bluuebunny/crossref_metadata_2025 huggingface dataset:
Perinatal Autoimmunity in Offspring of Diabetic Parents: The German Multicenter BABY-DIAB Study: Detection of Humoral Immune Responses to Islet Antigens in Early Childhood
Authors: Roll Ursula , Füchtenbusch Martin , Ziegler Anette-G , Christie Michael R , Payton Mark A , Hawkes Charlotte J
Published: September 2013

IDDM results from immune-mediated destruction of insulin-producing pancreatic (β-cells in individuals genetically susceptible for the disease. There is evidence that the 65-kDa isoform of GAD plays a critical role in the induction of autoimmune diabetes in NOD mice. In humans, it is still unclear when and to what β-cell antigens autoreactive lymphocytes become activated during early disease. We conducted a prospective study from birth, BABY-DIAB, among children of mothers with IDDM or gestational diabetes or fathers with IDDM, and we investigated the temporal sequence of antibody responses to islet cells (ICA), insulin (IAA), GAD (GADA), and the protein tyrosine phosphatase IA-2/ICA512 (IA-2A). Of 1,019 children included at birth, we have currently followed 513 to the age of 9 months, 214 to the age of 2 years, and 37 to the age of 5 years. At birth, all antibody specificities were frequent in newborns of diabetic mothers but not fathers and are suggested to be transplacentally acquired because they are strongly correlated with antibody levels in their diabetic mothers. In early childhood, antibody levels were <99th percentdle of control subjects in the majority of children. However, 37 children exhibited elevated antibody levels; these were most frequently detected at the age of 2 years. The antibody prevalence at age 2 years was 2.3% for ICA, 7% for IAA, 4.2% for GADA, and 2.8% for IA-2A (8.9% positive for at least one antibody). Children of diabetic fathers were positive for at least one antibody more frequently than were children of diabetic mothers (9 months of age: 8.5 vs. 3.6% 2 years of age: 16.7 vs. 7.9%). There was no specific sequence in the appearance of positive autoantibodies, but 13 (35%) antibody-positive cases already had more than one ICA before the age of 2 years and 7 (19%) showed reactivity to three islet cell antigens before age 5 years. The presence of multiple antibodies confers high risk for the future development of diabetes; three of six children who exhibited positive antibody responses to all four antibodies tested and another child with two positive antibodies developed clinical diabetes at the ages of 13, 21, and 27 months and 5 years. We conclude that loss of tolerance to β-cell autoantigens and appearance of autoimmune phenomena occur very early in life in individuals with genetic susceptibility for IDDM. Screening programs to identify candidates for disease-prevention therapies can therefore be focused on this young agegroup, in whom the disease process may be less advanced and who may therefore be best suited to such therapies.
Adipose Tissue Leptin Production and Plasma Leptin Kinetics in Humans
Authors: Klein Samuel , Coppack Simon W , Mohamed-Ali Vidya , Landt Michael
Published: September 2013

Abdominal adipose tissue leptin production was determined in vivo by arteriovenous balance in 14 lean and obese men (mean BMI 27.0 ± 1.9, range 21.4–45.2). Blood samples were taken simultaneously from an abdominal vein that drains subcutaneous adipose tissue and from a radial artery. Adipose tissue blood flow was measured by xenon washout. Abdominal vein leptin concentrations (mean 8.9 ± 2.4 ng/ml, range 2.1–36.5 ng/ml) were consistently greater than arterial values (mean 6.6 ± 1.9 ng/ml, range 1.7–28.2 ng/ml) (P < 0.001). The net rate of abdominal adipose tissue leptin production (mean 3.2 ± 0.5 ng · 100 g−1 · min−1) correlated directly with percentage body fat (rs = 0.59, P = 0.016). Estimated whole-body leptin production rate (797 ± 283 ng · person−1 · min−1) correlated directly with percent body fat (rs = 0.93, P < 0.0001) and with regional leptin production (rs = 0.81, P < 0.001). In contrast, the rate of leptin clearance from plasma (mean 1.50 ± 0.23 ml · kg−1 · min−1) and plasma leptin half-life (mean 24.9 ± 4.4 min) was unrelated to adiposity (rs = 0.06, P = 0.30; rs = 0.16, P = 0.30, respectively). These results provide direct evidence that leptin is produced by adipose tissue in humans and that the rate of production is directly related to adiposity. A combination of greater leptin production per unit of body fat and increased production from expanded total body fat mass, rather than alterations in leptin clearance, account for the increase in plasma leptin concentrations observed in obese humans.
Identification and Characterization of Vascular Endothelial Growth Factor Receptor (Flt) in Bovine Retinal Pericytes
Authors: Takagi Hitoshi , King George L , Aiello Lloyd Paul
Published: September 2013

Vascular endothelial growth factor (VEGF) plays an important role in the hypoxia-stimulated neovascularization of ischemic retinal diseases such as proliferative diabetic retinopathy. VEGF exerts its effect through two known high-affinity tyrosine kinase receptors, named kinase insert domain–containing receptor (KDR) and the fms-like tyrosine kinase (Flt). VEGF receptors are located primarily on endothelial cells, although receptors on a few other nonocular cell types also have been described. In the present study, we demonstrate the expression of Fit, but not KDR, in bovine retinal pericytes (BRPCs). Although KDR is expressed predominantly in retinal endothelial cells, Northern blot analysis demonstrated substantial expression of the Fit gene in BRPCs without detection of KDR despite using polyadenylated RNA. Hypoxia increased Fit gene expression in BRPCs (2.7-fold, P < 0.01). 125I-labeled VEGF binding analysis on BRPCs demonstrated two apparent high-affinity receptor subtypes (Kd = 14 and 215 pmol/1), with 2.9 × 104 and 1.4 × 105 receptors/cell, respectively. 125I-VEGF affinity cross-linking demonstrated VEGF-specific binding complexes at 150, 172, 187, and 200 kDa under reducing conditions. Western blot analysis using an anti-phosphotyrosine antibody demonstrated VEGF-induced tyrosine phosphorylation of several proteins. VEGF stimulation had little effect on initial BRPCs growth rates but significantly increased BRPCs number after 7 days. These results suggest that two classes of high-affinity VEGF receptors are present on BRPCs, at least one of which is analogous to Fit and is capable of intracellular protein phosphorylation. Thus, VEGF might regulate the function of both retinal endothelial cells and retinal pericytes to induce pathological angiogenesis and vascular remodeling during proliferative diabetic retinopathy and other ischemic retinal diseases.
Advanced Glycation End Products of the Maillard Reaction in Aortic Pepsin-Insoluble and Pepsin-Soluble Collagen From Diabetic Rats
Authors: Meng Jing , Sakata Noriyuki , Takebayashi Shigeo , Asano Takashi , Futata Tetsuhiro , Araki Norie , Horiuchi Seikoh
Published: September 2013

Recent immunohistological studies using antibodies against advanced glycation end products (AGEs) have demonstrated the presence of AGEs in several tissues. By an enzyme-linked immunosorbent assay using the monoclonal anti-AGE antibody, the present study aimed to determine AGEs in pepsin-insoluble collagen (PIC) as well as in pepsin-soluble collagen (PSC) from the aortas of streptozotocin (STZ)-induced diabetic rats (at 4, 16, and 28 weeks after STZ injection) and those of agematched control rats. Addition of EDTA to the immunoassay buffer has led us to successful determination of AGEs in the aortic PIC samples with following results: 1) in diabetic rats, there was a time-related increase in the AGE contents at 28 weeks (n = 9, 226.4 ± 13.5 ng/mg collagen [mean ± SE]), compared with that at 4 and 16 weeks (n = 6, 79.6 ± 9.5 ng/mg collagen, and n = 8, 149.4 ± 30.9 ng/mg collagen at 4 and 16 weeks, respectively; both P < 0.05, between 4 and 16 weeks and 28 weeks); 2) after 28 weeks of diabetes, the AGE contents in PIC of aortas were significantly higher in diabetic rats than in controls (n = 9, 226.4 ± 13.5 ng/mg collagen vs. n = 8, 129.6 ± 14.9 ng/mg collagen, P < 0.01, diabetic vs. control); and 3) the level of the AGE content was strongly correlated with the PIC/total collagen (TC) ratio (n = 45, r = 0.698, P = 0.0001). By treating the samples of PSC with alkaline solution, the AGE content of PSC was also determined. In the PSC fraction, the AGE levels in the diabetic rats tended to increase with time and to be higher than those of control rats at 28 weeks although these changes were not statistically significant (diabetic: n = 4, 19.4 ± 9.7; n = 6, 22.3 ± 6.2; n = 6, 39.6 ± 10.8; control: n = 4,19.7 ± 9.8; n = 6, 22.9 ± 7.3; n = 7, 30.7 ± 7.2; at 4, 16, and 28 weeks, respectively). Compared with the AGE levels of PSC, those of PIC were about four to seven times and four to five times higher in diabetic and control rats, respectively (PIC versus PSC in diabetic or control rats, all P < 0.001, at 4, 16, and 28 weeks, respectively). These findings provide the first immuno chemical evidence that AGE adducts are present in the materials extracted sequentially by pepsin and collagenase and that these adducts in PIC accumulated as a function of the increase in the aortic PIC/TC ratio.
Glucose Ingestion Causes GLUT4 Translocation in Human Skeletal Muscle
Authors: Goodyear Laurie J , Hirshman Michael F , Napoli Raffaele , Markuns Jeffrey F , Horton Edward S , Ljungqvist Olle , Calles Jorge
Published: September 2013

In humans, ingestion of carbohydrates causes an increase in blood glucose concentration, pancreatic insulin release, and increased glucose disposal into skeletal muscle. The underlying molecular mechanism for the increase in glucose disposal in human skeletal muscle after carbohydrate ingestion is not known. We determined whether glucose ingestion increases glucose uptake in human skeletal muscle by increasing the number of glucose transporter proteins at the cell surface and/or by increasing the activity of the glucose transporter proteins in the plasma membrane. Under local anesthesia, ∼1 g of vastus lateralis muscle was obtained from six healthy subjects before and 60 min after ingestion of a 75-g glucose load. Plasma membranes were isolated from the skeletal muscle and used to measure GLUT4 and GLUT1 content and glucose transport in plasma membrane vesicles. Glucose ingestion increased the plasma membrane content of GLUT4 per gram muscle (3,524 ± 729 vs. 4,473 ± 952 arbitrary units for basal and 60 min, respectively; P < 0.005). Transporter-mediated glucose transport into plasma membrane vesicles was also significantly increased (130 ± 11 vs. 224 ± 38 pmol · mg−1 · s−1; P < 0.017), whereas the calculated ratio of glucose transport to GLUT4, an indication of transporter functional activity, was not significantly increased 60 min after glucose ingestion (2.3 ± 0.4 vs. 3.0 ± 0.5 pmol · GLUT4 arbitrary units−1 · s−1; P < 0.17). These results demonstrate that oral ingestion of glucose increases the rate of glucose transport across the plasma membrane and causes GLUT4 translocation in human skeletal muscle. These findings suggest that under physiological conditions the translocation of GLUT4 is an important mechanism for the stimulation of glucose uptake in human skeletal muscle.
Treatment With Growth Hormone and Dexamethasone in Mice Transgenic for Human Islet Amyloid Polypeptide Causes Islet Amyloidosis and β-Cell Dysfunction
Authors: Couce Marta , Kane Laurie A , O'Brien Timothy D , Charlesworth Jon , Soeller Walter , McNeish John , Kreutter David , Roche Patrick , Butler Peter C
Published: September 2013

Islet amyloid derived from islet amyloid polypeptide (IAPP) is a well-recognized feature of type II diabetes. However, the mechanism of islet amyloidogenesis is unknown. In vitro studies suggest that amino acid residues 20–29 in human, but not mouse, IAPP confer amyloidogenicity consistent with the absence of spontaneous islet amyloidosis in mice. Several clinical and in vitro studies suggest that increased synthetic rates of IAPP predispose to IAPP-amyloidosis. In the present study, we sought to test the hypothesis that pharmacological induction of insulin resistance in a mouse transgenic (TG) for human IAPP would induce islet amyloid and β-cell dysfunction. TG and non-transgenic (N-TG) control mice were treated with both rat growth hormone (12 μg/day) and dexamethasone (0.24 mg/day) (dex/GH) or received no treatment for 4 weeks, after which animals were killed to examine islet morphology. Treatment with dex/GH caused hyperglycemia (7.3 ± 0.4 vs. 5.2 ± 0.1 mmol/l, TG vs. N-TG, P < 0.001) associated with a decreased plasma insulin concentration (595 ± 51 vs. 996 ± 100 pmol/1, TG vs. N-TG, P < 0.05) in TG versus control mice. Islet amyloid was induced in treated TG mice but not in control mice. Islet amyloid was identified in both intra- and extracellular deposits, the former being associated with evidence of β-cell degeneration. We conclude that dex/GH treatment in mice TG for human IAPP induces IAPP-derived islet amyloid, hyperglycemia, and islet dysfunction. The present model recapitulates the islet morphology and phenotype of type II diabetes.
Characterization of a Novel Glucose-Responsive Insulin-Secreting Cell Line, BRIN-BD11, Produced by Electrofusion
Authors: McClenaghan Neville H , Barnett Christopher R , Ah-Sing Eric , Abdel-Wahab Yasser H A , O'Harte Finbarr P M , Yoon Tai-Wook , Swanston-Flatt Sara K , Flatt Peter R
Published: September 2013

A novel insulin-secreting cell line (BRIN-BD11) was established after electrofusion of RINm5F cells with New England Deaconess Hospital rat pancreatic islet cells. Wells of cell fusion mixture with insulin output 5–10 times greater than parent RINm5F cells were subcultured with eventual establishment of clones, including BRIN-BD11. Morphological studies established that these cells grow as monolayers with epithelioid characteristics, maintaining stability in tissue culture for >50 passages. Culture of these cells for 24 h at 5.6–33.3 mmol/l glucose revealed a 1.8- to 2.0-fold increase of insulin output compared with 1.4 mmol/l glucose. Dynamic insulin release was recorded in response to 16.7 mmol/l glucose, resulting in a rapid threefold insulin secretory peak followed by a sustained output slightly above basal. In acute 20-min tests, 4.2–16.7 mmol/l glucose evoked a stepwise twoto threefold stimulation of insulin release. 3-Isobutyl-1-methylxanthine (1 mmol/l) served to increase basal and glucose-stimulated insulin release, shifting the threshold from 4.4 to 1.1 mmol/l glucose. Stimulation of insulin secretion with 16.7 mmol/l glucose was abolished by mannoheptulose or diazoxide (15 or 0.5 mmol/l). In contrast, glyceraldehyde (10 mmol/l) and 25 mmol/l K+ evoked 1.7- to 9.0-fold insulin responses. l-Alanine (10 mmol/l) evoked a twofold secretory response, which was potentiated 1.4-fold by increasing the Ca2+ concentration from 1.28 to 7.68 mmol/l. Forskolin (25 εmol/l) and phorbol 12-myristate 13-acetate (10 nmol/1) both increased insulin secretion in the presence of l-alanine (1.4- and 1.8-fold, respectively). Western blotting confirmed that BRIN-BD11 cells expressed the GLUT2 glucose transporter. This, coupled with a high glucokinase/hexokinase ratio in the cells, confirms an intact glucose sensing mechanism. High-performance liquid chromatography analysis demonstrated that insulin was the major product secreted under stimulatory conditions. Collectively, these data indicate that the BRIN-BD11 cell line represents an important stable glucose-responsive insulinsecreting β-cell line for future studies.
Phenotype of fatty Due to Gln269Pro Mutation in the Leptin Receptor (Lepr)
Authors: Chua Streamson C , White David W , Wu-Peng X Sharon , Liu Shun-Mei , Okada Norichika , Kershaw Erin E , Chung Wendy K , Power-Kehoe Loraine , Chua Melvin , Tartaglia Louis A , Leibel Rudolph L
Published: September 2013

The rat fatty (fa) mutation produces profound obesity of early onset caused by hyperphagia, defective nonshivering thermogenesis, and preferential deposition of energy into adipose tissue. Genetic mapping studies indicate that fa and diabetes (db) are homologous loci in the rat and mouse genomes, respectively. It has been shown that db alleles carry mutations in the Lepr (leptin receptor) gene. This paper describes a point mutation in the fatty allele of Lepr. A nucleotide substitution at position 880 (A→C) causes an amino acid substitution at position 269 (Gln → Pro). The mutation generates a novel Msp I site that cosegregates with fa in 1,028 meioses examined in obese F2 progeny from two crosses (BN×13M and WKY×13M) and is still segregating in three rat colonies. PCR-based mutagenesis was used to introduce the fa mutation into the mouse Lepr cDNA. Transient transfection studies indicate that the mutant Lepr cDNA has greatly reduced binding of leptin (Lep) at the cell surface. These data are strong evidence that the single nucleotide substitution in the fa allele of Lepr (Leprfa) is responsible for the obese phenotype.
Effect of Enterostatin on Insulin, Glucagon, and Somatostatin Secretion in the Perfused Rat Pancreas
Authors: Silvestre Ramona A , Rodríguez-Gallardo Jovita , Marco José
Published: September 2013

Enterostatin is a pentapeptide generated by trypic digestion of procolipase in the small intestine. Both peripheral and central administration of this peptide to rats has been shown to reduce food intake, this reduction being due to specific suppression of fat intake. In perifused pancreatic rat islets, enterostatin has been shown to inhibit the insulin response to a high glucose concentration. In the present study, we have investigated the effect of exogenous enterostatin on insulin, glucagon, and somatostatin secretion by the isolated perfused rat pancreas. Enterostatin, at 100 nmol/l, inhibited the insulin response to 9 mmol/l glucose (by 70%), 0.1 mmol/l tolbutamide (by 40%), and 5 mmol/l arginine (by 70%). Enterostatin had no effect on glucagon and somatostatin release at a maintained glucose level (5.5 mmol/l) or in response to 5 mmol/l arginine. Finally, preinfusion of the rat pancreas with a high enterostatin concentration (500 nmol/l) did not alter the insulin response to glucose, an observation that would rule out a toxic effect of this peptide on the β-cell. In summary, in the perfused rat pancreas, enterostatin, at putatively physiological concentrations, inhibits insulin secretion without affecting glucagon or somatostatin output, thus pointing to a direct effect of enterostatin on the β-cell and not through an α-cell or δ-cell paracrine effect. Because enterostatin is generated in the small intestine after feeding, it might play a role in the enteroinsular axis as an anti-incretin agent.
Anti-Endothelial Cell Autoantibodies in BB Rats With Spontaneous and Induced IDDM
Authors: Doukas John , Majno Guido , Mordes John P
Published: September 2013

Anti–endothelial cell (anti-EC) antibodies occur in several autoimmune diseases, including human IDDM, but the time course of their development and their importance in disease pathogenesis are unknown. To study such antibodies further, we investigated the BB rat model of autoimmunity. Diabetes-prone (DP) BB rats spontaneously develop autoimmune diabetes, whereas coisogenic diabetes-resistant (DR) BB rats are disease free but can be induced to become diabetic by the depletion of T-cells expressing the RT6 alloantigen. Anti-EC autoantibodies were readily detectable in both untreated DP-BB rats and RT6-depleted DR-BB rats before the onset of diabetes. Their concentration increased with time. The anti-EC antibodies in DP-BB rats were almost exclusively of the IgG2b subclass, whereas those in RT6-depleted DR-BB rats included both the IgGl/2a and the IgG2b subclasses. We also found that intravenous injections of purified immunoglobulins from RT6-depleted DR-BB rats induced abnormal pancreatic vascular leakage in mice. The preabsorption of immunoglobulins against cultured ECs abolished this activity. The pretreatment of mice with silica also abolished the ability of immunoglobulins of RT6- depleted DR-BB rats to induce pancreatic leakage, suggesting that monocytes are involved in the mechanism of anti-EC autoantibody-induced vascular leakage. We conclude that anti-EC autoantibodies are present in rat strains that are genetically predisposed to develop autoimmune diabetes. Their presence early in the disease process and their ability to induce pancreatic vascular leakage suggest that they may participate in diabetes pathogenesis.
Phlorizin or Vanadate Treatment Reverses Impaired Expression of Albumin and Hepatocyte Nuclear Factor 1 in Diabetic Rats
Authors: Barrera-Hernandez Gonzalo , Wanke Irene E , Wong Norman C W
Published: September 2013

Diabetes decreases transcription of the albumin gene. The role of hyperglycemia in mediating this suppression of albumin gene activity is unclear. To study the effect of glucose in vivo, we treated diabetic rats with phlorizin or vanadate, two agents that ameliorate hyperglycemia without increasing the levels of circulating insulin. When glucose was normalized in diabetic rats with either agent, the hepatic levels of albumin mRNA became indistinguishable from those in nondiabetic animals. In light of our previous observation that diabetes decreases the abundance of hepatocyte nuclear factor 1 (HNF1), the predominant factor increasing albumin gene transcription, we wondered whether glucose normalization in diabetes would alter HNF1. Both the levels and DNA binding activity of HNF1 were restored to control values when phlorizin or vanadate was administered to diabetic rats. These findings suggest that hyperglycemia is integrally involved in mediating the suppression of albumin gene expression in diabetes. The effect of hyperglycemia on HNF1 suggests that glucose affects albumin expression at the level of transcription.
Missense Mutation of Amylin Gene (S20G) in Japanese NIDDM Patients
Authors: Sakagashira Setsuya , Sanke Tokio , Hanabusa Tadashi , Shimomura Hiroko , Ohagi Shinya , Kumagaye Kumiko Y , Nakajima Kiichiro , Nanjo Kishio
Published: September 2013

Many studies suggest that amylin, which is cosecreted with insulin from islet β-cells, is a biologically active peptide and modulates plasma glucose levels. We therefore scanned the amylin gene for mutations in 294 Japanese NIDDM patients by single-strand conformational polymorphism, and we found a single heterozygous missense mutation (Ser→Gly at position 20: S20G mutation) in 12 NIDDM patients (frequency 4.1%). None of the 187 nondiabetic subjects or 59 IDDM patients had the mutation. Of 12 patients carrying the mutation, 8 were diagnosed as having NIDDM at a relatively early age (≤35 years), and they had severe diabetes and strong family histories of late-onset NIDDM. On the other hand, the remaining four patients were diagnosed as having NIDDM after age 51, and they had mild diabetes without family histories of diabetes. In high-performance liquid chromatography analysis, a small amount (16%) of amylin immunoreactivity appeared in the position corresponding to normal amylin and a much larger amount (84%) appeared in the position corresponding to mutant amylin. These findings suggest that the S20G mutation of the amylin gene may play a partial role in the pathogenesis of early-onset NIDDM in the Japanese population and may also provide an important model to investigate the true physiological action of amylin.
Expression of Human Hepatic Glucokinase in Transgenic Mice Liver Results in Decreased Glucose Levels and Reduced Body Weight
Authors: Hariharan Narayanan , Farrelly Dennis , Hagan Deborah , Hillyer Donna , Arbeeny Cynthia , Sabrah Talal , Treloar Allison , Brown Karen , Kalinowski Stephen , Mookhtiar Kasim
Published: September 2013

Glucokinase is the predominant hexokinase in pancreatic β-cells and liver parenchymal cells and functions as a critical component of the glucose-sensing apparatus in these glucose-responsive cell types. In the β-cells, the sensing leads to insulin secretion, while the role in hepatocytes is thought to be in hepatic glucose uptake. To determine the physiological response to an increase in hepatic glucokinase expression, transgenic mice expressing the human hepatic glucokinase gene under the control of a liver-specific human apolipoprotein A-I gene enhancer were generated. Transgenic mice had twofold higher total fasting hepatic glucokinase mRNA, which resulted in a modest 20% increase in fasting glucokinase activity. These animals showed lower fasting plasma glucose, insulin, and lactate levels and improved tolerance to glucose. In addition, glucokinase transgenic animals weighed less and had lower BMI than nontransgenic animals. Thus, glucokinase transgenic animals demonstrate that a modest change in hepatic glucokinase activity enhances the metabolism of glucose.
Macrovascular Disease Is Associated With Increased Plasma Apolipoprotein A-IV Levels in NIDDM
Authors: Vergès Bruno L , Lagrost Laurent , Vaillant Geneviève , Petit Jean M , Cohen Martine , Gambert Philippe , Brun Jean M
Published: September 2013

Apolipoprotein A-IV (apoA-IV) might play an important role in lipoprotein metabolism, including modulation of triglyceride-rich lipoprotein catabolism, reverse cholesterol transport and cholesteryl ester transfer protein (CETP) activity. Increased apoA-IV levels have been reported in plasma from NIDDM patients. The aim of the present study was to look for a possible association between plasma apoA-FV level and prevalence of macrovascular disease in NIDDM. One hundred and thirty-six NIDDM patients were studied (71 men, 65 women). Macrovascular disease was assessed in each patient by a standardized questionnaire, physical examination, resting electrocardiogram (ECG), and laboratory evaluation (ankle/arm blood pressure ratio, continuous wave Doppler velocimetry). Moreover, patients without any history of coronary heart disease and showing a normal resting ECG underwent a bicycle exercise test or a dipyridamole thallium scintigraphy to detect possible silent myocardial ischemia. Among the 136 NIDDM patients, 56 had macrovascular disease. ApoA-IV levels were significantly higher in NIDDM patients with macrovascular disease than in NIDDM patients without macrovascular disease (20.9 ± 8.6 vs. 13.3 ± 5.3 mg/dl; P < 0.001). The influence of different factors, such as age, BMI, cigarette smoking, hypertension, total cholesterol, triglycerides, HDL cholesterol, apoA-IV level, apoA-IV phenotype, fasting glycemia, fasting C-peptide, and microalbuminuria, on the prevalence of macrovascular disease was analyzed using a logistic regression model. In the univariate analysis, apoA-IV level (P < 0.00001), age (P = 0.0087), hypertension (P = 0.012), microalbuminuria (P = 0.018), triglycerides (P = 0.02), and fasting C-peptide (P = 0.03) were positively associated with macrovascular disease. In the multivariate analysis, macrovascular disease was positively associated only with apoA-IV (P < 0.0001) and age (P = 0.003) and negatively associated with HDL cholesterol (P = 0.013). These results indicate that increased plasma apoA-IV level is associated with an increased prevalence of macrovascular disease in NIDDM. Moreover, apoA-IV, in NIDDM patients, appears to be a better marker for macrovascular disease than triglycerides.
The Defective Glucagon Response From Transplanted Intrahepatic Pancreatic Islets During Hypoglycemia Is Transplantation Site–Determined
Authors: Gupta Vinendra , Wahoff David C , Rooney Desmond P , Poitout Vincent , Sutherland David E R , Kendall David M , Robertson R Paul
Published: September 2013

The optimal site for pancreatic islet cell transplantation is presently unclear, although the liver has been the most commonly used. However, glucagon secretion from islets that have been autotransplanted in liver has been reported to be unresponsive to hypoglycemia yet responsive to arginine. To determine whether this selective glucagon secretory defect is related to the intrahepatic site of islet implantation or to the process of transplantation per se, we studied counterregulatory responses to hypoglycemia in dogs with pancreatic islet autotransplantation in the hepatic parenchyma (the intrahepatic [IH] group, n = 9) or the peritoneal cavity (the intraperitoneal [IP] group, n = 9), following total pancreatectomy, and compared them with the responses in normal controls (n = 10). Dogs were subjected to a hypoglycemic hyperinsulinemic (5 mU · kg−1 min−1) clamp for 90 min under general anesthesia. Arterial glucose concentrations were clamped at 2.7 mmol/l for the final 45 min of the clamp. Immediately following the clamp, glucagon responses to IV arginine (5 g) were also assessed. During hypoglycemia, glucagon responses in the IH group (maximal incremental glucagon = 33 ± 21 ng/l; glucagon area under curve [AUC] = 713 ± 1,022 ng · 1−1 · min−1) were significantly lower than either the IP (maximal incremental glucagon = 92 ± 32 ng/l; glucagon AUC = 4,090 ± 1,600 ng · 1−1 · min−1) or control (maximal incremental glucagon = 154 ± 71 ng/l; glucagon AUC = 6,943 ± 2,842 ng · 1−1 · min−1) group (IH vs. IP group, P < 0.05; control vs. IH group, P < 0.01). Glucagon responses in the IP group did not differ significantly from the control group. Epinephrine responses to hypoglycemia were similar in all groups, whereas neither of the transplanted groups (IH and IP) had pancreatic polypeptide responses. There was a prompt rise in plasma glucagon after intravenous arginine in all groups. These data indicate that glucagon unresponsiveness to hypoglycemia is specific to intrahepatically transplanted islets, rendering the liver a disadvantageous site for optimal α-cell function.
Autoantibodies to IA-2 in IDDM: Location of Major Antigenic Determinants
Authors: Zhang Baowei , Lan Michael S , Notkins Abner Louis
Published: September 2013

Thirty-three IDDM sera that immunoprecipitated fulllength IA-2 were tested for reactivity with different fragments of the IA-2 molecule. The fragments were prepared by PCR amplification of IA-2 cDNA and by expression in a rabbit reticulocyte transcription/translation system. Whereas all 33 sera reacted with the intracellular domain (amino acid 604 to 979), none of the sera reacted with the extracellular domain of IA-2 (amino acid 31 to 577). Analysis of the reactivity of IDDM sera with the different regions of the intracellular domain showed that 94% (31 of the 33) reacted with the COOH-terminus (amino acid 771 to 979), 40% reacted with the NH2-terminus (amino acid 604 to 776), and 40% reacted with the middle portion (amino acid 692 to 875). Of the 31 sera that reacted with the COOH-terminus, 14 of these reacted only with the COOH-terminus and with no other region. Of the 13 sera that reacted with the NH2-terminus, only one reacted exclusively with the NH2-terminus. Treatment of the different domains of IA-2 with trypsin showed that only the COOH-terminus was resistant to trypsin, arguing that it is from this region of the IA-2 molecule that the 40-kDa tryptic fragment from insulinoma cells is derived. From these experiments, it is concluded that the major antigenic determinant of IA-2 is located at the COOH-terminus and that minor antigenic determinants are located at the NH2-terminus and middle portion of the intracellular domain.
Counterregulation in Peripheral Tissues: Effect of Systemic Hypoglycemia on Levels of Substrates and Catecholamines in Human Skeletal Muscle and Adipose Tissue
Authors: Maggs David G , Jacob Ralph , Rife Francis , Caprio Sonia , Tamborlane William , Sherwin Robert S
Published: September 2013

We used microdialysis to distinguish the effects hyperinsulinemia of and hypoglycemia on glucose, gluconeogenic substrate, and catecholamine levels in adipose and muscle extracellular fluid (ECF). Ten lean humans (six males and four females) were studied during baseline and hyperinsulinemic (3 mU · kg−1 · min−1 · for 3 h) euglycemia (5.0 mmol/l) and hypoglycemia (2.8 mmol/l). In muscle and adipose, basal ECF glucose was lower (muscle, 3.5 ± 0.2 mmol/l; adipose tissue, 3.3 ± 0.2 mmol/l) and lactate was higher (muscle, 2.2 ± 0.2 mmol/l; adipose, 1.5 ± 0.3 mmol/l) than respective plasma values (glucose, 4.9 ±0.1 mmol/l; lactate, 0.7 ± 0.1 mmol/l), whereas alanine was higher in muscle ECF (379 ± 22 μmol/l) than adipose tissue (306 ± 22 μmol/l) and plasma (273 ± 33 μmol/l). Plasma catecholamines (unchanged during euglycemia) rose during hypoglycemia with epinephrine, increasing approximately fivefold more than norepinephrine. In contrast, the hypoglycemia-induced increments in muscle dialysate norepinephrine and epinephrine were similar, suggesting local generation of norepinephrine. Compared with euglycemia, hypoglycemia produced a greater increase in lactate and a smaller reduction in alanine in muscle ECF, whereas hypoglycemia caused a greater relative fall in ECF glucose concentrations in muscle (72 ± 16%) and adipose tissue (69 ±9%) than in plasma (42 ± 3%) (P < 0.05). We conclude that hypoglycemia increases the generation of norepinephrine and gluconeogenic substrates in key target tissues, while increasing the plasma-tissue concentration gradient for glucose. These changes suggest the stimulation of glucose extraction by peripheral tissues, despite systemic counterregulatory hormone release and local sympathetic activation.
Interstitial Muscle Insulin and Glucose Levels in Normal and Insulin-Resistant Zucker Rats
Authors: Holmäng Agneta , Mimura Kazuo , Björntorp Per , Lsönroth Peter
Published: September 2013

To study interstitial insulin and glucose concentrations, microdialysis was performed in the medial femoral muscles in normal SD rats as well as in insulin-resistant obese Zucker rats during a euglycemic insulin clamp. [14C]inulin was given (0.1 mCi/rat) as a constant subcutaneous infusion 24 h before the insulin clamp. Insulin infusion rates were 5–8 mU · kg−1 · min−1 (low rate) for 140 min and 10–20 mU · kg−1 · min−1 (high rate) for another 100 min. The relationship between insulin and [14C]inulin dialysate recoveries was evaluated in vivo and in vitro in plasma to calculate interstitial insulin concentration. Relative microdialysis recovery of interstitial insulin in vivo was 3.0 ± 0.3% (mean ± SE, n = 68). In normal SD rats, plasma and interstitial insulin concentrations were identical when plasma insulin was ≤250 mU/ml, whereas interstitial insulin was lower when plasma insulin was ≥350 mU/ml. Half-maximal glucose infusion rate was achieved in the presence of plasma and interstitial insulin concentrations of ∼140 mU/ml, whereas maximal glucose disposal was seen at interstitial insulin concentrations of ∼325 mU/ml, corresponding to ∼500 mU/ml in plasma. In electrically stimulated and contracting (1 Hz) normal muscle with markedly increased blood flow, the dialysate insulin concentration was significantly higher at high rates, but not at low rates, of insulin infusion. In insulin-resistant obese Zucker rats, the interstitial insulin concentration was similar to that in plasma, even at pharmacological concentrations. The glucose infusion rate was significantly lower in the obese Zucker rats at both insulin infusion rates than in the lean animals. The glucose content in dialysates from skeletal muscle was equal in both obese and lean rats during the low insulin infusion rate. During the high insulin infusion rate, dialysate glucose concentrations decreased significantly in both groups but were significantly higher in the obese Zucker rats. The data suggest that transport of insulin and glucose diffusion across the capillary wall are rate limiting for insulin as well as for glucose metabolism in muscle in normal rats. This does not appear to be the case in the insulin-resistant obese Zucker rats, where the reduced insulin responsiveness in muscle is due to muscular cellular defects rather than an inhibited transcapillary delivery of insulin.
Defective Plasma Antioxidant Defenses and Enhanced Susceptibility to Lipid Peroxidation in Uncomplicated IDDM
Authors: Santini Stefano A , Marra Giampiero , Giardina Bruno , Cotroneo Patrizia , Mordente Alvaro , Martorana Giuseppe E , Manto Andrea , Ghirlanda Giovanni
Published: September 2013

Oxidative stress is postulated to be increased in patients with IDDM. Accumulating evidence suggests that oxidative cell injury caused by free radicals contributes to the development of IDDM complications. On the other side, a decreased efficiency of antioxidant defenses (both enzymatic and nonenzymatic) seems to correlate with the severity of pathological tissue changes in IDDM. Thus, we determined plasma antioxidant defenses, measuring the total radical-trapping antioxidant capacity (TRAP) and the two markers of oxidative stress, lipid hydroperoxides (ROOHs) and conjugated dienes, in 72 patients with well-controlled IDDM and without evident complications, compared with 45 nondiabetic subjects. Compared with control subjects, IDDM patients showed significantly reduced plasma TRAP (669 ±131 vs. 955 ± 104 μmol/1, P < 0.001) and significantly increased levels of ROOHs (7.13 ± 2.11 vs. 2.10 ± 0.71 μmol/1, P < 0.001) and conjugated dienes (0.0368 ± 0.0027 vs. 0.0328 ± 0.0023 arbitrary units [AU], P < 0.01), especially in the trans-trans conformation (0.0340 ± 0.0028 vs. 0.0259 ± 0.0022 AU, P < 0.001), with a concurrent reduction of conjugated dienes in the cis-trans conformation (0.0028 ± 0.0011 vs. 0.0069 ± 0.0012 AU, P < 0.001). The oxidative parameters studied did not appear to be correlated with metabolic control (HbA1c levels) and lipid profile (cholesterol or triglyceride levels). The reduced TRAP and the increased ROOH and conjugated diene plasma levels, together with the decreased ratio of cis-trans/trans-trans conjugated dienes, which reflects an altered redox status of plasma, indicate that in IDDM patients, oxidative stress is enhanced and antioxidant defenses are defective, regardless of diabetes duration, metabolic control, or presence of complications.
The Effect of HLA-B Allele on the IDDM Risk Defined by DRB1*04 Subtypes and DQB1*0302
Authors: Nejentsev Sergei , Reijonen Helena , Adojaan Bela , Kovalchuk Liliya , Sochnevs Arthur , Schwartz Eugene I , Åkerblom Hans K , Ilonen Jorma
Published: September 2013

The genes encoding the HLA-DQ heterodimer molecules, DQB1 and DQA1, have been found to have the strongest association with IDDM risk, although there is cumulative evidence for the effect of other gene loci within the major histocompatibility complex gene region. After the HLA-DQ locus, the HLA-DR locus has been suggested most often as contributing to the disease susceptibility. In this study we analyzed at the population level the effect of DR4 subtypes and class I, HLA-B alleles, on IDDM risk when the influence of the DQ locus was stratified. In all three populations studied (Estonian, Latvian, and Russian), DQB1*0302 haplotypes most frequently carried DRB1*0401 or DRB1*0404. DRB1*0401 was the most prevalent subtype in IDDM patients, whereas DRB 1*0404 was decreased in frequency. DRB 1*0402 was also prevalent among Russian haplotypes, but was not associated with IDDM risk. When HLA-B alleles were analyzed, strong associations between the presence of specific B alleles and DRB1*04 subtypes were detected. The HLA-B39 allele was found significantly more often in DRB1*0404-DQBl*0302-positive patients than in healthy control subjects positive for this haplotype: 27 of 54 (50%) vs. 4 of 49 (8.2%) (P < 0.0001). The results demonstrate that DQ and DR genes cannot explain all of the HLA-linked susceptibility to IDDM, and that the existence of a susceptibility locus telom-eric to DR is probable.
Augmentation of Insulin Release by Glucose in the Absence of Extracellular Ca2+: New Insights Into Stimulus-Secretion Coupling
Authors: Komatsu Mitsuhisa , Schermerhorn Thomas , Noda Mitsuhiko , Straub Susanne G , Aizawa Toru , Sharp Geoffrey W G
Published: September 2013

Glucose stimulates insulin secretion in the pancreatic β-cell by means of a synergistic interaction between at least two signaling pathways. One, the KATP channel-dependent pathway, increases the entry of Ca2+ through voltage-gated channels by closure of the KATP channels and depolarization of the β-cell membrane. The resulting increase in [Ca2+]i stimulates insulin exocytosis. The other, a KATP channel-independent pathway, requires that [Ca2+]i be elevated and augments the Ca2+-stimulated release. These mechanisms are in accord with the belief that glucose-stimulated insulin secretion has an essential requirement for extracellular Ca2+ and increased [Ca2+]i. However, when protein kinases A and C are activated simultaneously, a large effect of glucose to augment insulin release can be seen in the absence of extracellular Ca2+, under conditions in which [Ca2+]i is not increased, and even when [Ca2+]i is decreased to low levels by intracellular chelation with BAPTA. In the presence or absence of Ca2+, there are similarities in the characteristics of augmentation of insulin release that suggest that only one augmentation mechanism may be involved. These similarities include time course, glucose dose-responses, augmentation by nutrients other than glucose such as α-ketoisocaproate (α-KIC), and augmentation by the fatty acids palmitate and myristate. However, augmentation in the presence and absence of Ca2+ is distinctly different in GTP dependency. Therefore, exocytosis under these two conditions appears to be triggered differently—one by Ca2+ and the other by GTP or a GTPdependent mechanism. The augmentation pathways are likely responsible for time-dependent potentiation of secretion and for the second phase of glucose-stimulated insulin release.
Regulation of Hepatic Glutaminase in the Streptozotocin-Induced Diabetic Rat
Authors: Squires Stephen A , Ewart H Stephen , McCarthy Colin , Brosnan Margaret E , Brosnan John T
Published: September 2013

The liver of diabetic animals removes increased quantities of glutamine. We therefore examined factors that affect hepatic glutaminase activity in hepatocytes and mitochondria. Glutamine use, through glutaminase, was measured in isolated rat hepatocytes by monitoring the production of 14CO2 from [I-14C]glutamine. Hepatocytes from streptozotocin-induced diabetic rats use glutamine more rapidly than do hepatocytes from normal or insulin-maintained diabetic rats. Glutamine use in all of these hepatocytes was stimulated by glucagon and epinephrine. Glutaminase activity, assayed in broken mitochondrial membranes, was increased ∼2.5-fold in diabetic rats. The sensitivity of glutaminase, measured in intact liver mitochondria, to phosphate was markedly left-shifted in mitochondria from diabetic rats compared with those from controls. In fact, glutaminase was increased 10-fold at 2.5 mmol/l phosphate compared with controls. This increased sensitivity of glutaminase to physiological concentrations of phosphate is characteristic of its hormonal activation. Therefore, activation of glutaminase plays a major role in diabetes and is as important as increases in its total enzyme amount in determining the increased glutamine uptake in diabetes.
Effect of Captopril, Losartan, and Bradykinin on Early Steps of Insulin Action
Authors: Carvalho Carla RO , Thirone Ana Claudia P , Gontijo Jose A R , Velloso Licio A , Saad Mario J A
Published: September 2013

Insulin initiates its metabolic and growth-promoting effects by binding to the α subunit of its receptor, thereby activating the kinase in the β subunit. This event leads to tyrosyl phosphorylation of its cytosolic substrate, insulin receptor substrate 1 (IRS-1), which in turn associates with and activates phosphatidylinositol (PI) 3-kinase. The clinical use of ACE inhibitors has been associated with increased insulin sensitivity. However, the exact molecular mechanism is unknown. In the present study, we examined the phosphorylation status of the insulin receptor and IRS-1, as well as the association between IRS-1 and PI 3-kinase in the liver and muscle of 20-month-old rats treated acutely with captopril, using immunoprecipitation with antipeptide antibodies to the insulin receptor and IRS-1, and immunoblotting with antiphosphotyrosine and anti-PI 3-kinase antibodies. Insulin stimulation increased receptor autophosphorylation to 462 ± 253% (P < 0.05) in the liver and 697 ± 78% (P < 0.001) in the muscle of ACE inhibitor-treated rats. There were also increases to 250 ± 17% (P < 0.001) and 280 ± 50% (P < 0.05) in the insulin-stimulated IRS-1 phosphorylation levels in the liver and muscle, respectively, of animals treated with captopril. The insulin-stimulated IRS-1 association with PI 3-kinase rose to 305 ± 20% (P < 0.001) in liver and 267 ± 48% (P < 0.05) in muscle. Losartan, an ANG receptor blocker, had no significant effect on insulinstimulated IRS-1 phosphorylation in both tissues. The acute administration of bradykinin increased insulinstimulated tyrosine phosphorylation of the insulin receptor and IRS-1 in the liver and muscle. These data demonstrate that ACE inhibitors modulate the early steps of insulin signaling, and that this effect may be simulated by the administration of bradykinin.
Sensitization to Insulin Induced by β,β′-Methyl-Substituted Hexadecanedioic Acid (MEDICA 16) in Obese Zucker Rats In Vivo
Authors: Mayorek Nina , Kalderon Bella , Itach Etty , Bar-Tana Jacob
Published: September 2013

β,β′-methyl-substituted hexadecanedioic acid (MEDICA 16) consists of a nonmetabolizable long-chain fatty acid designed to probe the effect exerted by fatty acids on insulin sensitivity. The effect of MEDICA 16 was evaluated in insulin-resistant Zucker (fa/fa) rats in terms of liver, muscle, and adipose tissue response to clamped euglycemic hyperinsulinemia in vivo. Nontreated Zucker rats were insulin resistant, maintaining basal rates of total-body glucose disposal, glucose production in liver, free fatty acid (FFA) flux into plasma, and FFA reesterification in adipose tissue, irrespective of the insulin levels induced. MEDICA 16 treatment resulted in an insulin-induced decrease in hepatic glucose production, together with an insulin-induced increase in total-body glucose disposal. Intracellular reesterification of lipolysed FFA in adipose tissue was specifically activated by MEDICA 16, resulting in a pronounced decrease in FFA release, with a concomitant decrease in plasma FFA. In conclusion, MEDICA 16 treatment results in the sensitization of liver, muscle, and adipose tissue to insulin in an animal model for obesity-induced insulin resistance.