bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetics of Proinsulin Conversion in Human Islets
Authors: Sizonenko Stéphane , Irminger Jean-Claude , Buhler Léo , Deng Shaoping , Morel Philippe , Halban Philippe A
Published: September 2013

Islets isolated from human cadaver pancreas were pulse-labeled (10 min with [3H]leucine) and then incubated for a 180-min chase. Islets and chase medium were collected every 15 min and analyzed by reversed-phase HPLC to quantify the percentage of radioactively labeled proinsulin, conversion intermediates, and fully processed insulin. Release of proinsulin-related labeled products into the chase medium was < 10% of total. Whereas 50% of labeled proinsulin had been lost by conversion within 45 min, fully processed insulin only appeared with a half-time of 100 min. This discrepancy is attributable to accumulation of radioactive conversion intermediates. Des 64.65 split proinsulin was a minor component, reaching a maximum of 5.2 ± 1.7% (n = 4) at 60 min of chase. By contrast, des 31.32 split proinsulin—and a truncated form lacking the first three residues of C-peptide—rose progressively to 29.3 ± 1.4% by 75 min, and declined thereafter. The accumulation of des 31.32 split proinsulin rather than the des 64.65 split form during the conversion of human proinsulin reflects slower conversion at the C-peptide/A-chain than at the B-chain/C-peptide junction, and is consistent with the appearance of this particular conversion intermediate in the circulation.
Inhibition of Glucose-Induced Insulin Secretion Through Inactivation of Glucokinase by Glyceraldehyde
Authors: Murata Tomiyasu , Miwa Ichitomo , Toyoda Yukiyasu , Okuda Jun
Published: September 2013

D-Glyceraldehyde irreversibly inhibited rat liver glucokinase in a concentration-dependent manner. The inactivation of glucokinase by glyceraldehyde was blocked by the presence of its substrates such as glucose and mannose. Glucokinase was highly sensitive to glyceraldehyde compared with some other glycolytic enzymes (from animal tissues) including hexokinase, glucose-6-phosphate isomerase, 6-phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase. The amino acid analysis of untreated and glyceraldehyde-treated glucokinase suggested that glyceraldehyde-induced inactivation of glucokinase is caused by glycation of Lys residues of the enzyme by the triose. Treatment of pancreatic islets with 6 mM glyceraldehyde for 1 h at 37°C caused both inactivation of glucokinase and inhibition of glucose-induced insulin secretion. Another glucose-phosphorylating enzyme (hexokinase) in pancreatic islets, however, was little affected by glyceraldehyde. In addition, glyceraldehyde did not affect the insulin secretory responses of islets to nonglucose secretagogues such as glyceraldehyde and Leu. When pancreatic islets were cultured with a lower concentration (1 mM) of glyceraldehyde for a longer time (17 h) in the presence of 10 mM glucose to mimic the in vivo conditions, both glucokinase activity and glucose-induced insulin secretion were again decreased. This study demonstrates that glucose-induced insulin secretion is impaired by glyceraldehyde through the inactivation of glucokinase. The implication of this finding in the pathophysiology of type II diabetes is discussed.
Impairment of Coronary Vascular Reserve and ACh-Induced Coronary Vasodilation in Diabetic Patients With Angiographically Normal Coronary Arteries and Normal Left Ventricular Systolic Function
Authors: Nitenberg Alain , Valensi Paul , Sachs Regis , Dali Mustapha , Aptecar Eduardo , Attali Jean-Raymond
Published: September 2013

Evidence is increasing for small-vessel disease and disturbance of endothelium-dependent vasodilation in diabetic patients. The aim of this study was to compare coronary circulation in 11 diabetic patients (6 type I and 5 type II) and 7 control subjects. All patients had normal left ventricular systolic function and angiographically normal coronary arteries. To evaluate the maximal area of coronary microcirculation, coronary vascular reserve was determined by intracoronary Doppler and a maximally vasodilating dose of intracoronary papaverine (peak-to-resting coronary flow velocity ratio). To assess coronary endothelial function responses to stepwise intracoronary infusion of acetylcholine (10−8 to 10−5) M coronary estimated concentrations) were analyzed on four different segments in each patient by quantitative angiography. Peak-to-resting coronary flow velocity ratio was lower in diabetic patients than in control subjects (3.9 ± 0.9 and 5.0 ± 0.7, respectively, P < 0.02). Acetylcholine did not produce any diameter change at 10−8 and 10−7 M, but a progressive diameter reduction was observed at 10−6 and 10−5 M (−8.0 ± 15.2%, P < 0.02 and −24.0 ± 13.6%, P < 0.001, respectively). In control subjects, a progressive diameter dilation was produced from 10−8 to 10−6 M acetylcholine (5.1 ± 3.4, 12.1 ± 7.0, and 16.4 ± 7.3%, respectively, all P < 0.001), and a moderate reduction was observed at 10−5 M (−4.9 ± 7.5%, P < 0.02). In the two groups, all segments dilated similarly after intracoronary isosorbide dinitrate. In conclusion, these results provide evidence that diabetes mellitus may be responsible for both structural and functional alterations of the coronary circulation in patients with angiographically normal coronary arteries. These alterations of the coronary circulation in diabetic patients may contribute to the progressive deterioration of the myocardium and to the pathogenesis of diabetic cardiomyopathy.
Transport of Insulin Across Rabbit Nasal Mucosa In Vitro Induced by Didecanoyl-L-α-Phosphatidylcholine
Authors: Carstens Søren , Danielsen Gillian , Guldhammer Birgitte , Frederiksen Ole
Published: September 2013

To investigate the short-term effects of didecanoyl-L-α-phosphatidylcholine on the nasal mucosa and the mechanism by which didecanoyl-L-α-phosphatidylcholine enhances the nasal absorption of insulin, an in vitro model was developed. The mucosa from the posterior part of the rabbit nasal septum was mounted in an Ussing chamber and incubated in bicarbonate Ringer solution at 37°C. Potential difference, transmucosal conductance, and unidirectional tracer fluxes were measured across an exposed tissue area of 0.44 cm2. Morphological and physiological examinations revealed a typical respiratory epithelium containing amiloride-sensitive Na+ channels and diphenylamine-2-carboxylate–sensitive Cl− channels. Spontaneous potential difference (10.8 ± 0.4 mV [n = 50]; serosa positive) and transmucosal conductance (10.5 ± 0.4 mS/cm2 [n = 50]) were stable for several hours. Mucosal addition of 0.1–0.5% didecanoyl-L-α-phosphatidylcholine increased transmucosal conductance (by 43–53%) and decreased potential difference (to 0–2 mV) to new steady-state values within 10–15 min. Control unidirectional rate constants for permeation of sucrose, polyethylene glycol 4000, and insulin were low and varied according to the molecular size. After addition of didecanoyl-L-α-phosphatidylcholine, unidirectional rate constants for the three compounds all increased 3- to 5.5-fold. The didecanoyl-L-α-phosphatidylcholine effects on potential difference and transmucosal conductance were reversible after a recovery period of at least 40 min when didecanoyl-L-α-phosphatidylcholine had been applied to the mucosal side for 15 min. The results suggest that didecanoyl-L-α-phosphatidylcholine may increase the transepithelial absorption of insulin by facilitating a paracellular passage through a reversible opening of tight junctions.
GLUT2 Expression and Function in β-cells of GK rats with NIDDM: Dissociation Between Reductions in Glucose Transport and Glucose-Stimulated Insulin Secretion
Authors: Ohneda Makoto , Johnson John H , Inman Lindsey R , Chen Ling , Suzuki Ken-Ichi , Goto Yoshio , Alam Tausif , Ravazzola Mariella , Orci Lelio , Unger Roger H
Published: September 2013

GLUT2 underexpression has been reported in the +-cells of Zucker diabetic fatty rats and db/db mice, models of spontaneously occurring NIDDM with antecedent obesity. To determine whether the +-cells of a nonobese rodent model of NIDDM exhibit the same abnormalities in GLUT2, we studied Goto-Kakizaki rats. In these mildly diabetic animals glucose-stimulated insulin secretion was reduced at all ages examined from 8 to 48 wk. In normal control Wistar rats, immunostainable GLUT2 was present on all insulin-positive cells in the pancreatic islets. Only 85% of +-cells were GLUT2-positive in GK rats at 12 wk of age, and only 34% were positive at 48 wk of age. GLUT2 mRNA was 50% of normal in 12-wk-old GK rats. In the latter age-group, glucose-stimulated insulin secretion was only 28% of normal at a time when 85% of +-cells were GLUT2-positive and initial 3-O-methyl-D-glucose transport rate was 77% of the control value. We conclude that although GLUT2 is underexpressed, neither the magnitude of the underexpression of GLUT2 nor of the reduction in GLUT2 transport function in islets of GK rats is sufficient by itself to explain the profound reduction in glucose-stimulated insulin secretion.
Maternal Diabetes Induces Increased Expression of Extracellular Matrix Components in Rat Embryos
Authors: Cagliero Enrico , Forsberg Henrik , Sala Roberto , Lorenzi Mara , Eriksson Ulf J
Published: September 2013

The mechanisms responsible for the increased incidence of congenital malformations in offspring of diabetic mothers are poorly understood. Because the abnormal metabolic milieu of diabetes induces in the adult organism increased synthesis of basement membrane components, and these molecules play a prominent role in morphogenesis, we investigated whether maternal diabetes or high glucose levels disturb extracellular matrix synthesis in rat embryos. In gestational day 11 embryos, maternal diabetes induced a small but significant increase in laminin B1 (127 ± 40% of control, mean ± SD, P < 0.02) but not in fibronectin mRNA (101 ± 26% of control). Day 12 embryos from diabetic mothers showed a larger increment in laminin B1 (179 ± 91% of control, P < 0.02) and also an increase in fibronectin mRNA (172 ± 73% of control, P < 0.02). A similar increase in the expression of fibronectin was observed in the kidneys and hearts of day 20 fetuses dissected from diabetic rats. High glucose levels mimicked in vitro the effects of maternal diabetes. Day 9 embryos cultured for 48 h in 50 mM D-glucose showed, akin to the day 11 embryos in vivo, an increase in laminin B1 mRNA (129 ± 47% of control) and no changes in fibronectin mRNA (106 ± 35% of control). The finding that maternal diabetes induces increased expression of extracellular matrix components in developing embryos establishes a link with the abnormalities occurring in the chronic complications of diabetes and proposes a new path of investigation for the mechanism of teratogenicity of the diabetic milieu.
Upregulation of myo-Inositol Transport Compensates for Competitive Inhibition by Glucose: An Explanation for the Inositol Paradox?
Authors: Olgemöller Bernhard , Schwaabe Susanne , Schleicher Erwin D , Gerbitz Klaus D
Published: September 2013

High glucose concentrations inhibit the uptake of myo-inositol into cells. However, whether this leads to a depletion of intracellular myo-inositol levels has been debated, because unchanged, decreased, and increased cellular myo-inositol levels all have been reported for diabetic tissues. To evaluate whether cells are capable of counterregulating impaired uptake, we have investigated myo-inositol uptake in porcine aortic endothelial cells under short- and long-term hyperglycemic conditions. Although increasing glucose concentrations inhibited acute myo-inositol uptake competitively, the uptake was increased markedly, when cells were already preincubated in a high glucose medium for >6 h. The stimulation was maximal at 20 mM of glucose with no further increase at 40 mM glucose. The same stimulation of uptake could be induced by 5 mM of glucose plus 35 mM of raffinose, whereas 35 mM of sorbitol or mannitol, which do not compete for myo-inositol uptake, were ineffective. Lineweaver-Burk analysis revealed an increased Vmax for the induced myo-inositol transport activity, whereas the Km for myo-inositol remained constant (18 μM). The upregulated inositol transporter was still Na+ and ATP dependent, indicating that the same carrier system was operating. Uptake returned to control values when cells were again exposed to normoglycemic medium conditions for an additional 24 h. When endothelial cells were incubated with D-[U-14C]glucose and 10 μM myo-[2-3H]inositol of equal specific radioactivity for 24 h, no 14C radioactivity was found in intracellular myo-inositol, indicating that conversion of glucose to myo-inositol was rather low.
Sequence Variations of the Glucokinase Gene in Japanese Subjects With NIDDM
Authors: Eto Kazuhiro , Sakura Hiroshi , Shimokawa Kotaro , Kadowaki Hiroko , Hagura Ryoko , Akanuma Yasuo , Yazaki Yashio , Kadowaki Takashi
Published: September 2013

Mutations in the glucokinase gene have been identified recently in patients with maturity-onset diabetes of the young, a subtype of NIDDM. The proposed role of glucokinase as a glucose sensor, combined with the low insulin response to glucose found in most Japanese with NIDDM, prompted us to speculate that mutations in the glucokinase gene might be one of the major causes of NIDDM in Japanese subjects. To determine the prevalence of mutations and sequence variations in the glucokinase gene, we screened all 12 exons of the glucokinase gene, including exon/intron junctions, by polymerase chain reaction followed by single-strand conformation polymorphism in 209 Japanese NIDDM subjects. In addition to the mutation in exon 7, which substituted Arg (AGG) for Gly (GGG) at codon 261 (10), a silent mutation of Pro (CCC→CCG) in exon 4 at codon 145 and several new sequence variations in intervening sequences and the 5′-untranslated region of exon 1β (β-cell-specific exon 1) were identified. Because we identified only one subject who had a structurally abnormal glucokinase molecule, we conclude that the prevalence of structural mutations in the glucokinase gene responsible for NIDDM appears to be rare among Japanese patients. To our knowledge, this is the first thorough study describing the ethnic prevalence of mutations and sequence variations in the glucokinase gene in NIDDM.
Faulty Major Histocompatibility Complex Class II I-E Expression is Associated With Autoimmunity in Diverse Strains of Mice: Autoantibodies, Insulitis, and Sialadenitis
Authors: Li Xiangping , Golden Jeff , Faustman Denise L
Published: September 2013

Many regions and loci of the murine genome contribute to the pancreatic and salivary gland autoimmunity observed in the diabetic NOD mouse. Examination of the major histocompatibility complex region of the NOD mouse has revealed a defect in the expression of the major histocompatibility complex class II gene, I-E. To determine the isolated role of faulty I-E expression in abnormal self-recognition, we examined six commonly used inbred strains of mice on diverse genetic backgrounds that also do not express I-E, i.e., C57BL/10, SJL, ACA, DBA/1, NOD, and 129. Autoimmunity was assessed by the presence of inflammatory cell infiltrates (0,+/−,+,++,+++, ++++) within and among the pancreatic islets and salivary glands, and autoantibodies to self determinants. At 6 mo of age, inflammatory infiltrates in the pancreas (0, 3 mice; +/−, 3 mice; +, 7 mice; ++, 6 mice; +++ , 1 mouse; +++ +, 5 mice) and/or salivary glands (0, 0 mice; +/−, 3 mice; +, 1 mouse; ++, 4 mice; +++, 6 mice; ++++, 10 mice) were detected as well as autoantibodies in all 24 mice of all I-E– mouse strains on diverse genetic backgrounds. This indicates that defective expression of this single locus, in isolation, is sufficient for the spontaneous development of autoreactivity. In contrast, the simultaneous examination of 19 I-E+ mice on five commonly used inbred strains of mice (BALB/c, C67/KsJ, B10.BR, B10.A [2R], and B10.A [5R]) demonstrated no signs of humoral or cellular autoimmunity with target gland destruction or lymphocytic invasion. Our data suggest that many commonly used inbred strains of mice represent models of autoimmunity attributable to this single defective gene. The penetrance of pathology secondary to a defect in this gene appears to occur independently of a given major histocompatibility complex haplotype or background murine strain and for these murine models, does not lead to diabetic hyperglycemia.
MDL 29311: Antioxidant With Marked Lipid- and Glucose-Lowering Activity in Diabetic Rats and Mice
Authors: Johnson Marry B , Heineke Eric W , Rhinehart Barry L , Sheetz Matthew J , Barnhart Roger L , Robinson Keith M
Published: September 2013

MDL 29311, an analogue of probucol, administered to rats as a 1% dietary admixture for 2 wk before and 5 days after intravenous injection of 40 mg/kg of ALX significantly (P < 0.05) reduced plasma glucose (6.9 ± 0.3 vs. 19.2 ± 1.3 mM) and serum triglyceride (0.17 ± 0.01 vs. 1.82 ± 0.39 mM) levels in overnight-fasted ALX-plus-MDL 29311-administered rats vs. ALX-administered rats. A cross-over study indicated that MDL 29311 did not attenuate the diabetogenic action of ALX, but rather, directly lowered glucose and triglycerides. In rats injected intravenously with 45, 65, or 85 mg/kg of STZ and then administered control or MDL 29311 diet for 7 days, MDL 29311 decreased fasted plasma glucose to nondiabetic levels, decreased fasted and nonfasted plasma triglycerides by 49–79%, but did not affect plasma insulin levels. In STZ-induced (65 mg/kg) diabetic rats, MDL 29311 attenuated the increase in plasma nonesterified fatty acids during an 18-h fast; had little or no effect on glucagon, pyruvate, lactate, β-hydroxybutyrate, acetoacetate, or cholesterol; and did not induce hypoglycemia in rats fasted up to 64 h. In nonfasted hyperinsulinemic db/db mice treated for 10 wk, MDL 29311 significantly lowered glucose levels by 14–40%, triglyceride levels by 31–63% and GHb from 8.0 to 5.4%, and had no consistent effect on plasma insulin levels. Because of its marked glucose- and lipid-lowering activity in both nonfasted hyperinsulinemic and fasted insulinopenic animals, MDL 29311 merits additional investigation as a potential antidiabetic agent.
Characterization of Insulin Autoantibodies in Relatives of Patients With Type I Diabetes
Authors: Castaño Luis , Ziegler Anette G , Ziegler Ralph , Shoelson Steven , Eisenbarth George S
Published: September 2013

We studied in detail the anti-insulin autoantibodies in 29 nondiabetic relatives of patients with type I diabetes. The affinity of the autoantibodies for [125I]human insulin was high (1.34 × 109–20.71 × 109 L/mol), and the capacity was low (0.84 × 10−12–37.80 × 10−12 M). The product of affinity × capacity of each relative's antibodies directly correlated (r = 0.99) with the level of antibodies determined in our standard radioassay. The autoantibodies from each of the subjects studied had the same rank order of affinities for insulin from different species. Guinea pig, fish insulin, and insulin containing Trp rather than Leu in position 13 of the A-chain inhibited minimally the human insulin binding. Human proinsulin, insulin containing Gln rather than Glu in position 17 of the A-chain, and desoctapeptide insulin (des B23–30) all inhibited binding effectively. Insulin autoantibodies in relatives of patients with type I diabetes share common epitope(s), which suggests a common pathogenic mechanism for production of such antibodies. The epitopes from this initial analysis appear to include amino acids B1–B3 and A8–A13. The region recognized can be distinguished from the insulin receptor binding domain.
Glucagon-Like Peptide-I and the Control of Insulin Secretion in the Normal State and in NIDDM
Authors: Thorens Bernard , Waeber Gerard
Published: September 2013

Potentiation of glucose-induced insulin secretion by intestinal factors has been described for many years. Today, two major peptides with potent insulinotropic action have been recognized: gastric inhibitory peptide and truncated forms of glucagon-like peptide I, GLP-I(7–37) or the related GLP-I(7–36)amide. These hormones have specific β-cell receptors that are coupled to production of cAMP and activation of cAMP-dependent protein kinase. Elevation in intracellular cAMP levels is required to mediate the glucoincretin effect of these hormones: the potentiation of insulin secretion in the presence of stimulatory concentrations of glucose. In addition, circulating glucoincretins maintain basal levels of cAMP, which are necessary to keep β-cells in a glucose-competent state. Interactions between glucoincretin signaling and glucose-induced insulin secretion may result from the phosphorylation of key elements of the glucose signaling pathway by cAMP-dependent protein kinase. These include the ATP-dependent K+ channel, the Ca++ channel, or elements of the secretory machinery itself. In NIDDM, the glucoincretin effect is reduced. However, basal or stimulated gastric inhibitory peptide and glucagon-like peptide I levels are normal or even elevated, suggesting that signals induced by these hormones on the β-cells are probably altered. At pharmacological doses, infusion of glucagon-like peptide I but not gastric inhibitory peptide, can ameliorate postprandial insulin secretory response in NIDDM patients. Agonists of the glucagon-like peptide I receptor have been proposed as new therapeutic agents in NIDDM.
Counterregulation During Hypoglycemia Is Directed by Widespread Brain Regions
Authors: Frizzell R Tyler , Jones Elizabeth M , Davis Stephen N , Biggers David W , Myers Sharon R , Connolly Cynthia C , Neal Doss W , Jaspan Jonathan B , Cherrington Alan D
Published: September 2013

Previous studies have demonstrated the importance of the brain in directing counterregulation during insulin-induced hypoglycemia in dogs. The capability of selective carotid or vertebrobasilar hypoglycemia in triggering counterregulation was assessed in this study using overnight-fasted dogs. Insulin (21 pM · kg−1 · min−1) was infused for 3 h to create peripheral hypoglycemia in the presence of 1) selective carotid hypoglycemia (vertebral glucose infusion, n = 5), 2) selective vertebrobasilar hypoglycemia (carotid glucose infusion, n = 5), 3) the absence of brain hypoglycemia (carotid and vertebral glucose infusion, n = 4), or 4) total brain hypoglycemia (no head glucose infusion, n = 5). Glucose was infused via a leg vein as needed in each group to minimize the differences in peripheral glucose levels (2.6 ± 0.1, 3.0 ± 0.2, 2.7 ± 0.1, and 2.5 ± 0.1 mM, respectively). The humoral responses (cortisol, glucagon, catecholamines, and pancreatic polypeptide) to hypoglycemia were minimally attenuated (< 40%) by selective carotid or vertebrobasilar euglycemia. In addition, the increase in hepatic glucose production, as assessed using [3-3H]glucose, was attenuated by only 41 and 34%, respectively, during selective carotid or vertebrobasilar hypoglycemia. These observations offer support for the hypothesis that more than one center is important in hypoglycemic counterregulation in the dog and that they are located in brain regions supplied by the carotid and vertebrobasilar arteries, because significant counterregulation occurred when hypoglycemia developed in either of these circulations. Counterregulation during hypoglycemia, therefore, is probably directed by widespread brain regions that contain glucose-sensitive neurons such that the sensing sites are redundant.
Regulation of Apolipoprotein A-I–Containing Lipoproteins in IDDM
Authors: Kahri Juhani , Groop Per-Henrik , Viberti Giancarlo , Elliott Tom , Taskinen Marja-Riitta
Published: September 2013

In IDDM patients, serum high-density lipoprotein cholesterol concentrations have been reported to be normal or elevated. The spectrum of high-density lipoprotein particles is highly heterogeneous, but no data are available on the subpopulations of high-density lipoprotein in IDDM. We, therefore, studied the spectrum of high-density lipoprotein particles in 86 IDDM patients (51 men and 35 women) 37 ± 10 yr of age and in 74 sex-, age-, and body mass index-matched healthy nondiabetic subjects. The concentrations of high-density lipoprotein and HDL2 cholesterol were higher in the IDDM group than in the control subjects (P < 0.01). The apoA-I-to-apoA-II ratio was higher in the IDDM patients than in the nondiabetic subjects (P < 0.001) because of an increased concentration of LpA-I particles (61 ± 17 vs. 53 ± 15, P < 0.01). LpA-I particles correlated positively with high-density lipoprotein and HDL2 cholesterol in the two groups. Postheparin plasma lipoprotein lipase activity was significantly higher in the IDDM group than in the control group (P < 0.001), whereas postheparin plasma hepatic lipase activities were similar in both groups. Plasma cholesteryl ester transfer protein activity was estimated in an in vitro isotopic assay using exogenous labeled donor (low-density) and acceptor (high-density) lipoproteins in the absence of native lipoproteins. We observed no difference in cholesteryl ester transfer protein activity between the groups, and no significant correlations existed between cholesteryl ester transfer protein activity and high-density lipoprotein subpopulations. A positive correlation existed between HDL2 cholesterol and lipoprotein lipase-to-hepatic lipase ratio in IDDM patients (r = 0.35, P = 0.001) and in nondiabetic subjects (r = 0.55, P < 0.001). A positive correlation existed between LpA-I particles and lipoprotein lipase-to-hepatic lipase ratio in both groups (r = 0.34, P < 0.01; r = 0.38, P < 0.01, respectively) suggesting that lipolytic enzymes participate in the regulation of the metabolism of LpA-I particles. In conclusion, the elevation of high-density lipoprotein cholesterol in IDDM patients is mainly caused by a rise of LpA-I particles, which are suggested to have a key role in reverse cholesterol transport.
Endocytosis is Inhibited in Hepatocytes From Diabetic Rats
Authors: Krischer Joachim , Gilbert Anne , Gorden Phillip , Carpentier Jean-Louis
Published: September 2013

The consequences of type I diabetes on cellular endocytosis were investigated by comparing [125I]insulin, [125I]α2-macroglobulin, and Lucifer yellow uptake in hepatocytes freshly isolated from control and STZ-induced diabetic rats. In addition to the previously described reversible inhibition of ligand-induced internalization of the insulin receptor, we report a decrease in the constitutive receptor-mediated endocytosis of α2-macroglobulin and a near abolition of fluid-phase endocytosis of Lucifer yellow in cells from diabetic animals. Despite decreased receptor autophosphorylation and internalization, the ligand-induced surface redistribution of the insulin receptor was normal in the diabetic cell population. By contrast, the insulin receptor association with clathrin-coated pits was impaired in diabetic cells as a result of a decreased concentration of these specialized invaginations on the nonvillous cell surface. The morphology and diameter of clathrin-coated pits were similar in both conditions under study. These results demonstrate a general impairment of endocytosis in hypoinsulinemic diabetes: receptor-mediated endocytosis was less affected than fluid-phase endocytosis. Impaired endocytosis of specific ligands or other macromolecules could be an important mechanism underlying the accumulation of extracellular matrix or even blood cholesterol removal in diabetes.
Effect of Elevated Glucose Concentration on Membrane Voltage Regulation in Retinal Capillary Pericytes
Authors: Berweck Susanne , Thieme Hagen , Helbig Horst , Lepple-Wienhues Albrecht , Wiederholt Michael
Published: September 2013

The influence of elevated glucose concentration on resting membrane voltage, electrogenic Na+-K+-ATPase, and ATP-sensitive potassium channels (KATP channels) was studied in cultured bovine retinal capillary pericytes using conventional microelectrodes. The resting membrane voltage in cells grown in medium containing 5 mM glucose (control) averaged –27 ± 1.2 mV (mean ± SE, n = 26) and was not different from cells grown in medium containing 22.5 mM glucose (–26 1.2 mV, n = 26). Addition of ouabain (10−4 M), a specific inhibitor of the Na+-K+-ATPase, depolarized the membrane potential by 3.6 ± 0.4 mV (n = 10) in cells grown under control conditions and 0.7 ± 0.2 mV (n = 6) in cells grown under elevated glucose conditions. Thus, electrogenic activity of the Na+-K+-ATPase was significantly (P < 0.0001) reduced to 19% compared with control conditions. Electrogenic Na+-K+-ATPase activity could be partially restored (ouabain-induced depolarization ΔV = 2.0 ± 0.2 mV, n = 6) in cells grown with high glucose in the presence of the aldose reductase inhibitor tolrestat (10−5 M). The potassium channel opener Hoe 234 (10−6 M) induced membrane potential hyperpolarization in control cells (ΔV = 7.3 ± 1.2 mV, n = 13), which could be completely inhibited by the KATP channel blocker glibenclamide (10−7 M, n = 5). This indicates that pericytes possess KATP channels. The effect of KATP channels on membrane voltage was not significantly changed (P = 0.16) in cells cultured under high-glucose conditions (ΔV = 9.6 ± 2.0 mV, n = 6). Acute changes of glucose concentration did not affect the membrane voltage (n = 6). We conclude that high glucose concentrations alter the activity of the Na+-K+-ATPase in retinal pericytes via a mechanism involving the polyol metabolism. Therefore, hyperglycemia may alter regulation of membrane voltage and contractility of pericytes and, hence, the regulation of retinal microcirculation in diabetes. Altered microcirculation could be an important factor in the pathogenesis of diabetic retinopathy.
Insulin Resistance in Spontaneously Hypertensive Rats: Difference in Interpretation Based on Insulin Infusion Rate or on Plasma Insulin in Glucose Clamp Studies
Authors: Rao R Harsha
Published: September 2013

Isotopic glucose turnover was measured during euglycemic glucose clamp studies in spontaneously hypertensive rats and normotensive Wistar-Kyoto control rats, under pentobarbital sodium anesthesia. At an insulin infusion rate of 4 mU · kg−1 · min−1, glucose disposal rate and hepatic glucose output were not significantly different in either group, at a plasma glucose of 6 mM. However, steady-state plasma insulin levels in spontaneously hypertensive rats were more than double those in Wistar-Kyoto rats (2.34 ± 0.16 [mean ± SE] vs. 6.78 ± 0.58 nM, P < 0.00005, n = 7 in each group). Additional studies (n = 30 in spontaneously hypertensive rats and n = 32 in Wistar-Kyoto control rats) were conducted to match insulin levels in the two groups over a wide range, using infusion rates of 0.5–10 mU · kg−1 · min−1. When the responses of glucose disposal rate and hepatic glucose output to insulin were analyzed from the standpoint of plasma insulin levels, a highly significant difference was seen in the response to insulin between spontaneously hypertensive rats and Wistar-Kyoto rats, for both glucose disposal rate (P < 0.000005) and hepatic glucose output (P = 0.00007). The slope of the regression line for glucose disposal rate versus plasma insulin was lower in spontaneously hypertensive rats compared with Wistar-Kyoto rats (2.652 vs. 4.864, P < 0.001), indicating that insulin stimulation of glucose disposal rate was reduced by 50% in spontaneously hypertensive rats. The slope of hepatic glucose output versus plasma insulin was 0.739 in spontaneously hypertensive rats, indicating impaired suppression of hepatic glucose output, compared with Wistar-Kyoto rats, where the slope was –10.3, (P < 0.001 vs. spontaneously hypertensive rats). The clearance of insulin showed great variability in both groups but was much lower in spontaneously hypertensive rats compared with Wistar-Kyoto rats (6.7 ± 0.9 vs. 14.9 ± 1.3 ml · kg−1 · min−1, P < 0.000005). Post hoc power analysis showed that the within-group variation resulted in low power and high risk of type II errors when comparisons were restricted to a small sample size (n = 8). We conclude that insulin sensitivity is impaired in spontaneously hypertensive rats when expressed as a function of the ambient plasma insulin level, but not when expressed as a function of the administered dose of insulin, because of the risk of type II errors caused by the great variability in insulin concentrations at any given insulin infusion rate when the sample size is small.
Major Histocompatibility Complex–Encoded Antigen Processing Gene Polymorphism in IDDM
Authors: Van Endert Peter M , Liblau Roland S , Patel Salil D , Fugger Lars , Lopez Theresa , Pociot Flemming , Nerup Jørn , McDevitt Hugh O
Published: September 2013

Susceptibility to insulin-dependent diabetes mellitus (IDDM) is greatly influenced by polymorphisms in the genes of the class II region of the human leukocyte antigen (HLA) complex. The complexity of this genetic association and the lack of a direct proof of involvement of HLA class II genes in human IDDM have continued to support speculation on a possible role of genes encoded in the close vicinity of these loci in IDDM. Because the recently discovered transporter associated with antigen processing (TAP) and large multifunctional protease (LMP) genes are encoded in the HLA class II region and are implicated in the processing of antigenic proteins for presentation by HLA class I molecules, they are additional candidates for a role in IDDM pathogenesis. We have analyzed genomic and coding sequence polymorphisms in the LMP2, TAP1, and TAP2 genes of 77 Danish IDDM patients and 102 control subjects. Although patients and control subjects did not differ in TAP1 and LMP2 alleles, we found a striking absence of the TAP2 allele B (long form) in IDDM patients. An analysis of the TAP2 alleles in individual DR types, however, revealed that this phenomenon is likely to be caused by linkage disequilibrium between the two loci. Thus, polymorphisms in the TAP and LMP genes are unlikely to be associated with IDDM.
Isolation of Nonobese Diabetic Mouse T-Cells That Recognize Novel Autoantigens Involved in the Early Events of Diabetes
Authors: Gelber Cohava , Paborsky Lisa , Singer Steven , McAteer Deborah , Tisch Roland , Jolicoeur Christine , Buelow Roland , McDevitt Hugh , Fathman C Garrison
Published: September 2013

Insulin-dependent diabetes mellitus (IDDM) is thought to result from chronic, cell-mediated, autoimmune islet damage. Our aim was to identify the earliest T-cell autoantigen in IDDM, reasoning that this antigen could be causally involved in the initiation of the disease. Identification of the earliest β-cell-specific autoantigen is extremely important in allowing advances in prevention and treatment of initial events in the development of inflammatory insulitis that precedes β-cell destruction and overt diabetes. Therefore, we analyzed the proliferative responses of peripheral β-cells from young, female nonobese diabetic (NOD) mice to extracts of pancreatic β-cell lines. We were able to demonstrate that T-cells responsive to β-cell antigens exist in the peripheral lymphoid tissue of these mice in the absence of deliberate priming before the manifestation of histologically detectable insulitis. T-cell lines and clones isolated from the peripheral lymphatic tissues of young, unimmunized, female NOD mice were also shown to react with extracts of β-cells. Fractionation of the β-cell extracts showed that these T-cell clones recognized multiple β-cell-specific autoantigens but none of the previously reported putative autoantigens (glutamic acid decarboxylase [GAD]65, GAD67, Hsp65, insulin, ICA 69, car boxy peptidase-H, and peripherin). Thus, we can conclude that these responses are specific for novel β-cell autoantigens. Finally, NOD T-cell proliferative responses were also seen to an extract of human islets suggesting potential shared antigenic determinants between human and mouse β-cells. Our observation that human and murine β-cell-specific antigens are conserved offers the possibility that identification of these murine autoantigens may lead to the discovery of the human homologue. This will pave the way toward effective diagnosis and/or immunotherapy to prevent diabetes.
Biological Actions of Insulin Are Differentially Regulated by Glucose and Insulin in Primary Cultured Adipocytes: Chronic Ability to Increase Glycogen Synthase Activity
Authors: Lima Fabio B , Bao Shichun , Garvey W Timothy
Published: September 2013

We have shown previously that prolonged exposure to insulin and glucose impairs the insulin-responsive glucose transport system in primary cultured adipocytes. To assess the ability of insulin and glucose to regulate other cellular insulin actions, epididymal rat adipocytes were cultured in media containing 0–15 mM D-glucose and with or without insulin (50 ng/ml). After 24 h, cells were washed and basal and maximally insulin-stimulated rates of 2-deoxy-D-glucose uptake, L-leucine incorporation into protein, glucose oxidation to CO2, glucose incorporation into lipids, and glycogen synthase activity were measured. The results confirmed that glucose potentiates insulin's chronic ability to decrease basal and maximal glucose transport rates by ∼ 50% at 5 mM glucose and by ∼70% at 15 mM glucose compared with control cells. However, neither glucose nor insulin, alone or in combination, affected rates of leucine incorporation into protein. In addition, basal and maximal rates of glucose oxidation and of glucose incorporation into lipids were not regulated by glucose, and maximal responses declined ∼50% over 24 h only when insulin was not present during preincubation (i.e., chronic insulin exposure was necessary to maintain full maximal responses). Glycogen synthase activity was measured in a cell-free system (0.5 mM UDP-glucose, with 10 or 0.01 mM glucose-6-phosphate) after exposing intact cells to glucose and insulin. Both short-term (1 h) and long-term (24 h) exposure to glucose alone led a dose-dependent increase in I-form and D-form glycogen synthase activity. Chronic exposure to insulin also increased total glycogen synthase activity (I- plus D-form) but did not affect absolute rates of maximally stimulated I-form activity. Glucose (but not insulin) increased the cellular content of immunoreactive glycogen synthase by 70% after 1 h. These results show that 1) chronic exposure to glucose and insulin impairs insulin responsiveness of the glucose transport system but does not affect rates of amino acid incorporation into protein; 2) the chronic presence of insulin is necessary for the maintenance of normal maximally stimulated rates of glucose oxidation and of glucose incorporation into lipids in cultured cells; and 3) glucose increases both D-form and I-form glycogen synthase activity, in part by increasing the amount of synthase protein, whereas chronic insulin exposure increases total glycogen synthase activity without altering maximal absolute rates of I-form activity. In conclusion, glucose and insulin differentially regulate the multiple biological actions of insulin in cultured adipocytes, indicating that underlying mechanisms involve the hormone effector systems or components of signal transduction unique to individual action pathways. In NIDDM, defects in glucose transport but not other insulin actions may be acquired as a result of high circulating blood glucose and insulin concentrations.
Maternal Effect and Familial Aggregation in NIDDM: The CODIAB Study
Authors: Thomas Frederique , Balkau Beverley , Vauzelle-Kervroedan Francoise , Papoz Laure
Published: September 2013

Non-insulin-dependent diabetes mellitus (NIDDM) is known to have a strong genetic basis, but the mode of inheritance is still unknown. Recent studies have suggested that maternal inheritance is important; this complicates the transmission pattern of NIDDM. In our study, the familial aggregation of diabetes and the maternal effect were investigated through three generations. The CODIAB Study recruited 536 NIDDM patients between 35 and 74 years of age from 10 diabetes centers in France. Familial aggregation was confirmed: among 218 NIDDM patients, 66% had at least one diabetic relative. Mothers were implicated 2 times more frequently than fathers (P < 0.001). This maternal effect was confirmed because more diabetic cases were noted among maternal than paternal aunts and uncles (P < 0.02). When we considered the next generation, women had more diabetic offspring than men (P < 0.01). Other factors susceptible to modify the familial aggregation were considered. The maternal effect was not significantly related to the patients' ages (P > 0.2). The genetic component was more important when the diagnosis was made earlier, but the maternal effect was homogeneous (P > 0.3). In conclusion, we found a familial aggregation of diabetes that suggests a strong genetic component with a mode of inheritance that may be influenced by a maternal environment.
Effects of Chronic Vanadate Administration in the STZ-Induced Diabetic Rat: The Antihyperglycemic Action of Vanadate Is Attributable Entirely to Its Suppression of Feeding
Authors: Malabu Usman H , Dryden Simon , Mccarthy H David , Kilpatrick Anne , Williams Gareth
Published: September 2013

Vanadate treatment can lower glycemia in diabetic rats. This action is generally attributed to vanadate's insulinomimetic properties, but vanadate also inhibits feeding, which could lower blood glucose. We therefore assessed the contribution of hypophagia to vanadate's antihyperglycemic action in a 3-week study of streptozocin-induced (STZ) diabetic rats. Untreated diabetic rats (n = 8) ate 54% more food than nondiabetic control rats (P < 0.001). Diabetic rats given sodium metavanadate (0.5 mg in 0.5 ml of water by gavage twice daily; n = 8) had significantly lower food intakes (P < 0.001) than untreated diabetic rats. In vanadate-treated diabetic rats, blood glucose levels were significantly lower than in untreated diabetic rats (P < 0.001). Untreated diabetic rats pair-fed to the food intake of the vanadate-treated diabetic rats (n = 8) showed virtually identical blood glucose falls (P > 0.05 vs. vanadate-treated diabetic rats). Vanadate treatment did not affect plasma insulin concentrations in diabetic rats. In nondiabetic rats (n = 8), vanadate treatment significantly reduced food intake (P < 0.05) and also lowered plasma insulin concentrations (P < 0.05) without significantly affecting glycemia. To investigate the mechanism of vanadate's hypophagic effect, we also measured regional hypothalamic levels of neuropeptide Y (NPY), a potent central appetite stimulant that is thought to drive hyperphagia in STZ-induced diabetes. Hypothalamic NPY concentrations rise markedly in diabetes and are normalized by insulin replacement. Unlike insulin, vanadate treatment did not normalize regional hypothalamic NPY concentrations in diabetic rats. Vanadate does not therefore appear to exert an insulin-like action at the hypothalamic level; its hypophagic action does not appear to involve inhibition of NPYergic pathways in the hypothalamus. We conclude that the glucose-lowering effect of vanadate in STZ-induced diabetic rats can be explained by its inhibition of feeding. Although vanadate has certain insulinomimetic effects in vitro and in vivo, the role of these effects in vanadate's antidiabetic actions must be critically reexamined.
Synthesis of Type VI Collagen by Cultured Glomerular Cells and Comparison of Its Regulation by Glucose and Other Factors With That of Type IV Collagen
Authors: Wakisaka Masanori , Spiro Mary Jane , Spiro Robert G
Published: September 2013

Homogeneous cultures of calf glomerular mesangial and endothelial cells were found to be active in the synthesis of type VI as well as type IV collagen in contrast to the epithelial cells that were devoted primarily to the production of the latter collagen. Studies with rat mesangial cells indicated that they responded to high glucose (20 mM) in the medium by a significant (P < 0.001) increase in type VI collagen synthesis as measured by the production of the protein and its mRNA level, both of which were closely correlated to each other and to glucose consumption. Similar observations were made with type IV collagen, but the enhanced formation of this protein was not as rapidly apparent as that of type VI and, moreover, could not be as readily reversed on restoration of the glucose to a physiological level (5 mM). Evaluation of a number of other agents indicated that although mannitol had no effect, L-glucose and NaCI significantly stimulated synthesis of both type VI and IV collagens and glucose consumption. Insulin-like growth factor I and aldosterone, on the other hand, also increased glucose consumption but brought about an enhancement of only type IV collagen production, suggesting that the two collagens are independently regulated. This possibility was supported by our observation that pyruvate, which was actively taken up by the cells, selectively stimulated type IV collagen production. Although the details of the regulation of these two major glomerular extracellular matrix components have not yet been defined, it is apparent that mesangial cells in culture reflect the enhanced formation of type VI and type IV collagen, which is evident in the diabetic glomerular lesions.
Diabetes-Associated Mesenteric Vascular Hypertrophy Is Attenuated by Angiotensin-Converting Enzyme Inhibition
Authors: Cooper Mark E , Rumble Jonathan , Komers Radko , He-Cheng Du , Jandeleit Karin , Sheung-To Chou
Published: September 2013

In experimental diabetes, the mesenteric vascular tree undergoes hypertrophy, and this is associated with an increase in mesenteric angiotensin-converting enzyme (ACE) levels. The aim of this study was to determine if inhibition of mesenteric ACE by ACE inhibition would influence diabetes-associated mesenteric vascular hypertrophy. Control or streptozocin-induced diabetic rats were randomized to receive no drug or the ACE inhibitor perindopril. In addition, other diabetic rats were randomized to receive either low-dose insulin that does not alter glycemic control or high-dose insulin, administered as a silastic pellet to achieve euglycemia. After 3 weeks, animals were killed for measurement of mesenteric ACE, vessel weight, and wall:lumen ratio. Diabetes was associated with increased mesenteric ACE levels, increased vessel weight, and an increase in the wall:lumen ratio. ACE inhibition, despite no effect on glycemic control, food intake, urinary urea excretion, or gut weight, prevented the increase in mesenteric ACE levels and attenuated mesenteric vascular hypertrophy as assessed by weight or wall:lumen ratio. The increase in staining by an antibody to the endothelial product, von Willebrand factor, in diabetic rats was totally prevented by perindopril treatment. Euglycemia but not low-dose insulin therapy in the diabetic rats normalized mesenteric vessel ACE, weight, and wall:lumen ratio. In conclusion, ACE inhibition may have a specific role in preventing diabetes-associated vascular hypertrophy, an important process in the genesis of micro- and macrovascular diabetic complications.
The Risk to Develop NIDDM Is Related to the Fatty Acid Composition of the Serum Cholesterol Esters
Authors: Vessby Bengt , Aro Antti , Skarfors Einar , Berglund Lars , Salminen Irma , Lithell Hans
Published: September 2013

This investigation was undertaken to study whether the risk to develop non-insulin-dependent diabetes mellitus (NIDDM) among 50-year-old men during a 10-year follow-up period was related to the fatty acid composition of their serum cholesterol esters. There were highly significant differences in the initial health survey between the fatty acid composition in serum in subjects who remained normoglycemic (w = 1,753) and in those who later developed NIDDM (n = 75). The main differences were that the latter had higher proportions of saturated fatty acids and palmitoleic acid (16:ω-7), a low proportion of linoleic acid (18:2ω-6), and a relatively high content of γ-linolenic (18:3ω-6) and dihomo-γ-linolenic (20:3ω-6) acids in the serum cholesterol esters. The picture was similar also after adjusting for differences in body mass index. In a logistic model, a high proportion of dihomo-γ-linolenic acid remained a significant contributor to the development of diabetes, along with the height of the insulin index, the blood glucose concentration at 60 min, and the fasting insulin concentration. The increased risk to develop NIDDM related to the serum cholesterol ester fatty acid composition may be mediated by diet and/or genetic factors.