predictors of insulin-stimulated glucose transport. Muscle PC-1 content accounted for 42% and insulin receptor content for 17% of the variance in glucose transport values. These studies raise the possibility that increased expression of PC-1 and a decreased insulin receptor content in skeletal muscle may be involved in the insulin resistance of obesity.
Control of Islet Intercellular Adhesion Molecule-1 Expression by Interferon-α and Hypoxia
Authors: Chakrabarti Debasis , Huang Xiaojian , Beck Joanne , Henrich Jill , McFarland Nancy , James Roger F L , Stewart Timothy A
Published: September 2013

The ability of interferon-α (IFN-α) to induce the adhesion molecules that characterize the islets of patients with type I diabetes has been investigated. We have found that all tested recombinant IFN-as will induce major histocompatibility complex (MHC) class I on arterial endothelial cells. Some but not all IFN-as will induce intercellular adhesion molecule-1 (ICAM-1). However, there is only a transient and modest increase in VCAM on arterial endothelial cells. IFN-α has very little effect on endothelial MHC class II expression but will induce these proteins on monocytes. Thus, there is a close concordance between the biological actions of IFN-α and the appearance of those adhesion molecules induced in the islets of patients with type I diabetes. IFN-α is also produced in normal human islets during short-term cultures, probably as a result of the ischemia present at the center of the islet. This induction of IFN-α by hypoxia may explain the previously reported spontaneous induction of ICAM-1 in human islets and may also be a contributing factor to the failure of islet grafts.
Chronic Activation of Protein Kinase C in Soleus Muscles and Other Tissues of Insulin-Resistant Type II diabetic Goto-Kakizaki (GK), Obese/Aged, and Obese/Zucker Rats: A Mechanism For Inhibiting Glycogen Synthesis
Authors: Avignon Antoine , Yamada Kouji , Zhou Xiaoping , Spencer Barbara , Cardona Osvaldo , Saba-Siddique Shereen , Galloway Lamar , Standaert Mary L , Farese Robert V
Published: September 2013

We examined the possibility that protein kinase C (PKC) is chronically activated and may contribute to impaired glycogen synthesis and insulin resistance in soleus muscles of hyperinsulinemic type II diabetic Goto-Kakizaki (GK) rats. Relative to nondiabetic controls, PKC enzyme activity and levels of immunoreactive PKC-α, β, є, and delta were increased in membrane fractions and decreased cytosolic fractions of GK soleus muscles. In addition, PKC-θ levels were decreased in both membrane and cytosol fractios, whereas PKC-ζ levels were not changed in either fraction in GK soleus muscles. These increases in membrane PKC (α, β, є, and δ) could not be accounted for by alterations in PKC mRNA or total PKC levels but were associated with increases in membrane diacylglycerol (DAG) and therefore appeared to reflect translocative activation of PKC. In evaluation of potential causes for persistent PKC activation, membrane PKC levels were decreased in soleus muscles of hyperglycemic streptozotocin (STZ)-induced diabetic rats; thus, a role for simple hyperglycemia as a cause of PKC activation in GK rats was not evident in the STZ model. In support of the possibility that hyperinsulinemia contributed to PKC activation in GK soleus muscles, we found that DAG levels were increased, and PKC was translocated, in soleus muscles of both (1) normoglycemic hyperinsulinemic obese/aged rats and (2) mildly hyperglycemic hyperinsulinemic obese/Zucker rats. In keeping with the possibility that PKC activation may contribute to impaired glycogen synthase activation in GK muscles, phorbol esters inhibited, and a PKC inhibitor, RO 31-8220, increased insulin effects on glycogen synthesis in soleus muscles incubated in vitro. Our findings suggested that: (1) hyperinsulinemia, as observed in type II diabetic GK rats and certain genetic and nongenetic forms of obesity in rats, is associated with persistent translocation and activation of PKC in soleus muscles, and (2) this persistent PKC activation may contribute to impaired glycogen synthesis and insulin resistance.
Diazoxide Treatment at Onset Preserves Residual Insulin Secretion in Adults with Autoimmune Diabetes
Authors: Björk E , Berne C , Kämpe O , Wibell L , Oskarsson P , Karlsson F A
Published: September 2013

Twenty islet cell antibody (ICA)-positive patients, aged 19–38 years, with IDDM were randomized at onset to treatment with either diazoxide, a K+ channel opener that inhibits the release of insulin, or placebo for 3 months, in addition to multiple insulin injection therapy. The patients who were given diazoxide displayed higher residual insulin secretion than the placebo group after 1 year (basal C-peptide level, 0.40 ± 0.04 vs. 0.25 ± 0.04 [mean ± SE] nmol/l; P < 0.021) and at an 18-month follow-up (0.37 ± 0.06 vs. 0.20 ± 0.01 nmol/l, P < 0.033). Metabolic control did not differ between the two groups. During the course of the study, no differences in islet cell or GAD autoantibodies were detected between the two groups. The results of this study warrant further trials to explore the potential of inducing target cell rest in order to halt the loss of insulin-producing cells during the early course of the disease.
Muscle Subcellular Localization and Recruitment by Insulin of Glucose Transporters and Na+-K+-ATPase Subunits in Transgenic Mice Overexpressing the GLUT4 Glucose Transporter
Authors: Ramlal Toolsie , Ewart H Stephen , Somwar Romel , Deems Rhonda O , Valentin Michele A , Young Douglas A , Klip Amira
Published: September 2013

Insulin-stimulated glucose uptake in skeletal muscle is mediated through the GLUT4 glucose transporter. Transgenic (TG) mice overexpressing human GLUT4 in skeletal muscle show an increased ability to handle a glucose load. Here, the participation of the overexpressed GLUT4 in the response to insulin was examined. In TG mouse muscle, the GLUT4 protein content was 10-fold higher in crude membrane (CM), sevenfold higher in internal membrane (IM), and 15-fold higher in a plasma membrane (PM)-rich fraction, relative to non-TG littermates. This suggested partial saturation of the normal sorting mechanisms. The distribution and abundance of the GLUT1 glucose transporter was not affected. Insulin injection (4.3 U/kg body wt) increased GLUT4 in the PM-rich fraction; the increase was threefold higher in TG than in non-TG mice. Insulin decreased the GLUT4 content of the IM in both animal groups and of a second, heavier intracellular membrane fraction only in TG mice. The net content of Na+-K+-pump subunits was 40–65% lower in CM from TG compared with non-TG littermates. In spite of this, insulin caused a three- to sixfold higher translocation of the α2 and β1 subunits of the Na+-K+-pump in TG compared with non-TG animals. The results suggest that overexpression of GLUT4 confers to the muscle increased ability to translocate subunits of the Na+-K+-pump either as a direct consequence of the recruitment of glucose transporters or as an adaptation to the more demanding metabolic state.
Inhibition of Mitochondrial Complex I May Account for IDDM Induced by Intoxication With the Rodenticide Vacor
Authors: Esposti Mauro D , Ngo Anna , Myers Mark A
Published: September 2013

Human intoxication with the rodenticide Vacor [N-3-pyridylmethyl-N′-p-nitrophenyl urea or 1-(4-nitrophenyl)-3-(3-pyridylmethyl) urea] induces acute IDDM. We report here that Vacor specifically inhibits the NADH:ubiquinone reductase activity of complex I in mammalian mitochondria. The activity of other respiratory enzymes of mitochondria is unaffected by Vacor at concentrations that completely inhibit the redox and energetic function of complex I. Vacor inhibition of complex I activity quantitatively correlates with the inhibition of insulin release in insulinoma cells and pancreatic islets and is also consistent with the doses reported in cases of human poisoning. These results indicate that the toxic and diabetogenic action of Vacor primarily derives from the inhibition of mitochondrial respiration of NAD-linked substrates in the high-energy demanding cells of the pancreatic islets. This newly identified mechanism of the pathological effects resulting from Vacor intoxication could constitute a paradigm in which to understand environmental or metabolic causes of IDDM.
Genetic Control of Giant Perivascular Space Formation in the Thymus of NOD Mice
Authors: Colomb Evelyne , Savino Wilson , Wicker Linda , Peterson Larry , Dardenne Mireille , Carnaud Claude
Published: September 2013

The immune system of NOD mice exhibits several anomalies, one being the intrathymic formation of giant perivascular spaces (PVSs) filled with mature thymocytes and some B-cells, intermingled within a network of extracellular matrix. The abnormal retention of thymocytes on their way to the periphery could have a profound impact on the nature of the exported cells and the regulation of autoimmune events. In the present study, we evaluated the appearance of this defect into F1 hybrids, the association with some of the known diabetes susceptibility loci (Idd genes) in a panel of NOD and reciprocal C57BL congenic strains, and the relative contribution of epithelial versus hematopoietic stroma. The analysis of F1 hybrid thymuses reveals a dominant expression of thymic giant PVS that is only marginally influenced by the outcross strain. Moreover, giant PVS expression in major histocompatibility complex (MHC) and Idd congenic mice is determined by the genetic background. All of the NOD congenics express the anomaly, irrespective of the Idd resistance alleles that have been introgressed, whereas none of the C57BL congenic mice present abnormal PVS. Finally, the expression of giant PVS in parental → F1 bone marrow chimeras is predominantly controlled by the thymic NOD-derived hematopoietic microenvironment. In conclusion, the giant PVS formation in the NOD mouse thymus is a dominantly inherited anomaly associated with hematopoietic-derived tissue and with non-MHC genes. The exact contribution of PVS to the autoimmune process remains to be definitively established.
Regulation of Rat Liver Glucose-6-Phosphatase Gene Expression in Different Nutritional and Hormonal States: Gene Structure and 5′-Flanking Sequence
Authors: Argaud Doriane , Zhang Qing , Pan Wansong , Maitra Subir , Pilkis Simon J , Lange Alex J
Published: September 2013

The mRNA level of the catalytic subunit of rat liver glucose-6-phosphatase (Glu-6-Pase) was regulated by hormones commensurate with activity changes in vivo. Insulin exerts a dominant negative effect on the mRNA levels of Glu-6-Pase. Both mRNA levels and activities of the enzyme are low in the fed and refed state where insulin levels are elevated. Insulin administration to diabetic rats also decreases levels of mRNA and Glu-6-Pase activity. Insulin at a concentration of 1 nmol/l completely overcomes the stimulatory effect of glucocorticoids on Glu-6-Pase message levels in FAO hepatoma cells. The stimulatory response to glucocorticoid in FAO cells is biphasic, with maxima seen at 3 and 18 h after hormone addition (respectively 1.6- and 3.3-fold). 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) causes a fourfold increase in Glu-6-Pase mRNA at 3 h in FAO cells. The gene of rat liver Glu-6-Pase is 13 kilobases in length and comprised of 5 exons. The exon-intron structure is completely conserved when compared with the mouse and human genes. A 0.5-kb 3′-untranslated region, which is present in rat and mouse liver Glu-6-Pase cDNA, is absent in the Glu-6-Pase gene reported here, indicating the possible duplication of either the terminal fifth exon or the entire gene. The promoter region contains a consensus core CCAAT element at position –207 and a TATAAA at position –31. Several possible response elements have been identified in the 5′-flanking region (from a HindIII site at position –1641). A consensus glucocorticoid response element is located at base pair –1552, a 9/10 match of the insulin response sequence is located at position –1449, and a 7/8 match of the cAMP response element is located at position –164.
Effect of Troglitazone on Insulin Sensitivity and Pancreatic β-Cell Function in Women at High Risk for NIDDM
Authors: Berkowitz Kathleen , Peters Ruth , Kjos Siri L , Goico Jose , Marroquin Aura , Dunn Meleana E , Xiang Anny , Azen Stanley , Buchanan Thomas A
Published: September 2013

We conducted a randomized placebo-controlled study to determine the effects of the thiazolidinedione compound troglitazone on whole-body insulin sensitivity (SI), pancreatic β-cell function, and glucose tolerance in 42 Latino women with impaired glucose tolerance (IGT) and a history of gestational diabetes mellitus (GDM), characteristics that carry an 80% risk of developing NIDDM within 5 years. After baseline oral (OGTT) and intravenous (IVGTT) glucose tolerance testing, subjects were assigned to take placebo or 200 or 400 mg troglitazone daily for 12 weeks (14 subjects per treatment group). An OGTT and IVGTT were repeated during the 12th week of treatment. Five subjects failed to complete the trial for personal reasons, and medication compliance averaged 90% in the remaining subjects, none of whom experienced a serious adverse event. SI, calculated by minimal model analysis of IVGTT results, changed by only 4 ± 14% during 12 weeks of placebo administration, but increased 40 ± 22 and 88 ± 22% above basal during treatment with 200 and 400 mg troglitazone, respectively (P = 0.01 among groups). Troglitazone administration was also associated with a dose-dependent reduction in the total insulin area during IVGTTs, which was highly significant (P < 0.001), and with a reduction during OGTTs, which approached statistical significance (P = 0.09). Glucose tolerance improved slightly in all groups, but the magnitude of change did not differ significantly among groups, whether it was assessed as the number of subjects who continued to manifest IGT at 12 weeks (P = 0.64 among groups), the change in total glucose area during OGTTs (P = 0.58), or the change in fractional glucose disappearance rates during IVGTTs (P = 0.28). Among the women who received troglitazone, the greatest improvement in SI occurred in the women who had the highest diastolic blood pressures and the best IVGTT insulin responses during baseline testing. Our findings indicate that troglitazone improved whole-body insulin sensitivity and lowered circulating insulin concentrations in women with prior GDM who are at very high risk for NIDDM. The lack of improvement in glucose tolerance despite improved insulin sensitivity may be a manifestation of the β-cell defect that predisposes the women to NIDDM. The overall pattern of response to troglitazone in our high-risk patients indicates that the drug is an ideal agent with which to test whether the amelioration of insulin resistance can delay or prevent diabetes in women with limited β-cell reserve.
Metabolic Consequences of a Family History of NIDDM (The Botnia Study): Evidence for Sex-Specific Parental Effects
Authors: Groop Leif , Forsblom Carol , Lehtovirta Mikko , Tuomi Tlinamaija , Karanko Samuel , Nissén Michael , Ehrnström Bjorn-Olof , Forsén Björn , Isomaa Bo , Snickars Börje , Taskinen Marja-Riitta
Published: September 2013

Although a strong genetic susceptibility has been established for NIDDM and a maternal transmission of the disease predominates in some populations, a relationship between parental diabetes status and metabolic abnormalities in nondiabetic offspring has not been shown in humans. To address this question, we studied 2,152 first-degree relatives of patients with NIDDM (FH+) and 528 age- and weight-matched spouses without a family history of NIDDM (FH−) in Western Finland (the Botnia study). A subset of the subjects underwent a euglycemic insulin clamp combined with indirect calorimetry to measure insulin sensitivity and energy expenditure. Despite similar amounts of total body fat, persons with a family history of NIDDM had a greater waist-to-hip ratio (WHR) than spouses without a family history of diabetes (P < 0.003). They also had a decreased resting metabolic rate (P = 0.005), but this difference disappeared when adjusted for the difference in WHR. Insulin-stimulated glucose metabolism (P = 0.002), particularly nonoxidative glucose metabolism (P = 0.009), was reduced in FH+ compared with FH− subjects, and this difference remained after adjustment for WHR. A parental history of NIDDM influenced the insulin response to the oral glucose load, with male offspring of diabetic mothers showing the lowest insulin values (P = 0.011). Moreover, a parental effect was also observed on HDL and HDL2 cholesterol concentrations with female offspring of diabetic mothers showing lower values than female offspring of diabetic fathers (both P < 0.002). We conclude that abdominal obesity, insulin resistance, and decreased resting metabolic rate are characteristic features of first-degree relatives of patients with NIDDM and that the decrease in resting metabolic rate is partially related to the degree of abdominal obesity. A sex-specific paternal effect was observed on insulin and HDL cholesterol concentrations. Therefore, one has to consider the possibility of unprecedented maternal or paternal inheritance of different NIDDM phenotypes.
The Pituitary-Gonadal Axis and Health in Elderly Men: A Study of Men Born in 1913
Authors: Tibblin Gösta , Adlerberth Annika , Lindstedt Göran , Björntorp Per
Published: September 2013

The results of recent studies suggest that a relative hypogonadism in men is associated with several established risk factors for prevalent diseases. Therefore, we determined total and free testosterone, luteinizing hormone (LH), and sex-hormone binding globulin (SHBG) in a cohort of randomly selected men (n = 659) at 67 years of age. These data were analyzed cross-sectionally in relation to blood glucose and serum insulin, which were measured while fasting and after an oral glucose tolerance test, in addition to plasma lipids and blood pressure. The data were also analyzed in relation to impaired glucose tolerance (IGT) and diabetes, which were discovered at examination or earlier diagnosis. Risk factors for the development of diabetes up to 80 years of age were analyzed with univariate and multivariate statistics. Total and free testosterone and SHBG concentrations correlated negatively with glucose and insulin values; total testosterone and SHBG, with triglycerides; and SHBG, with blood pressure (from P < 0.05 to P < 0.01). Men with IGT or newly diagnosed diabetes had higher BMI values (26.2 ± 0.31 and 27.0 ± 0.59 [mean ± SE], respectively) and waist circumference (99.0 ± 1.03 and 100.5 ± 1.57) than nondiabetic men (BMI, 25.1 ± 0.14; waist circumference, 95.4 ± 0.47; P < 0.05), indicating abdominal obesity. Such men and men with previously diagnosed diabetes had, in general, lower total and free testosterone and SHBG levels, while those for LH were not different. In multivariate analyses that included BMI, waist-to-hip ratio, total and free testosterone, and SHBG, the remaining independent predictors for the development of diabetes were low total testosterone (P = 0.015) and, on the borderline, low SHBG (P = 0.053). In relation to nondiabetic men, the risk ratio for mortality, myocardial infarction, and stroke increased gradually and significantly from 1.18 to 1.68, from 1.51 to 1.78, and from 1.72 to 2.46 in men with IGT, newly diagnosed diabetes, and previously known diabetes, respectively. It was concluded that low testosterone and SHBG concentrations in elderly men are associated with established risk factors for diabetes and in established diabetes. Moreover, low testosterone levels independently predict the risk of developing diabetes. In different degrees of expression, the diabetic state predicts strongly (and gradually mortality from) myocardial infarction and stroke. It has been suggested that a relative hypogonadism might be a primary event, because other studies have shown that testosterone deficiency is followed by insulin resistance, which is ameliorated by testosterone substitution. The data suggest that the relative hypogonadism involved might be of both central and peripheral origin.
Evidence for Leptin Binding to Proteins in Serum of Rodents and Humans: Modulation With Obesity
Authors: Houseknecht Karen L , Mantzoros Chris S , Kuliawat Regina , Hadro Edward , Flier Jeffrey S , Kahn Barbara B
Published: September 2013

Many hormones circulate bound to serum proteins that modulate ligand bioactivity and bioavailability. To understand the biology of leptin action, we investigated the presence of leptin binding proteins in serum. 125I-labeled leptin binds competitively to at least three serum macromolecules with molecular masses of ∼ 85, ∼ 176, and ∼240 kDa in rodents and ∼176 and ∼240 kDa in humans. The ability to bind appears to involve sulfhydryl/disulfide interactions because it is inhibited under reducing conditions. When serum is added to recombinant 125I-leptin, there is a shift in sedimentation of 125I-leptin as analyzed by sucrose gradient centrifugation from ∼ S1.9 to ∼ S4.3. This shift is markedly attenuated in serum from obese mice (ob/ob, db/db, brown-fat ablated, gold-thioglucose treated, high-fat fed) compared with that from nonobese controls. The size distribution of endogenous serum leptin as determined by radioimmunoassay (RIA) after sucrose gradient centrifugation is also consistent with saturation of binding in hyperleptinemic obesity. In humans, free leptin increases with BMI. Thus, in lean rodents and humans a large proportion of leptin circulates bound to several serum proteins. Free leptin is increased in serum of obese subjects, which may alter leptin bioactivity, transport, and/or clearance.
Heterologous Expression and Characterization of Rat Liver Glucokinase Regulatory Protein
Authors: Mookhtiar Kasim A , Kalinowski Stephen S , Brown Karen S , Ha Tsay Yim , Smith-Monroy Constance , Robinson Gordon W
Published: September 2013

Glucokinase is a critical component of the physiological glucose sensor found in cell types that are responsive to changes in plasma glucose levels. The acute regulation of glucokinase activity has been shown to occur via a regulatory protein found in liver parenchymal cells (Van Schaftingen E, Detheux M, Da Cunha MV. Faseb J 8:414–419, 1994). The action of this protein is modulated by phosphate esters of fructose. In the presence of fructose-6-phosphate, the protein inhibits glucokinase in an allosteric competitive manner, while fructose-1-phosphate reverses this inhibition. A cDNA potentially encoding the rat liver regulatory protein has been cloned, but its identity is uncertain because of the small amounts of soluble protein obtained by expression in bacteria. We report the heterologous expression of the regulatory protein in Escherichia coli and its purification to homogeneity and high specific activity in a single chromatographic step. The properties of this recombinant protein are very similar to those of the liver protein. Direct demonstration of the binding of the recombinant protein to glucokinase has been obtained in vitro using coprecipitation experiments and in vivo, using the yeast two-hybrid system. These studies establish that the protein encoded by the cDNA is identical to the glucokinase regulatory protein and also validate tools with which to carry out structure-function studies on the interaction of the regulatory protein with glucokinase.
Relationship of Generalized and Regional Adiposity to Insulin Sensitivity in Men With NIDDM
Authors: Abate Nicola , Garg Abhimanyu , Peshock Ronald M , Stray-Gundersen James , Adams-Huet Beverley , Grundy Scott M
Published: September 2013

Abdominal obesity, particularly excess intraperitoneal fat, is considered to play a major role in causing insulin resistance and NIDDM. To determine if NIDDM patients accumulate excess intraperitoneal fat, and whether this contributes significantly to their insulin resistance, 31 men with mild NIDDM with a wide range of adiposity were compared with 39 nondiabetic, control subjects for insulin sensitivity (measured using euglycemic-hyperinsulinemic clamp technique with [3-3H]glucose turnover) and total and regional adiposity (assessed by hydrodensitometry and by measuring subcutaneous abdominal, intraperitoneal, and retroperitoneal fat masses using magnetic resonance imaging [MRI], and truncal and peripheral skinfold thicknesses using calipers). MRI analysis revealed that intraperitoneal fat was not increased in NIDDM patients compared with control subjects; in both groups it averaged 11% of total body fat. NIDDM patients, however, had increased truncal-to-peripheral skinfolds thickness ratios. In NIDDM patients, as in control subjects, amounts of truncal subcutaneous fat showed a stronger correlation with glucose disposal rate than intraperitoneal or retroperitoneal fat; however, NIDDM patients were more insulin resistant at every level of total or regional adiposity. Further, no particular influence of excess intraperitoneal fat on hepatic insulin sensitivity was noted. We conclude that NIDDM patients do not have excess intraperitoneal fat, but that their fat distribution favors more truncal and less peripheral subcutaneous fat. Moreover, for each level of total and regional adiposity, NIDDM patients have a heightened state of insulin resistance.
Reduced Expression of the Leptin Gene (ob) by Catecholamine Through a GS Protein-Coupled Pathway in 3T3-L1 Adipocytes
Authors: Kosaki Atsushi , Yamada Kazunori , Kuzuya Hideshi
Published: September 2013

Leptin, a recently identified hormone, is believed to reduce appetite and maintain body weight. The mRNA of leptin is expressed only in mature adipose cells. To clarify the regulation of leptin gene expression in adipocytes, 3T3-L1 adipocytes were treated for 16 h with various agents known to modulate lipid metabolism, and then the leptin mRNA was measured by the reverse transcription-polymerase chain reaction method. Interestingly, both norepinephrine and isoproterenol reduced the level of leptin mRNA to about 20% of that found in untreated control cells in a dose-dependent fashion. The maximum reduction occurred at 100 nmol/l of either norepinephrine or isoproterenol, and the half-maximal effect was observed at ∼ 3 nmol/l norepinephrine and ∼ 1 nmol/l isoproterenol. Propranolol reversed about 50% of the reduction by either norepinephrine or isoproterenol. In contrast, phentolamine did not inhibit the reduction by either norepinephrine or isoproterenol. Moreover, both cholera toxin and dibutyryl cAMP decreased the level of leptin mRNA to about 10% of that in control cells in a dose-dependent fashion. The maximum effect was elicited at 100 ng/ml cholera toxin and 100 micromol/l dibutyryl cAMP. The concentration producing the half-maximal effect was ∼ 1 ng/ml cholera toxin and ∼ 50 μmol/l dibutyryl cAMP. Dibutyryl cGMP, however, did not affect leptin gene expression. These results suggest that a signaling pathway that results in the activation of protein kinase A regulates leptin gene expression in 3T3-L1 adipocytes.
Diminished Fraction of Blockable ATP-Sensitive K+ Channels in Islets Transplanted Into Diabetic Mice
Authors: Soria Bernat , Martín Franz , Andreu Etelvina , Sanchez-Andrés Juan V , Nacher Victor , Montana Eduard
Published: September 2013

The reasons for the poor outcome of islet transplantation in diabetic patients are not well known; a better understanding of the pathophysiology of transplanted islets is needed. To study the mechanism coupling secretagogue stimuli with insulin release in transplanted islets, we determined the effects of glucose, tolbutamide, and carbamylcholine on the β-cell membrane potential and cytosolic calcium concentrations ([Ca2+]i) of islets syngeneically transplanted into normal and streptozocin-induced diabetic mice. In both groups, normoglycemia was maintained after transplantation. Islets transplanted into normal recipients showed similar changes in β-cell membrane potential and [Ca2+]i oscillations to those in control islets. In contrast, when islets were transplanted into diabetic mice, bursts of electrical activity were triggered at lower glucose concentrations (5.6 mmol/l) than in control islets (11 mmol/l), and maximal electrical activity was achieved at lower glucose concentrations (11 mmol/l) than in control islets (22 mmol/l). When membrane potential was plotted as a function of glucose concentration, the dose-response curve was shifted to the left. Compared with control islets, glucose-induced [Ca2+]i oscillations were broader in duration (22.3 ± 0.6 s vs. 118.1 ± 12.6 s; P < 0.01) and higher in amplitude (135 ± 36 nmol/l vs. 352 ± 36 nmol/l; P < 0.01). Glucose supersensitivity was attributed to a resting decrease in the fraction of blockable ATP-sensitive K+ (K+ATP) channels in transplanted islets that maintained normoglycemia with a limited β-cell mass.
Stimulation of Glucose Uptake by the Natural Coenzyme α-Lipoic Acid/Thioctic Acid: Participation of Elements of the Insulin Signaling Pathway
Authors: Estrada D Elizabeth , Ewart H Stephen , Tsakiridis Theodoros , Volchuk Allen , Ramlal Toolsie , Tritschler Hans , Klip Amira
Published: September 2013

Thioctic acid (α-lipoic acid), a natural cofactor in dehydrogenase complexes, is used in Germany in the treatment of symptoms of diabetic neuropathy. Thioctic acid improves insulin-responsive glucose utilization in rat muscle preparations and during insulin clamp studies performed in diabetic individuals. The aim of this study was to determine the direct effect of thioctic acid on glucose uptake and glucose transporters. In L6 muscle cells and 3T3-L1 adipocytes in culture, glucose uptake was rapidly increased by (R)-thioctic acid. The increment was higher than that elicited by the (S)-isomer or the racemic mixture and was comparable with that caused by insulin. In parallel to insulin action, the stimulation of glucose uptake by thioctic acid was abolished by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, in both cell lines. Thioctic acid provoked an upward shift of the glucose-uptake insulin dose-response curve. The molar content of GLUT1 and GLUT4 transporters was measured in both cell lines. 3T3-L1 adipocytes were shown to have >10 times more glucose transporters but similar ratios of GLUT4:GLUT1 than L6 myotubes. The effect of (R)-thioctic acid on glucose transporters was studied in the L6 myotubes. Its stimulatory effect on glucose uptake was associated with an intracellular redistribution of GLUT1 and GLUT4 glucose transporters, similar to that caused by insulin, with minimal effects on GLUT3 transporters. In conclusion, thioctic acid stimulates basal glucose transport and has a positive effect on insulin-stimulated glucose uptake. The stimulatory effect is dependent on phosphatidylinositol 3-kinase activity and may be explained by a redistribution of glucose transporters. This is evidence that a physiologically relevant compound can stimulate glucose transport via the insulin signaling pathway.
Long-Term Metabolic Control by Rat Islet Grafts Depends on the Composition of the Implant
Authors: Keymeulen Bart , Korbutt Greg , De Paepe Monique , Gorus Frans , Klöppel Gönter , Pipeleers Daniel G
Published: September 2013

This study examines whether the loss of metabolic control in initially normalized islet transplants can result from the inadequate composition of the donor tissue. Streptozotocin-induced diabetic rats were followed for 64 weeks after the intraportal injection of islet isografts with different composition. The implantation of 2.3 million β-cells (107/kg body wt) as particles (>100 microm diameter) of primarily insulin-positive (70%) and glucagon-positive (20%) cells succeeded in a long-term normalization of 2-h fasting glycemia, glucose tolerance, and serum fructosamine. The same metabolic control was achieved in animals with short and long durations of diabetes or when grafts were implanted under the kidney capsule. At posttransplantation (PT) week 64, insulin reserves were 60% lower than those in age-matched controls, which may account for the glucose intolerance in a few old recipients. The same type of graft containing 0.7 million β-cells (4 × 106/kg body wt) corrected these metabolic parameters for more than 12 weeks; the proportionally lower insulin reserves were sufficient for the long-term correction of 2-h fasting glycemia, but did not avoid glucose intolerance in older recipients. When the higher β-cells number (107/kg body wt) was injected as smaller particles (<100 mpm diameter) of lower purity (55% insulin-positive) and negligible glucagon content (<5% glucagon-positive), the metabolic parameters were also corrected for 12 weeks PT but then progressively returned to overt diabetes (6 of 10) or glucose intolerance (4 of 10). We concluded that long-term metabolic normalization can be achieved by islet implants in the liver or under the kidney capsule. The loss of metabolic control in older animals can be caused by the inadequate composition of the graft, with the number of β-cells, the proportion of other endocrine and nonendocrine cells, and the particle size as influential variables.
Troglitazone Attenuates High-Glucose–Induced Abnormalities in Relaxation and Intracellular Calcium in Rat Ventricular Myocytes
Authors: Ren Jun , Dominguez Ligia J , Sowers James R , Davidoff Amy J
Published: September 2013

Diabetes is associated with impaired cardiac diastolic dysfunction. Isolated ventricular myocytes from diabetic animals demonstrate impaired relaxation concomitant with prolonged intracellular Ca2+ transients. We have recently shown that maintaining normal adult rat ventricular myocytes in a “diabetic-like” culture medium (low insulin and high glucose) produces abnormalities in excitation-contraction coupling similar to in vivo diabetes. Troglitazone (TRO), a novel insulin-sensitizing agent, significantly lowers blood pressure and modestly increases cardiac output in vivo, but its direct impact on cardiac function is unknown. To determine whether TRO could prevent high-glucose-induced dysfunction, normal myocytes were maintained in culture for 1–2 days in either normal medium containing 5 mmol/l glucose or high-glucose medium containing 25 mmol/l glucose. TRO (5 μmol/l) was added to both normal and high-glucose media. Mechanical properties were evaluated using a high-resolution video-edge detection system, and Ca2+ transients were recorded in fura-2–loaded myocytes. Relaxation from peak contraction was significantly longer in myocytes cultured in high glucose. Treating cells with TRO either attenuated or prevented the high-glucose effects, without changing the mechanical properties of myocytes cultured in normal medium. TRO also prevented the abnormally slow rates of Ca2+ transient decay induced by high glucose. Collectively, these data demonstrate that TRO can protect against the high-glucose-induced relaxation defects, perhaps through changes in intracellular Ca2+ handling. If TRO has both vasodilatory actions and beneficial cardiac properties (e.g., improvement of diastolic function) in the presence of hyperglycemia, this antidiabetic agent may prove to have significant salutary cardiovascular effects in type II diabetes.
Hepatic Insulin Resistance After Pancreas Transplantation in Type I Diabetes
Authors: Rooney Desmond P , Robertson Rooney P
Published: September 2013

Hyperinsulinemia and peripheral insulin resistance caused by systemic insulin delivery and prednisone therapy are recognized consequences of pancreas transplantation. However, there is little information about insulin action on the liver. To investigate hepatic insulin sensitivity in recipients of pancreas transplants, we devised a staged euglycemic hyperinsulinemic clamp to measure hepatic glucose production (HGP) in 10 type I diabetic pancreas transplant recipients, 10 pair-matched healthy control subjects, and 6 nondiabetic kidney transplant recipients. Clamps were performed in two sequential stages. In stage 1, a 2-h low-dose insulin infusion (0.4 mU · kg−1 · min−1) was used to partially suppress HGP. In stage 2, insulin-mediated suppression of HGP was challenged by a 1.5-h glucagon infusion (0.8 ng · kg−1 · min−1), while continuing the hyperinsulinemic euglycemic-clamp conditions. During both stages, somatostatin (250 μg/h) was infused to suppress endogenous insulin secretion. All subjects underwent stage 1, and all except one pancreas recipient and a respective matched healthy control subject completed stage 2. Fasting HGP was greater in pancreas recipients than in healthy control subjects (15.1 ± 0.7 vs. 12.0 ± 0.4 μmol · l−1 · kg−1 · min−1, P < 0.005) but similar in healthy control subjects and in kidney recipients. During stage 2, both total (706 ± 28 vs. 469 ± 31 μmol · l−1 · kg−1, P < 0.005) and incremental (62 ± 20 vs. −21 ± 16 μmol · l−1 · kg−1, P < 0.005) HGP responses to glucagon infusion were significantly greater in pancreas recipients than in healthy control subjects. Changes in HGP in kidney recipients during stage 2 were not significantly different from those in healthy control subjects. In conclusion, fasting HGP is increased in pancreas transplant recipients. Furthermore, recipients have hepatic insulin resistance as demonstrated by an enhanced stimulatory effect of glucagon on HGP during insulin-mediated HGP suppression. Because the magnitude of hepatic insulin resistance was a significant (P < 0.01) predictor of HbA1c level, we suggest that variable hepatic insulin resistance may be responsible for some of the variance observed in glycemic levels after successful pancreas transplantation.
Identification of Trinucleotide Repeat–Containing Genes in Human Pancreatic Islets
Authors: Aoki Minoru , Koranyi Laszlo , Riggs Andrew C , Wasson Jon , Chiu Ken C , Vaxillaire Martine , Froguel Philippe , Gough Stephen , Liu Li , Donis-Keller Helen , Permutt Alan
Published: September 2013

In the search for diabetes genes, the combined approaches of positional cloning with random markers and subsequent evaluation of candidate genes mapping to areas of interest will be increasingly used. For islet candidate genes of unknown function, expressed trinucleotide (triplet) repeats represent a unique subset. It is unlikely that abnormal expansion of expressed islet triplet repeats would be a major cause of diabetes, yet the triplet repeats are frequently polymorphic and can thus be used to map the genes in the human genome. In this study, a human islet cDNA library was screened with (CGG)7 and (CAG)7, and 23 triplet repeats were isolated. Sequencing revealed four known and six novel islet genes containing 4–15 triplet repeats. The four known cDNAs included ferritin, the major iron-binding protein in cells; HSGSA2R, a full-length clone of the α-subunit of the G-regulatory protein; HUMSATB1A, a DNA-binding protein expressed predominantly in thymus; and HUMPPA-PRO, a ribosomal protein. The triplet repeats in ferritin and HUMPPAPRO were found to be monomorphic. Characterization of the six unique novel expressed islet triplet cDNAs revealed that they were 0.6–1.5 kb in size, contained 4–15 triplet repeats, and were expressed in islets and all other tissues examined. Four of the novel clones, CGG-isl 10, CGG-isl 11, CAG-isl 6, and CAG-isl 7, were mapped to human chromosomes 19, 16, 12, and 3, respectively, via somatic cell hybrids. One islet cDNA, CAG-isl 7, contained a repeat that was highly polymorphic, with 14 alleles (4–18 triplets) in African-Americans (heterozygosity = 0.86) and 6 alleles (heterozygosity = 0.77) in whites. Northern analysis indicated that the mRNA was abundant in pancreatic islets. A putative full-length clone contained an open reading frame encoding 213 amino acids with a variable number of alanines (4–18) within the COOH-terminal. The gene was uniquely mapped with odds > 1,000:1 on chromosome 3p in Centre d'Etude du Polymorphisme Humain pedigrees. There were no differences in CAG-isl 7 allele frequencies between African-American patients with NIDDM (n = 108) and control subjects (n = 116), nor was expansion above 18 repeats noted. Linkage analysis in 14 nonglucokinase maturity-onset diabetes of the young pedigrees showed a cumulative logarithm of odds score of –33.19 at θ = 0.00. Abnormal expansion was not observed in 20 IDDM patients with one NIDDM parent. While these data suggest no major role for CAG-isl 7 in diabetes, at least four of the six novel islet triplet genes are coexpressed in pancreatic islets and neural tissue, and these genes can now be considered as candidates for diabetes and/or neuropsychiatric diseases.
Effects of Cilazapril and Amlodipine on Kidney Function in Hypertensive NIDDM Patients
Authors: Velussi Mario , Brocco Enrico , Frigato Francesco , Zolli Mario , Muollo Bruno , Maioli Mario , Carraro Andrea , Tonolo Giancarlo , Fresu Pietro , Cernigoi Anna M , Fioretto Paola , Nosadini Romano
Published: September 2013

Contrasting information has been reported concerning the course of renal function in NIDDM with hypertension alone or in association with renal damage. The aim of the present study was to elucidate the course of the glomerular filtration rate (GFR) in hypertensive NIDDM patients during antihypertensive therapy. Furthermore, we compared the effects of ACE inhibitors (cilazapril, Inibace, Roche, Milan, Italy) and Ca2+-channel blockers (amlodipine, Norvasc, Pfizer, Rome, Italy). Of the hypertensive NIDDM patients attending the outpatient's clinic of the internal medicine departments of the University of Padova and Sassari, 44 participated in the present study. Of these patients, 26 were normoalbuminuric and 18 microalbuminuric. They were randomly treated with either cilazapril or amlodipine. The target of antihypertensive treatment was a value <140 mmHg for systolic and 85 mmHg for diastolic blood pressure (BP). Microalbuminuria was defined as an albumin excretion rate (AER) between 20 and 200 μg/min. GFR was measured by plasma clearance of 51Cr-labeled EDTA at baseline and every 6–12 months during a 3-year follow-up interval. A significant decrease was observed in the values of GFR, AER, and systolic and diastolic BP in normoalbuminuric and microalbuminuric patients during antihypertensive therapy. The GFR fall in the overall population of NIDDM patients was significantly and inversely related to the decrease of mean BP (diastolic + 1/3 pulse pressure) (r = −0.80, P < 0.0001) but not to that of HbA1c, triglycerides, and BMI. The GFR decline (mean ± SE) per year in the normoalbuminuric patient was 2.03 ± 0.66 ml · min−1 · 1.73 m−2 (95% CI 0.92–3.17) during cilazapril and 2.01 ± 0.71 ml · min−1 · 1.73 m−2 (95% CI 0.82–3.11) during amlodipine therapy. The GFR decline per year in the microalbuminuric patient was 2.15 ± 0.69 ml · min−1 · 1.73 m−2 (95% CI 0.86–3.89) during cilazapril and 2.33 ± 0.83 ml · min−1 · 1.73 m−2 per year (95% CI 1.03–3.67) during amlodipine therapy. Cilazapril and amlodipine lowered AER to a similar extent in normoalbuminuric and microalbuminuric patients. No significant changes were observed concerning other clinical and biochemical features between the two antihypertensive therapies and particularly HbA1c, BMI, triglycerides, and cholesterol plasma values. These results support the tenet that arterial hypertension plays a pivotal role in contributing to renal damage in NIDDM, even when AER is normal. However, the degree of BP control, with both cilazapril and amlodipine, can successfully delay the slope of GFR decline in hypertensive NIDDM patients with or without incipient nephropathy.