bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucose Regulates the Maximal Velocities of Glucokinase and Glucose Utilization in the Immature Fetal Rat Pancreatic Islet
Authors: Tu Jian , Tuch Bernard E
Published: September 2013

The cause of the poor secretion of insulin in response to glucose by the β-cell in the fetal rat pancreas is thought to be immaturity of the metabolism of glucose. Glucokinase (GK), a key enzyme in glycolysis, is the glucose sensor that maintains glucose homeostasis in the adult β-cell; its role in the fetal β-cell has not been determined. The aim of this study was to examine whether GK was functional in phosphorylation of glucose in the fetal islet, and if so, to determine what factors regulated this activity. Similar Km values were found in both fetal and adult islets: 7.4 vs. 7.7 mmol/l. The maximal GK velocity (Vmax) of the fetal islet and the contribution of GK to total glucose phosphorylation were also not significantly different from their adult counterparts. Western blot analysis of protein extracts from fetal and adult islets confirmed the presence of GK at 52 kDa. To determine if glucose had any effect on the Vmax of GK, islets were cultured for 7 days in medium containing low (1.4 or 2.8 mmol/l), normal (5.6 mmol/l), or high (11.2 or 16.8 mmol/l) concentrations of glucose. The maximal GK velocity increased linearly with increasing concentrations of glucose (r = 0.93; P < 0.01). To determine whether it was possible to up- and downregulate Vmax of GK, islets were cultured in either a low (1.4 mmol/l) or high (30 mmol/l) concentration of glucose for 7 days and then switched to the opposite concentration for a further 3 days. The Vmax of GK in the fetal islet was upregulated 3.8-fold when the glucose concentration was raised. Conversely, the Vmax was downregulated 3.6-fold when the glucose concentration was lowered. The same phenomenon was also observed in the adult islet. These data indicate that GK is the glucose sensor for the fetal rat islet, just as it is for the adult islet. Since glucose did not cause insulin secretion from the fetal islet, it was important to examine whether this substrate had any effect on its own metabolism. Glucose utilization was estimated, and its Vmax was found to increase linearly with increasing concentrations of glucose (r = 0.96; P < 0.01). We conclude that the inability of the fetal rat β-cell to secrete insulin in response to glucose cannot be explained by immaturity of GK or the glycolytic pathway.
Vulnerability of Islets in the Immediate Posttransplantation Period: Dynamic Changes in Structure and Function
Authors: Davalli Alberto M , Scaglia Luisa , Zangen David H , Hollister Jennifer , Bonner-Weir Susan , Weir Gordon C
Published: September 2013

To learn more about islet vulnerability in the immediate posttransplant period, 400 syngeneic islets were transplanted under the kidney capsule of B6AF1 mice. Three groups of recipients were used: normal mice (normal), streptozotocin (STZ)-diabetic (diabetic), and STZ-diabetic kept hypo- or normoglycemic with insulin pellets (diabetic-normalized). Normoglycemia was achieved in all three groups 14 days after transplantation; however, in the diabetic and diabetic-normalized groups, blood glucose levels throughout the posttransplantation period were respectively higher and lower than in the normal group. Grafts were harvested 1, 3, 7, and 14 days after transplantation and analyzed for morphology, β-cell death, β-cell mass, insulin content, and insulin mRNA expression. In all groups, substantial damage in islet grafts was found on days 1 and 3 with apoptotic nuclei and necrotic cores; on day 3, β-cell death was significantly higher in the diabetic group than in the other groups. Tissue remodeling occurred in all groups with stable graft appearance on day 14; the actual β-cell mass of the grafts was lowest in the diabetic group. Graft insulin content decreased in all groups on day 1 and fell even further on days 3 and 7. Insulin mRNA levels of grafts retrieved from both the diabetic and diabetic-normalized group were lower than those from the normal group already by day 1 and remained lower on day 14. In conclusion, the first few days of islet transplantation, even under the most advantageous circumstances of excellent metabolic control, are characterized by dynamic changes, with substantial islet cell dysfunction and death followed by tissue remodeling and then stable engraftment.
Mucosa-Associated (βT-integrinhigh) Lymphocytes Accumulate Early in the Pancreas of NOD Mice and Show Aberrant Recirculation Behavior
Authors: Hänninen Arno , Salmi Marko , Simell Olli , Jalkanen Sirpa
Published: September 2013

The mucosal addressin cell adhesion molecule-1 (MAd CAM-1) becomes expressed on islet vessels of NOD mice early during lymphocyte accumulation in islets. Because MAdCAM-1 preferentially mediates the homing of mucosal lymphocytes, islet-associated MAdCAM-1 could favor the accumulation of mucosal lymphocytes in pancreatic islets. Therefore, we investigated the relative frequency of islet-infiltrating lymphocytes with a mucosal phenotype (α4/β7-integrinhigh and L-selectinlow) at early and advanced stages of insulitis. We found that until the age of 12 weeks, lymphocytes with a mucosal phenotype were particularly frequent in the pancreas (percentage of β7- integrinhigh-lymphocytes was 48% at 8 weeks and 73% at 12 weeks), whereas in diabetic mice older than 16 weeks, their relative number was smaller (26% of pancreas- infiltrating lymphocytes). To define the origin and homing behavior of β7-integrinhigh-lymphocytes before their accumulation in pancreatic islets in NOD mice, we sought for such lymphocytes in different lymphoid organs and studied their recirculation. We found that compared with lymphocytes in several other strains, in NOD mice such lymphocytes were most frequent in nonmucosal lymphoid tissues (peripheral and pancreatic lymph nodes, spleen) from an early age. When injected to blood circulation, mucosal β7high-lymphocytes failed to home efficiently back to mucosal lymphoid tissue in NOD mice but homed aberrantly to nonmucosal lymphoid tissues. After adoptive transfer of diabetogenic splenocytes, the first lymphocytes accumulating in the pancreas were predominantly β7-integrinhigh. We conclude that mucosa-associated lymphocytes are involved in the early phases of islet-inflammation in NOD mice and that the aberrant homing behavior of lymphocytes expressing high levels of β7-integrin may associate with their accumulation in pancreatic islets early during insulitis.
Autoimmunity to the GM2-1 Islet Ganglioside Before and at the Onset of Type I Diabetes
Authors: Dotta Francesco , Gianani Roberto , Previti Marcello , Lenti Luisa , Dionisi Sabrina , D'Erme Maria , Eisenbarth George S , Di Mario Umberto
Published: September 2013

Recently, the GM2-1 pancreatic islet ganglioside, proposed as a potential autoantigen in type I diabetes autoimmunity, has been biochemically characterized and found to be a novel ganglioside structure. In the present study, we aimed to determine whether an autoimmune response toward this novel islet molecule is 1) present in type I diabetes and is specifically directed against this molecule and not to gangliosides in general and 2) predictive of disease in high-risk subjects. To this end, the following patients have been studied: 1) 24 newly diagnosed type I diabetic subjects, 20 islet cell autoantibody (ICA)-negative first-degree relatives of type I diabetic subjects, and 25 age-matched normal control individuals; and 2) 31 prospectively evaluated ICA+ first-degree relatives of type I diabetic subjects who were followed for up to 10 years, during which 14 of them developed type I diabetes. A direct assay for autoantibodies to GM2-1 and to other pancreatic gangliosides (GM3, GD3, GDla) was developed using an indirect immunoperoxidase technique performed directly on thin layer chromatography plates. Anti-GM2-1 autoantibodies (all belonging to the IgG class) were expressed in a high percentage of newly diagnosed type I diabetic subjects (71%), while no significant difference was found in the expression of antibodies directed against other pancreatic gangliosides (GM3, GD3, GDla) among the different groups studied. Anti-GM2-1 autoantibodies were also present in ICA+ relatives (64%) (P < 0.001 vs. control subjects and ICA relatives): in this group, life table analysis of progression to diabetes showed that anti–GM2-l autoantibodies were significantly (P < 0.001) associated with disease, occurring in all relatives developing type I diabetes within 5 years and thus identifying a cohort of ICA+ subjects with markedly increased diabetes risk.
A Molecular Variant of Angiotensinogen Is Associated With Diabetic Nephropathy in IDDM
Authors: Fogarty Damian G , Harron J Camille , Hughes Anne E , Nevin Norman C , Doherty Ciaran C , Maxwell A Peter
Published: September 2013

Recent studies have suggested that an inherited predisposition to essential hypertension may increase susceptibility to nephropathy for patients with IDDM. Essential hypertension has been linked to the angiotensinogen (AGT) gene in genetic linkage studies in American and European populations. A molecular variant (M235T), which has a functional effect, has been described with highest plasma AGT levels being associated with the TT genotype. In a case-control study, we have evaluated the role of this functional genetic marker in patients with IDDM and nephropathy and in IDDM patients without nephropathy. We studied 195 IDDM patients, of whom 95 had established diabetic nephropathy; the remaining 100 patients, who had no evidence of microalbuminuria, served as control subjects. All patients were whites born in Northern Ireland. The point mutation in the AGT gene was analyzed using restriction typing. The background frequency of the M235T variant was assessed in 80 healthy blood donors, and the TT genotype was present in 9%. This genotype occurred in 8% of control IDDM patients without nephropathy and 19% of IDDM patients with nephropathy (P = 0.025). The odds ratio for diabetic nephropathy associated with the TT genotype was 2.7 (95% CI 1.04–7.52). There was no relationship between blood pressure and AGT genotypes in the control group. We cannot exclude the possibility that the observed association in the nephropathy group is due to an association between AGT genotype and hypertension. This evidence may help to explain the predisposition to diabetic nephropathy afforded by hypertension and merits further investigation.
Early Atherosclerosis Is Retarded by Improved Long-Term Blood Glucose Control in Patients With IDDM
Authors: Jensen-Urstad Kerstin J , Reichard Per G , Rosfors J Stefan , Lindblad Lars Erik L , Jensen-Urstad Mats T
Published: September 2013

Microangiopathy is retarded by improved blood glucose control in patients with IDDM. Whether or not this is true for macroangiopathy (atherosclerosis) has remained unclear. A total of 59 patients (44 ± 1.5 years, previous HbA1c 9.4 ± 0.2%, mean ± SE) with IDDM were investigated. Of the 59 patients, 31 had been randomized to long-term intensified conventional insulin treatment (ICT), and the remaining 28 had received standard insulin treatment (ST). Blood glucose control was significantly better in the ICT patients with an HbA1c value (mean of 29 values during 10 years) of 7.1 ± 0.1% compared with the ST patients' 8.2 ± 0.2% (P < 0.0001). With high-frequency ultrasound, endothelial function was measured as flow-mediated dilation of the right brachial artery. The carotid arteries were scanned for plaques, intima-media thickness was measured, and arterial wall stiffness was calculated in the right common carotid artery. These measurements correlate with manifest and/or risk factors for coronary atherosclerosis. The patients in the ST group had stiffer arteries (P = 0.011) and thicker intima-media in the left common carotid artery (P = 0.009) than those in the ICT group. Patients with lower HbA1c generally had better endothelial function (P = 0.028) and less stiff arteries (P = 0.009). Better blood glucose control in patients with IDDM is related not only to less microangiopathy but also to a slower development of atherosclerosis.
HLA-Encoded Genetic Predisposition in IDDM: DR4 Subtypes May Be Associated With Different Degrees of Protection
Authors: Undlien Dag E , Friede Thomas , Rammensee Hans-Georg , Joner Geir , Dahl-Jörgensen Knut , Sövik Oddmund , Akselsen Hanne E , Knutsen Ingebjörg , Rönningen Kjershi S , Thorsby Erik
Published: September 2013

Recent studies have shown that the risk conferred by the high-risk DQAl*03-DQBl*0302 (DQ8) haplotype is modified by the DRB1*O4 allele that is also carried by this haplotype. However, many of these studies suffer from lack of sufficient numbers of DQ-matched control subjects, which are necessary because there is a strong linkage disequilibrium between genes in the HLA complex. In the present study, using a large material of IDDM patients and DQ-matched control subjects, we have addressed the contribution of DR4 subtypes to IDDM susceptibility. Our data, together with recent data from others, clearly demonstrate that some DR4-DQ8 haplotypes are associated with disease susceptibility, while others are associated with protection, depending on the DRB1*O4 allele carried by the same haplotype. In particular, our data demonstrate that DRBl*0401 confers a higher risk than DRBl*0404. Based on combined available data on the genetic susceptibility encoded by various DR4-DQ8 haplotypes and the amino acid composition of the involved DRβ*04 chains as well as the ligand motifs for these DR4 subtypes, we have developed a unifying hypothesis explaining the different risks associated with different DR4-DQ8 haplotypes. We suggest that disease susceptibility is mainly conferred by DQ8 while DR4 subtypes confer different degrees of protection. Some DR4 subtypes (i.e., DRB1*0405, 0402, and 0401) confer little or no protection, while others (i.e., DRB 1*0404, 0403, and 0406) cause an increasing degree of protection, possibly by binding a common protective peptide. Features of a protective peptide that fit such a model are briefly discussed.
The Effect of Leptin Is Enhanced by Microinjection Into the Ventromedial Hypothalamus
Authors: Jacob Ralph J , Dziura James , Medwick Matthew B , Leone Paola , Caprio Sonia , During Matthew , Shulman Gerald I , Sherwin Robert
Published: September 2013

To determine whether changes in food intake produced by leptin involve targeting the hormone to distinct central nervous system regions, guide cannulas were positioned stereotaxically into three brain regions—the ventromedial hypothalamus (VMH) (bilaterally, n = 6), the dorsal raphe nucleus (n = 3), and the lateral ventricle (n = 3)—of nonobese male rats (400–500 g). Daily food intake and body weight changes were measured during twice-daily injections of saline (0.1 μl) followed by recombinant human leptin (0.05 μg) for 3 days via the brain cannulas. VMH–injected rats also were followed during a postleptin saline recovery interval. This small dose of leptin did not change food intake or body weight from that during the preceding saline injection period in ventricle-injected or dorsal raphe–injected rats. In sharp contrast, VMH-injected rats ate much less food (56 ± 8% basal) and lost 9 ± 3 g/day or 5% of their body weight during 3 days of leptin administration. VMH-injected animals fully recovered from leptin-induced effects within 3 days. We conclude that small doses of leptin that do not effect eating behavior when delivered to the ventricle or the dorsal raphe (another brain region believed to regulate feeding), suppress food intake when injected into the VMH. These data suggest that the VMH or a brain region in close proximity to it is a key target for the biological actions of leptin.
Role of Fatty Acids in the Pathogenesis of Insulin Resistance and NIDDM
Authors: Boden Guenther
Published: September 2013

Evidence is reviewed that free fatty acids (FFAs) are one important link between obesity and insulin resistance and NIDDM. First, plasma FFA levels are elevated in most obese subjects. Second, physiological elevations in plasma FFA concentrations inhibit insulin stimulated peripheral glucose uptake in a dose-dependent manner in normal controls and in patients with NIDDM. Two possible mechanisms are identified: 1) a fat-related inhibition of glucose transport or phosphorylation, which appears after 3–4 h of fat infusion, and 2) a decrease in muscle glycogen synthase activity, which appears after 4–6 h of fat infusion. Third, FFAs stimulate insulin secretion in nondiabetic individuals. Some of this insulin is transmitted in the peripheral circulation and is able to compensate for FFA-mediated peripheral insulin resistance. FFA-mediated portal hyperinsulinemia counteracts the stimulation of FFAs on hepatic glucose production (HGP) and thus prevents hepatic glucose overproduction. We speculate that, in obese individuals who are genetically predisposed to develop NIDDM, FFAs will eventually fail to promote insulin secretion. The stimulatory effect of FFAs on HGP would then become unchecked, resulting in hyperglycemia. Hence, continuously elevated levels of plasma FFAs may play a key role in the pathogenesis of NIDDM in predisposed individuals by impairing peripheral glucose utilization and by promoting hepatic glucose overproduction.
Effects of Free Fatty Acids and Glucose on Splanchnic Insulin Dynamics
Authors: Hennes Magda M I , Dua Arnavaz , Kissebah Ahmed H
Published: September 2013

The mechanism of hyperinsulinemia that accompanies insulin resistance in some abdominally obese and diabetic individuals is poorly understood. Both increased secretion of insulin and decreased clearance have been demonstrated. The present study was undertaken to examine the role of free fatty acids (FFAs) and glucose in regulating splanchnic insulin dynamics in vivo. Plasma FFA levels were raised approximately twofold via an intralipid/heparin infusion in eight lean women. Insulin dynamics were assessed using the individual's C-peptide kinetic coefficients. Studies were performed in the basal state and during two levels of glycemia, 7 and 11 mmol/l. Studies were repeated using saline, and thus each subject served as her own control. Under basal conditions, raising FFA flux resulted in a modest increase in plasma insulin concentration (PIC) secondary to an increase in insulin secretion rate (ISR); however, endogenous insulin clearance (EIC) was not influenced. During the 7 mmol/l hyperglycemic clamp, maintaining a high FFA flux resulted in a 30% increase in PIC above the effect produced by glucose alone. This represents the cumulative effects of stimulation of ISR and inhibition of EIC. Clamping plasma glucose at 11 mmol/l while maintaining a high FFA flux increased PIC twofold above that produced by glucose alone. This increase in PIC was mainly due to a significant reduction in EIC without an accompanying increase in ISR (392 ± 159 and 787 ± 187 ml/min with and without intralipid infusion, respectively). Analysis of variance indicated that the suppressive effect of FFA on EIC was independent of the effect of glucose. The effect of the two substrates seems to be additive.
Insulin Sensitivity and Acute Insulin Response in African-Americans, Non-Hispanic Whites, and Hispanics With NIDDM: The Insulin Resistance Atherosclerosis Study
Authors: Haffner Steven M , Howard George , Mayer Elizabeth , Bergman Richard N , Savage Peter J , Rewers Marian , Mykkänen Leena , Karter Andrew J , Hamman Richard , Saad Mohammed F
Published: September 2013

NIDDM is usually characterized by β-cell failure decreased and insulin sensitivity. It has been reported that a high proportion of African-American NIDDM subjects are insulin sensitive. To examine this issue, we determined insulin sensitivity (SI) in 479 NIDDM subjects by minimal model analyses of frequently sampled intravenous glucose tolerance (FSIGT) from the Insulin Resistance Atherosclerosis Study (IRAS), a large multicenter study of insulin sensitivity and cardiovascular risk in African-Americans, Hispanics, and non-Hispanic whites. The African-Americans and non-Hispanic whites were sampled in Los Angeles and Oakland, California. The non-Hispanic whites and Hispanics were sampled in San Antonio, Texas, and San Luis Valley, Colorado. We defined the proportion of insulin-sensitive (SI) subjects as ≥1.61 min−1 · μU−1 · ml−1, which is above the median for nondiabetic subjects of all ethnic groups in the IRAS. Using this definition, the proportion of insulin-sensitive diabetic subjects was very low in all ethnic groups (non-Hispanic whites [14.3%] vs. African- Americans [6.5%], P = 0.039 in Los Angeles and Oakland; non-Hispanic whites [6.8%] vs. Hispanics [4.9%], P = 0.737 in San Luis Valley and San Antonio). These results were also similar in newly diagnosed mildly hyperglycemic diabetic subjects. In addition, these results were not affected by the adjustment for differences in obesity, body fat distribution, and severity of hyperglycemia. Even in nonobese subjects (with BMI <30 kg/m2), the proportion of insulin-sensitive subjects (S1 ≥1.61 min−1 · μU−1 · ml−1) was low (3.6–9.7%). The acute insulin response (AIR) was significantly higher in African-Americans than in non-Hispanic whites; there were no ethnic differences in AIR between Hispanics and non–Hispanic whites. There were no significant ethnic differences for non-insulin-mediated glucose disposal (SG). We conclude that the number of insulinsensitive NIDDM subjects is low and similar among non-Hispanic whites, Hispanics, and African-Americans in the U.S.
Polymorphism of the β3-Adrenergic Receptor Gene Affects Basal Metabolic Rate in Obese Finns
Authors: Sipilä Raisa , Uusitupa Matti , Heikkinen Sami , Rissanen Aila , Laakso Markku
Published: September 2013

Low basal metabolic rate (BMR) is a risk factor for weight gain and obesity. The polymorphism at codon 64 of the β3-adrenergic receptor gene has been suggested to be associated with BMR. We investigated the frequency of the Trp64Arg of the β3-adrenergic receptor gene and the effects of this polymorphism on BMR in obese Finns. Altogether, 170 obese subjects (29 men, 141 women, BMI 34.7 ± 3.8 kg/m2, mean ± SD) participated in the study. The frequency of the Trp64Arg polymorphism was 19%. None of the obese subjects were homozygous for the Arg-encoding allele. The frequency of the Trp64Arg polymorphism in obese Finns did not differ from nonobese and normoglycemic control subjects. BMR adjusted for lean body mass and age was lower in subjects with the Trp64Arg polymorphism (n = 20) than in normal homozygotes Trp64Trp (n = 99) (1,569 ± 73 vs. 1,635 ± 142 kcal/day, P = 0.004). For the female group (n = 98), the respective values were 1,501 ± 66 kcal/day vs. 1,568 ± 127 kcal/day (P = 0.004). There were no significant differences in weight, BMI, waist-tohip ratio, lean body mass, percentage of fat, and respiratory quotient between the groups with or without the Trp64Arg polymorphism. Neither serum glucose nor insulin levels differed between the two groups. We conclude that the Trp64Arg polymorphism of the β3-adrenergic receptor gene affects basal metabolic rate in obese Finns but does not have significant effect on glucose metabolism.
Discordant Effects of Guanidines on Renal Structure and Function and on Regional Vascular Dysfunction and Collagen Changes in Diabetic Rats
Authors: Nyengaard Jens R , Chang Katherine , Berhorst Susan , Reiser Karen M , Williamson Joseph R , Tilton Ronald G
Published: September 2013

We examined the effects of aminoguanidine and methylguanidine on vascular dysfunction, glomerular structural changes, and indexes of early and late nonenzymatic glycation in 7-month streptozotocininduced diabetic rats. Kidney weight, glomerular volume, fractional mesangial volume, glomerular capillary basement membrane width, and urinary albumin excretion were increased in diabetic rats. Diabetes also 1) increased vascular albumin permeation twofold in retina, sciatic nerve, aorta, skin, and kidney; 2) decreased renal collagenase-soluble collagen; 3) increased collagen-associated fluorescence in kidney and skin but not in aorta; and 4) increased glycated hemoglobin levels and aortic pentosidine levels. Aminoguanidine reduced albuminuria by 70% after 4 months, and both guanidines 1) normalized aortic pentosidine levels and renal collagenase-soluble collagen, 2) had no effect on glycated hemoglobin levels or collagen-associated fluorescence (in aorta, kidney, or skin), and 3) had little or no effect on regional albumin permeation. These discordant effects of aminoguanidine on diabetes-induced vascular changes versus parameters of nonenzymatic glycation are consistent with a multifactorial pathogenesis of diabetic complications, including roles for metabolic imbalances independent of nonenzymatic glycation. To the extent that glomerular matrix accumulation and increased regional albumin permeation in chronically diabetic rats are sequelae of nonenzymatic glycation, these findings point to an important role for early glycation reactions and products.
Vanadate Activates Membranous Nonreceptor Protein Tyrosine Kinase in Rat Adipocytes
Authors: Elberg Gerard , He Zhibin , Li Jinping , Sekar Natesampillai , Shechter Yoram
Published: September 2013

The insulin-like effects of vanadate are independent of the insulin receptor and insulin receptor substrate 1 (IRS-1) phosphorylation. A cytosolic protein tyrosine kinase (CytPTK), sensitive to inhibition by nanomolar concentrations of staurosporine (concentration at which 50% inhibition occurs [IC50], 1–2 nmol/1), has been implicated in some (i.e., glucose oxidation, lipogenesis) but not all (i.e., hexose uptake, inhibition of lipolysis) of the insulin-like effects of vanadate. We report here the existence of another nonreceptor protein tyrosine kinase in rat adipocytes, located exclusively in the plasma membranes (MembPTK), which we suggest is associated with hexose uptake and the antilipolytic activity of vanadate. MembPTK is a nong-lycoprotein with an estimated molecular weight of 55–60 kDa. In a cell-free experiment, vanadate activates MembPTK seven- to ninefold (median effective dose, 17 ± 2 μmol/l). Vanadate-activated MembPTK is inhibited by staurosporine (IC50, 60 ± 5 nmol/l). In intact adipocytes, staurosporine antagonized vanadate-induced hexose uptake (IC50, 6.0 ± 0.3 μmol/l) and significantly reversed the antilipolytic effect of vanadate (IC50, 5.0 ± 0.4 μmol/l). After vanadate treatment, a phosphorylated P55 protein is immunoprecipitated by antibodies to both phosphotyrosine and phosphatidyli-nositol (PI) 3-kinase. In conclusion, rat adipocytes contain an additional vanadate-activatable nonreceptor membranous protein tyrosine kinase that may participate in the effects of vanadate not carried out by CytPTK. We also suggest that after treatment with vanadate, MembPTK is activated by autophosphorylation and interacts with PI 3-kinase. This may explain how vanadate activates PI 3-kinase without involving receptor activation and IRS-1 phosphorylation.
GLP-I(7–36) Amide Augments Ba2+ Current Through L-Type Ca2+ Channel of Rat Pancreatic β-Cell in a cAMP-Dependent Manner
Authors: Suga Sechiko , Kanno Takahiro , Nakano Kyoko , Takeo Teruko , Dobashi Yoshifusa , Wakui Makoto
Published: September 2013

The whole-cell patch-clamp method was used to examine the effect of glucagon-like peptide I (GLP-I)(7–36) amide on the activation process of L-type Ca2+ channels of rat pancreatic β-cells. After depolarization, GLP-I (1–100 nmol/l) caused action potentials in cells exposed to a glucose-free solution for 10 min. The percentage of cells producing action potential depended on the concentration of GLP-I. In some cells, GLP-I caused action potentials without the prior depolarization of the membrane. In cells exposed to the glucose-free solution for longer than 30 min, or in cells that were deprived of ATP by a means of the conventional whole-cell configuration, GLP-I (20 nmol/l) did not cause the electrical excitation. Application of GLP-I augmented the maximum Ba2+ current (IBa) through L-type Ca2+ channels and shifted the current voltage curve to the left. Values of changes in the maximum IBa depended on GLP-I concentration. Application of dibutyryl cAMP (dbcAMP, 1 mmol/1) also augmented IBa. In cells pretreated with Rp-cAMP, dbcAMP did not change the magnitude of IBa. Also in cells pretreated with Rp-cAMP, GLP-I failed to augment IBa. These results suggest that in pancreatic β-cells, GLP-I, by a cAMP-dependent mechanism, increases opening of L-type Ca2+ channels. cAMP-dependent augmentation of Ca2+ entry as well as cAMP production itself by GLP-I plays a crucial role in controlling insulin secretion.
Mechanisms of Liver and Muscle Insulin Resistance Induced by Chronic High-Fat Feeding
Authors: Oakes Nicholas D , Cooney Gregory J , Camilleri Souad , Chisholm Donald J , Kraegen Edward W
Published: September 2013

To elucidate cellular mechanisms of insulin resistance induced by excess dietary fat, we studied conscious chronically high-fat–fed (HFF) and control chow diet-fed rats during euglycemic-hyperinsulinemic (560 pmol/1 plasma insulin) clamps. Compared with chow diet feeding, fat feeding significantly impaired insulin action (reduced whole body glucose disposal rate, reduced skeletal muscle glucose metabolism, and decreased insulin suppressibility of hepatic glucose production [HGP]). In HFF rats, hyperinsulinemia significantly suppressed circulating free fatty acids but not the intracellular availability of fatty acid in skeletal muscle (long chain fatty acyl-CoA esters remained at 230% above control levels). In HFF animals, acute blockade of β-oxidation using etomoxir increased insulin-stimulated muscle glucose uptake, via a selective increase in the component directed to glycolysis, but did not reverse the defect in net glycogen synthesis or glycogen synthase. In clamp HFF animals, etomoxir did not significantly alter the reduced ability of insulin to suppress HGP, but induced substantial depletion of hepatic glycogen content. This implied that gluconeo-genesis was reduced by inhibition of hepatic fatty acid oxidation and that an alternative mechanism was involved in the elevated HGP in HFF rats. Evidence was then obtained suggesting that this involves a reduction in hepatic glucokinase (GK) activity and an inability of insulin to acutely lower glucose-6-phos-phatase (G-6-Pase) activity. Overall, a 76% increase in the activity ratio G-6-Pase/GK was observed, which would favor net hepatic glucose release and elevated HGP in HFF rats. Thus in the insulin-resistant HFF rat 1) acute hyperinsulinemia fails to quench elevated muscle and liver lipid availability, 2) elevated lipid oxidation opposes insulin stimulation of muscle glucose oxidation (perhaps via the glucose-fatty acid cycle) and suppression of hepatic gluconeogenesis, and 3) mechanisms of impaired insulin-stimulated glucose storage and HGP suppressibility are not dependent on concomitant lipid oxidation; in the case of HGP we provide evidence for pivotal involvement of G-6-Pase and GK in the regulation of HGP by insulin, independent of the glucose source.
The Blood Contribution to Early Myocardial Reperfusion Injury Is Amplified in Diabetes
Authors: McDonagh Paul F , Hokama Jason Y , Copeland Jack G , Reynolds James M
Published: September 2013

Cardiovascular disease is excessive in diabetes, and blood cell function is altered. It is not clear, however, if alterations in the blood contribute to the excessive cardiovascular complications of this disease. In this study, we compared the contribution of nondiabetic and diabetic blood to myocardial reperfusion injury. The recovery of cardiac contractile function following no-flow ischemia was studied in isolated diabetic and nondiabetic rat hearts perfused with diabetic or nondiabetic diluted whole blood. Hearts were isolated from 10- to 12-week-old diabetic (streptozotocin, 65 mg/kg, i.v.) and nondiabetic rats and perfused with a Krebs-albumin-red cell solution (K2RBC, Hct 20%). After a 30-min pre-ischemic control period, during which cardiac pump function was evaluated, diabetic and nondiabetic hearts were perfused for 5 min with diluted whole blood (DWB; Hct 20%) collected from either diabetic or nondiabetic donor animals. Coronary flow was then stopped and the hearts subjected to 30 min of no-flow ischemia. Following ischemia, the hearts were reperfused with the K2RBC perfusate. Cardiac contractile function was evaluated throughout the 60-min reperfusion period. Six groups were studied: diabetic and nondiabetic hearts perfused before ischemia with either K2RBC, nondiabetic DWB (NDDWB), or diabetic DWB (DDWB). Perfusion with DWB prior to ischemia impaired the recovery of contractile function in all cases. The impairment to recovery was greater with DDWB than with NDDWB. Although diabetic hearts perfused with K2RBC throughout recovered quite well, the effect of DDWB perfusion in the diabetic hearts was dramatic. In an effort to determine why diabetic blood impaired functional recovery, measures of blood filterability and the generation of reactive oxygen species (ROS) were made. We found that diabetic blood was less filterable than nondiabetic blood; that is, the diabetic blood cells tended to plug the 5-μm filter pores more readily than the nondiabetic blood cells. Also, we found that the diabetic blood was capable of generating significantly greater ROS (oxygen free radicals) than nondiabetic blood (P < 0.05). These findings suggest that the blood contribution to myocardial reperfusion injury is amplified in diabetes. A tendency for diabetic blood cells to plug capillary-sized pores and show enhanced oxygen free radical production may account for the excessive contribution of diabetic blood to reperfusion injury in the heart.
Upregulation of Mesangial Growth Factor and Extracellular Matrix Synthesis by Advanced Glycation End Products Via a Receptor-Mediated Mechanism
Authors: Pugliese Giuseppe , Pricci Flavia , Romeo Giulio , Pugliese Francesco , Mené Paolo , Giannini Stefano , Cresci Barbara , Galli Gianna , Rotella Carlo M , Vlassara Helen , Mario Umberto Di
Published: September 2013

Enhanced advanced glycosylation end product (AGE) formation has been shown to participate in the patho-genesis of diabetes-induced glomerular injury by mediating the increased extracellular matrix (ECM) deposition and altered cell growth and turnover leading to mesangial expansion. These effects could be exerted via an AGE-receptor-mediated upregulation of growth factors, such as the IGFs and transforming growth factor-β (TGF-β). We tested this hypothesis in human and rat mesangial cells grown on nonglycated or native bovine serum albumin (BSA), glycated BSA with AGE formation (BSA-AGE), or glycated BSA in which AGE formation was prevented by the use of aminoguanidine (BSA-AM), in the presence or absence of an antibody, α-p60, directed against the p60/OST protein named AGE-receptor 1 (AGE-R1), or normal control (pre-immune) serum. The mRNA and/or protein levels of IGF-I, IGF-II, IGF receptors, IGF binding proteins (IGFBPs), TGF-β1 and the ECM components fibronectin, laminin, and collagen IV were measured, together with cell proliferation. Both human and rat mesangial cells grown on BSA-AGE showed increased IGF-I and total and bioac-tive TGF-β medium levels and enhanced IGF-I, IGF-II, and TGF-β1 gene expression, compared with cells grown on BSA, whereas total IGFBP and IGFBP-3 medium content, IGF receptor density and affinity, and IGF-I receptor transcripts were unchanged. Moreover, cells grown on BSA-AGE showed increased ECM protein and mRNA levels versus cells cultured on BSA, whereas cell proliferation was unchanged in human mesangial cells and slightly reduced in rat mesangial cells. Growing cells on BSA-AM did not affect any of the measured parameters. Co-incubation of BSA-AGE with anti-AGE-R1, but not with pre-immune serum, prevented AGE-induced increases in IGF-I, TGF-β1, and ECM production or gene expression; anti-AGE-R1 also reduced growth factor and matrix synthesis in cells grown on BSA. These results demonstrate that mesangial IGF and TGF-β1 synthesis is upregulated by AGE-modified proteins through an AGE-receptor-mediated mechanism. The parallelism with increased ECM production raises the speculation that the enhanced synthesis of these growth factors resulting from advanced nonen-zymatic glycation participates in the pathogenesis of hyperglycemia-induced mesangial expansion.
Progressive Islet Graft Failure Occurs Significantly Earlier in Autoantibody-Positive Than in Autoantibody-Negative IDDM Recipients of Intrahepatic Islet Allografts
Authors: Jaeger Clemens , Brendel Mathias D , Hering Bernhard J , Eckhard Michael , Bretzel Reinhard G
Published: September 2013

Alloimmunity has been uncovered to be a cause of graft loss representing a major barrier for clinical islet transplantation, and several studies are designed to evaluate new strategies for immunosuppression to prevent alloimmunity. In contrast, the significance for autoimmune destruction of transplanted β-cells has remained somewhat controversial. Recently, two case reports based on histological findings have suggested recurrent autoimmune insulitis despite immunosup-pressive therapy both in clinical pancreas and in islet transplantation. In the present study, in 23 islet-grafted patients with IDDM receiving standard immunosuppressive therapy, we demonstrate that progressive impairment of islet graft function occurs significantly earlier in those individuals positive for autoantibodies as a typical stigma of diabetes-associated autoimmunity that is well established in the pre-diabetic periods of IDDM. Intraportal infusion of allo-geneic islets was performed in 23 C-peptide-negative IDDM patients, according to the clinical transplantation categories defined as islet after kidney (IAK) or simultaneous islet and kidney (SIK). Complete islet graft failure was defined as the 1st day of permanent C-peptide negativity in the serum (<0.2 ng/ml) and C-peptide negativity in the urine (<2 μg/dl). The median observation period following islet transplantation was 12 months (range 1–50) with a cumulative follow-up of 336 months. Islet cell antibodies (ICAs) and GAD65 antibodies were monitored before and regularly after islet transplantation. Kaplan-Meier survival analysis and log-rank statistics revealed a significant (P < 0.05) difference in cumulative islet graft survival depending on the presence of islet cell and/or GAD65 antibodies. These results strongly suggest that recurrent autoimmunity directed to transplanted β-cells contributes to islet graft failure despite sustained immunosuppression. For successful clinical islet transplantation in the future, new immunosuppressive therapies are needed to prevent both alloimmunity and autoimmunity.
Long-Term Function (6 Years) of Islet Allografts in Type 1 Diabetes
Authors: Alejandro Rodolfo , Lehmann Roger , Ricordi Camillo , Kenyon Norma S , Angelico M Clara , Burke George , Esquenazi Violet , Nery Jose , Betancourt Arthur E , Kong Shen S , Miller Joshua , Mintz Daniel H
Published: September 2013

Eight type 1 diabetic patients, ages 29–41 years, with mean diabetes duration of 23 years (range 18–29 years) received islet transplants from 1 to 5 donors. Seven patients had stable kidney allografts 1–11 years before the islet transplant, and one patient had a simultaneous islet-kidney allograft. Patients' blood glucose control was poor as reflected by the mean ± SD HbA1c of 9.1 ± 1.7% before transplant. Of the first three patients, two (1 and 3) achieved insulin independence for 36 and 38 days, respectively. Two recipients rejected their islet grafts within 1 month (2 and 8) and therefore were excluded from analysis. The HbA1c and insulin requirement of the six remaining patients who had persistent islet function for more than 60 days was significantly reduced from 9.3 ± 1.9 to 6.4 ± 1.0% (P = 0.002) and from 0.75 ± 0.15 to 0.35 ± 0.12 U · kg−1 · day−1 (P < 0.001), respectively. The two patients with the longest graft survival (4 and 6) achieved a normalization or near-normalization of their HbA1c levels during 6 years in the absence of severe episodes of hypoglycemia. As demonstrated by a decline in C-peptide response during Sustacal challenge tests over a 6-year period, there was a diminution of islet allograft function over time, despite persistence of normal or near normal HbA1c. We concluded that transplantation of allogeneic islets with an islet mass comparable with whole or segmental pancreas transplants in type 1 diabetic patients can result in long-term islet allograft function; further, we concluded that, in conjunction with small dosages of exogenous insulin, a functioning islet allograft can result in near-normalization of blood glucose levels and significant improvement in HbAlc. The occurrence of severe hypoglycemic episodes observed for patients in the Diabetes Control and Complications Trial was not observed in recipients with functioning islet transplants, despite the continuous need for exogenous insulin therapy to sustain normal HbA1c over the 6-year follow-up. The significant improvement in metabolic control observed for the patients described in this study, and the potential to significantly decrease or halt the progression of diabetic complications, support the continued application of islet allotransplantation as a treatment modality for type 1 diabetic patients.
Oscillations in Activities of Enzymes in Pancreatic Islet Subcellular Fractions Induced by Physiological Concentrations of Effectors
Authors: MacDonald Michael J , Al-Masri Hussam , Jumelle-Laclau Muriel , Cruz Meredith O
Published: September 2013

Glucose, the most potent insulin secretagogue, stimulates insulin secretion by aerobic glycolysis, but other secretagogues stimulate insulin release exclusively by mitochondrial metabolism. It is well known that in the intact pancreatic β-cell, either kind of secretagogue can induce oscillations in metabolism (e.g., glycolysis, ATP/ADP, NAD(P)/NAD(P)H ratios) that occur with a periodicity similar to oscillations in membrane electrical potential and insulin secretion. In this study, pancreatic islet cytosol or mitochondrial fractions were incubated in the presence of physiological concentrations of substrates. Repeated additions of physiological effectors caused oscillations in the activities of the three enzymes studied. Succinate dehydrogenase activity in islet mitochondrial extracts was made to oscillate by adding oxaloacetate (5 μmol/l) to inhibit the enzyme. The enzyme was reactivated by adding acetyl-CoA (3 μmol/l), which combines with oxaloacetate in the citrate synthase reaction and lowers the concentration of oxaloacetate, thus beginning another oscillation. Pyruvate kinase activity was made to oscillate by adding fructose bisphosphate (10 μmol/l). Fructose bisphosphate was degraded to triose phosphates fairly rapidly, and, as it was degraded, there was a parallel decrease in pyruvate kinase activity. The enzyme was reactivated and made to oscillate with subsequent additions of fructose bisphosphate. The mitochondrial glycerol phosphate dehydrogenase was made to oscillate by adding EGTA to chelate calcium, which activates the enzyme. When the concentration of free calcium was raised to >0.1 μmol/l by adding more calcium, the activity of the enzyme increased. Repeated additions of chelator and calcium caused the enzyme activity to oscillate. The results with these three enzymes and physiological concentrations of naturally occurring effectors raise the possibility that the activities of not only these enzymes but of numerous enzymes oscillate in vivo in response to levels of allosteric effectors and substrates. If this is the case, pacemaker activity may result from complex effects distributed across multiple regulatory sites in both the cytosol and mitochondria, rather than from a single enzyme acting as a primary pacemaker.
Effects of Insulin on Blood Flow and Volume in Skeletal Muscle of Patients With IDDM: Studies Using [15O]H2O, [15O]CO, and Positron Emission Tomography
Authors: Raitakari Maria , Nuutila Pirjo , Knuuti Juhani , Raitakari Olli T , Laine Hanna , Ruotsalainen Ulla , Kirvelä Olli , Takala Teemu O , Iida Hidehiro , Yki-Järvinen Hannele
Published: September 2013

Exaggerated vasoconstriction and blunted vasodilation of peripheral resistance arteries to various vasoactive agents characterize patients with IDDM. We characterized the hemodynamic effects of insulin in skeletal muscle in patients with IDDM. Muscle blood flow and blood volume were measured basally and during a highdose insulin infusion (5 mU · kg−1 · min−1) in seven normotensive patients with IDDM (age, 30 ± 6 years; BMI, 24.5 ± 2.0 kg/m2; blood pressure, 124 ± 12/78 ± 11 mmHg) and nine matched normal subjects, using [15O]H2O, [15O]CO, and positron emission tomography (PET). Whole-body insulin sensitivity was determined using the euglycemic insulin clamp technique. Insulinstimulated whole-body glucose uptake was significantly lower in the patients with IDDM (45 ± 15 μmol · kg−1 · min−1) than in the normal subjects (62 ± 14 umol · kg−1 · min−1) (P < 0.05). Insulin increased muscle blood flow by 111 ± 69% above basal from 3.0 ± 2.0 to 5.8 ± 3.0 ml · 100 g−1 muscle · min−1 (P < 0.005) in the normal subjects, but only by 42 ± 30% from 2.0 ± 0.9 to 2.9 ± 1.4 ml · 100 g−1 muscle · min−1 (P < 0.005) in patients with IDDM (P < 0.05 for change in flow in IDDM vs. normal subjects). The calculated muscle vascular resistances were comparable basally, but higher during hyperinsulinemia in the patients with IDDM (37 ± 17 mmHg · 100 g · min · ml−1) than in the normal subjects (16 ± 7 mmHg · 100 g · min · ml−1) (P < 0.05). Muscle blood volume increased significantly by insulin in both groups without any difference between the groups. We conclude that the ability of supraphysiological concentrations of insulin to stimulate muscle blood flow is blunted in patients with IDDM, because of the inability of insulin to stimulate linear flow velocity rather than blood volume in skeletal muscle. This defect adds yet another defect to the list of abnormalities in vascular function in IDDM, which might predispose these patients to develop hypertension.
Leptin Sensitivity in Nonobese Glucagon-Like Peptide I Receptor −/− Mice
Authors: Scrocchi Louise A , Brown Theodore J , Drucker Daniel J
Published: September 2013

Glucagon-like peptide I (GLP-I) stimulates glucosedependent insulin secretion and inhibits food intake in the central nervous system. Because leptin reduces food intake but inhibits insulin secretion, we examined leptin action in mice with a null mutation in the GLP-I receptor. Intracerebroventricular leptin administration inhibited food intake in both wild-type and GLP-I receptor (GLP-IR) −/− mice, and daily intraperitoneal administration of leptin for 2 weeks produced comparable reductions in food intake and body weight in control and GLP-IR −/− mice. Glucose tolerance was improved in both wild-type and GLP-IR −/− mice, whether pair fed or leptin treated; however, blood sugars were significantly lower in the leptin-treated GLPIR −/− mice following oral glucose challenge (P < 0.01). Glucose-stimulated insulin was reduced in both pair-fed and leptin-treated mice (P < 0.01–0.001); however, insulin levels were significantly lower in leptin-treated versus pairfed GLP-IR −/− mice (P < 0.01). A single leptin injection had no effect on glucose tolerance in GLPIR −/− mice, but decreased hepatic PEPCK mRNA in both wild-type and GLP-IR −/− mice. The improvement in blood glucose excursion, despite lower levels of glucose-stimulated insulin in lean leptin-treated GLP-IR −/− mice, suggests that leptin may have beneficial effects on control of blood glucose in the absence of obesity. Furthermore, the greater effects of leptin on glucose and insulin in leptin-treated versus pair-fed GLP-IR −/− mice raises the possibility that disruption of GLP-I signaling modifies the sensitivity to leptin in vivo.