bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Maternal Diabetes on the Expression of Genes Regulating Fetal Brain Glucose Uptake
Authors: Schroeder Robert E , Devaskar Uday P , Trail Susen E , Demello Daphne E , Cole Daniel P , Devaskar Sherin U
Published: September 2013

Diabetes alters adult brain glucose uptake and glucose transporter 1 gene expression. To investigate the effect of diabetes on genes regulating fetal brain glucose uptake, we examined the effect of moderate (blood glucose 10–16.7 mM, normoinsulinemia) and severe (blood glucose >16.8 mM, hypoinsulinemia) maternal diabetes on the expression of genes regulating fetal brain glucose uptake in the genetically nonobese diabetic mouse. In the moderately diabetic state, a 50% decline in fetal brain GLUT1 mRNA levels was associated with a 20% increase in the corresponding GLUT1 protein levels. Simultaneously, although fetal brain GLUT3 mRNA and protein levels were barely detectable, no change in hexokinase I enzyme mRNA, protein (115,000 and 100,000 Mr) or activity, was noted. In the severe form of maternal diabetes GLUT1 protein was unchanged, GLUT3 protein levels remained low, and a 2- to 3-fold increase in the lower molecular form of the hexokinase I protein (100,000 Mr) and enzyme activity occurred. These observations suggest that moderate and severe forms of maternal diabetes do not affect the fetal brain glucose transporter levels to a physiologically significant extent. The severe form of maternal diabetes, however, enhances 1.5- to 3-fold the expression and activity of hexokinase I. This enzyme mediates the rate-limiting step in brain glucose metabolism, namely the intracellular conversion of glucose to glucose–6-phosphate.
Exposure To Elevated D-Glucose Concentrations Modulates Vascular Endothelial Cell Vasodilatory Response
Authors: Graier Wolfgang F , Wascher Thomas C , Lackner Lydia , Toplak Hermann , Krejs Günter J , Kukovetz Walter R
Published: September 2013

The possible role of endothelial dysfunction in early stages of uncomplicated diabetes mellitus was investigated in porcine aortic endothelial cells. Prolonged exposure to various D-glucose concentrations resulted in concentration-dependent amplification of agonist-induced Ca2+ mobilization, whereas L-glucose and D-mannitol failed to mimic the effect of D-glucose. This stimulatory effect of high D-glucose on endothelial Ca2+ mobilization could be antagonized by coincubation with cytochalasin B, which prevented D-glucose uptake into the cells. In agreement with its effect on agonist-induced Ca2+ response, prolonged preincubation with pathological D-glucose concentrations amplified formation of endothelium-derived relaxing factor, which is well established to be strictly attributable to increases in endothelial free Ca2+. In contrast to endothelium-derived relaxing factor formation stimulated by receptor-interacting autacoids, preincubation with high D-glucose failed to modulate A 23,187-induced endothelium-derived relaxing factor formation, which is attributable to unphysiological increases in endothelial free Ca2+ by this ionophore. Similar to its effect on D-glucose-mediated amplification of agonist-stimulated Ca2+ mobilization, cytochalasin B abolished the stimulatory effect of high D-glucose on endothelium-derived relaxing factor formation. We therefore suggest that prolonged exposure to pathological high D-glucose concentrations results in an enhanced endothelium-derived relaxing factor formation caused by amplification of agonist-stimulated Ca2+ mobilization in endothelial cells. This mechanism may be of particular importance representing a possible basis of pathological vasodilation and reduced peripheral resistance in early stages of diabetes mellitus.
Major Histocompatibility Complex Class I Deficiency Prolongs Islet Allograft Survival
Authors: Osorio Robert W , Ascher Nancy L , Jaenisch Rudolf , Freise Chris E , Roberts John P , Stock Peter G
Published: September 2013

Because of islet allograft rejection, nonimmunosuppressed pancreatic islet allotransplantation has been unsuccessful for the treatment of type I diabetes. The role of major histocompatibility complex class I antigen expression on islet allograft survival was evaluated with the use of mice homozygous for a β2-microglobulin gene disruption. These mice express little if any functional major histocompatibility complex class I antigen. When these major histocompatibility complex class I-deficient islets were used as donors in an allogenic murine transplantation model, islet allograft survival was markedly prolonged. These results demonstrate a major importance for the alloresponse directed against major histocompatibility complex class I antigen.
Effects of High Glucose Concentrations and Epalrestat on Sorbitol and myo-Inositol Metabolism in Cultured Rabbit Aortic Smooth Muscle Cells
Authors: Sakakibara Fumihiko , Hotta Nigishi , Koh Naoki , Sakamoto Nobuo
Published: September 2013

To clarify the relationship between abnormality of sorbitol and/or myo-inositol metabolism caused by hyperglycemia and diabetic macroangiopathy, we investigated the effects of high glucose concentrations and epalrestat, an aldose reductase inhibitor, on the metabolism of sorbitol and myo-inositol in cultured rabbit aortic smooth muscle cells. In cells incubated in the presence of 30 mM glucose for 72 h, the sorbitol content increased ∼4.5-fold, and the myo-inositol level decreased by 55% compared with control values. Kinetic analysis of high-affinity myo-inositol uptake suggested that smooth muscle cells exposed to high glucose concentrations exhibited a noncompetitive type of inhibition characterized by ouabain-sensitive, energy-dependent active transport. Epalrestat blocked glucose-induced changes in sorbitol and myo-inositol metabolism, suggesting that these changes were caused by the accumulation of sorbitol in the cells. These metabolic changes may impair function of smooth muscle cells, contributing to the pathology of diabetic atherosclerosis, especially Mönckeberg's calcific medial sclerosis. The use of an aldose reductase inhibitor may prevent these glucose-induced changes.
Functional Activity of Plasma Fibronectin in Patients With Diabetes Mellitus
Authors: Di Girolamo Nick , Underwood Anne , McCluskey Peter J , Wakefield Denis
Published: September 2013

Decreased wound healing and increased infection are major problems in patients with diabetes mellitus. Fibronectin plays a fundamental role in wound healing and acts as an opsonin for the phagocytosis of foreign antigens. The aim of this study was to ascertain the functional activity of plasma fibronectin from patients with diabetes mellitus. Initially, a modified Boyden chamber technique was used to measure cell migration on fibronectin purified from patient's plasma and an enzyme-linked immunosorbent assay was used to measure the binding of gelatin. A sandwich assay was developed that enabled the capture of fibronectin directly from patient's plasma without prior purification. With the use of a 96-well format, the binding of two different monoclonal antibodies could be compared simultaneously with the binding of gelatin and cell adhesion. In this way, differences in the function of particular domains of fibronectin from diabetic patients and control subjects could be measured. Results showed no difference between fibronectin from diabetic patients and control subjects with respect to the monoclonal antibodies binding in 1) the cell adhesion domain and 2) the heparin-binding domain. Furthermore, no detectable differences were noted with respect to cell adhesion, cell migration, or gelatin binding. These results suggest that diabetic patients receiving insulin treatment show no modulation of plasma fibronectin function, despite raised levels of circulating glucose.
Endothelial Cell-Binding Properties of Lymphocytes Infiltrated Into Human Diabetic Pancreas: Implications for Pathogenesis of IDDM
Authors: Hänninen Arno , Salmi Marko , Simell Olli , Jalkanen Sirpa
Published: September 2013

In IDDM, mononuclear cells accumulate in the islets of Langerhans and destroy insulin-producing β-cells. To study the mechanisms that control extravasation of circulating mononuclear cells into the pancreas, we examined the phenotype of vascular endothelium of the pancreas, propagated a T-cell line from pancreatic islets at the onset of the disease and compared endothelial binding of this cell line in vitro to vascular endothelium in different body regions. The adhesion molecules expressed on the resulting T-cell line and the functional binding capacity of these cells to the endothelium of the normal and diabetic pancreas, mucosa-associated lymphatic tissues, and regional and peripheral lymph nodes were studied. We present evidence of pancreatic endothelial activation in diabetes, leading to endothelial morphology typical for HEVs and accompanying local increase in extravasation of mononuclear cells into the pancreas. Endothelial-cell binding experiments with the T-cell line showed strong adherence of the cells to the endothelium of diabetic pancreas and mucosal lymphoid tissue. The cell line was uniformly CD4-positive, TCR Vβ5.1-positive, LFA-1-positive (CD 11a/CD18), VLA-4α-positive (CD 49d), and CD 44-positive but negative for L-selectin (peripheral lymph node homing receptor). The pancreatic or control cell lines showed no binding to vessels of normal pancreas, and the binding of the pancreatic cell line to the endothelium of peripheral lymph node was weak. Our results suggest that lymphocyte-endothelial cell interactions are important for the accumulation of inflammatory mononuclear cells into the pancreas and imply that lymphocytes derived from the mucosal lymphoid tissue may be involved in the pathogenesis of IDDM.
Quantification of the Relationship Between Insulin Sensitivity and β-Cell Function in Human Subjects: Evidence for a Hyperbolic Function
Authors: Kahn Steven E , Prigeon Ronald L , McCulloch David K , Boyko Edward J , Bergman Richard N , Schwartz Micheal W , Neifing James L , Ward W Kenneth , Beard James C , Palmer Jerry P
Published: September 2013

To determine the relationship between insulin sensitivity and β-cell function, we quantified the insulin sensitivity index using the minimal model in 93 relatively young, apparently healthy human subjects of varying degrees of obesity (55 male, 38 female; 18–44 yr of age; body mass index 19.5–52.2 kg/m2) and with fasting glucose levels <6.4 mM. SI was compared with measures of body adiposity and β-cell function. Although lean individuals showed a wide range of SI, body mass index and SI were related in a curvilinear manner (P < 0.0001) so that on average, an increase in body mass index was associated generally with a lower value for SI. The relationship between the SI and the β-cell measures was more clearly curvilinear and reciprocal for fasting insulin (P < 0.0001), first-phase insulin response (AIRglucose; P < 0.0001), glucose potentiation slope (n = 56; P < 0.005), and β-cell secretory capacity (AIRmax; n = 43; P < 0.0001). The curvilinear relationship between SI and the β-cell measures could not be distinguished from a hyperbola, i.e., SI × β-cell function = constant. This hyperbolic relationship described the data significantly better than a linear function (P < 0.05). The nature of this relationship is consistent with a regulated feedback loop control system such that for any difference in SI, a proportionate reciprocal difference occurs in insulin levels and responses in subjects with similar carbohydrate tolerance. We conclude that in human subjects with normal glucose tolerance and varying degrees of obesity, β-cell function varies quantitatively with differences in insulin sensitivity. Because the function governing this relationship is a hyperbola, when insulin sensitivity is high, large changes in insulin sensitivity produce relatively small changes in insulin levels and responses, whereas when insulin sensitivity is low, small changes in insulin sensitivity produce relatively large changes in insulin levels and responses. Percentile plots based on knowledge of this interaction are presented for evaluating β-cell function in populations and over time.
Cloning and Functional Expression of the Human Islet GLP-1 Receptor: Demonstration That Exendin-4 Is an Agonist and Exendin-(9–39) an Antagonist of the Receptor
Authors: Thorens Bernard , Porret Andrée , Bühler Léo , Deng Shao-Ping , Morel Philippe , Widmann Christian
Published: September 2013

A complementary DNA for a glucagon-like peptide-1 receptor was isolated from a human pancreatics islet cDNA library. The isolated clone encoded a protein with 90% identity to the rat receptor. In stably transfected fibroblasts, the receptor bound [125I]GLP-1 with high affinity (Kd = 0.5 nM) and was coupled to adenylate cyclase as detected by a GLP-1-dependent increase in cAMP production (EC50 = 93 pM). Two peptides from the venom of the lizard Heloderma suspectum, exendin-4 and exendin-(9–39), displayed similar ligand binding affinities to the human GLP-1 receptor. Whereas exendin-4 acted as an agonist of the receptor, inducing cAMP formation, exendin-(9–39) was an antagonist of the receptor, inhibiting GLP-1–induced cAMP production. Because GLP-1 has been proposed as a potential agent for treatment of NIDDM, our present data will contribute to the characterization of the receptor binding site and the development of new agonists of this receptor.
Intensive Insulin Therapy and Weight Gain in IDDM
Authors: Carlson Michael G , Campbell Peter J
Published: September 2013

Intensive insulin therapy is frequently complicated by excessive weight gain. The purpose of this study was to determine the cause and composition of this weight gain. Therefore, changes in body composition, energy expenditure, glycosuria, and substrate kinetics were evaluated in patients with IDDM who transferred from conventional insulin therapy to intensive insulin therapy. Six adult patients with IDDM were studied on conventional insulin therapy and after 2 mo of intensive insulin therapy while maintaining constant caloric intake and were compared with a group of 6 matched nondiabetic volunteers. Body composition was determined by underwater weighing. Energy expenditure was measured during 24-h stays in a whole-room calorimeter. Whole-body turnover rates of glucose, glycerol, palmitate, and leucine were determined by isotope dilution methods. Intensive insulin therapy lowered the mean daily blood glucose concentration and HbA1 (14.8 ± 1.6 to 7.7 ± 0.6 mM and 12.9 ± 0.9 to 9.6 ± 0.6%, both P < 0.01) and almost eliminated glycosuria (428 ± 116 to 39 ± 22 mmol/day, P < 0.05). Body weight increased 2.6 ± 0.8 kg with intensive insulin therapy (P < 0.05) as a result of an increase in fat mass (2.4 ± 0.8 kg, P < 0.05). Daily energy expenditure decreased 5% (118 ± 32 kcal/day) with intensive insulin therapy (P < 0.05). The rates of glucose, glycerol, free fatty acid, and leucine turnover, triglyceride/free fatty acid cycling, and nonoxidative glucose and protein disposal were reduced in the diabetic volunteers during intensive insulin therapy. Thus, intensive insulin therapy causes an increase in body fat as a result of the elimination of glycosuria and reduction in 24-h energy expenditure. The elimination of glycosuria contributed 70% to the positive energy balance during intensive insulin therapy, and the reduction in 24-h energy expenditure contributed the remainder. The reduction in 24-h energy expenditure was the result of the decrease in triglyceride/free fatty acid cycling and nonoxidative glucose and protein metabolism.
Localization of a Type I Diabetes Susceptibility Locus to the Variable Tandem Repeat Region Flanking the Insulin Gene
Authors: Owerbach David , Gabbay Kenneth H
Published: September 2013

A susceptibility gene for type I diabetes is present on chromosome 11p15.5, but its location, identity, and mechanism of action are unknown. We have sequenced 14 kilobases of DNA flanking the human insulin gene and found new DNA polymorphisms and determined their frequencies in the general population and in families of type I diabetic subjects. A DNA polymorphism located 3123 base pairs downstream from the initiation site of transcription of the insulin gene, when present in the homozygous state, provides a relative risk for type I diabetes of 5.2 (P = 0.006). However, this DNA polymorphism as well as other diabetes-associated 3′ markers are in linkage-disequilibrium with the actual susceptibility region, because these polymorphisms are found on haplotypes both positively and negatively associated with type I diabetes susceptibility. Nucleotide sequence analysis of the variable tandem repeat region flanking the 5′ end of the insulin gene shows variable tandem repeat elements associated with these haplotypes to differ greatly in composition, i.e., an ATAGGGGTGTGGGG repeat element is absent on a haplotype associated with type I diabetes susceptibility, but is found in 6–10 copies on two haplotypes negatively associated with the disease. These findings suggest that the type I diabetes susceptibility locus on chromosome 11p15.5 is probably located in the 5′ variable tandem repeat region rather than in the 3′ region of the insulin gene.
Mechanisms for D-Glucose Inhibition of myo-Inositol Influx Into Rat Lens
Authors: Mistry Kishor P , Beyer-Mears Annette , Diecke Friedrich P J
Published: September 2013

myo-Inositol depletion as a result of hyperglycemia is considered one of the leading contributors to chronic diabetic complications. We investigated the possible mechanisms through which elevated extracellular glucose levels affect the loss of intracellular myo-inositol in rat lens. Short-term incubation (up to 4 h) in solutions with elevated glucose concentrations revealed a concentration-dependent inhibition of myo-inositol influx. This inhibition was caused by both an increase of the transport coefficient and a decrease of maximal flux and thus was a mixed competitive and noncompetitive inhibition. If polyol accumulation was prevented with sorbinil, an aldose reductase inhibitor, the inhibition of myo-inositol influx was partially reduced. The remaining inhibition was the result of an increased transport coefficient without a change in maximal flux and therefore represents a strictly competitive inhibition. A similar competitive inhibition was observed with the nonmetabolizable glucose analogue L-glucose, which cannot be converted to polyol. Longer exposure (16 h) to solutions with high glucose concentrations resulted in an inhibition that correlated with high lens polyol levels. This inhibition persisted after the lenses were returned to solutions with normal glucose concentrations and was the result of a decrease of maximal flux without a significant change in transport coefficient, a strictly noncompetitive inhibition. The noncompetitive inhibition associated with polyol accumulation and the competitive inhibition due to extracellular glucose were additive. Lens myo-inositol depletion after exposure to elevated glucose concentrations thus resulted from a competitive inhibition caused by the interaction of extracellular glucose with the myo-inositol carrier and a noncompetitive inhibition associated with polyol accumulation.
Early Introduction of Dairy Products Associated with Increased Risk of IDDM in Finnish Children
Authors: Virtanen Suvi M , Räsänen Leena , Ylönen Katrina , Aro Antti , Clayton David , Langholz Bryan , Pitkäniemi Janne , Savilahti Erkki , Lounamaa Raisa , Tuomilehto Jaakko , Åkerblom Hans K
Published: September 2013

Associations between infant-feeding patterns and risk of IDDM were investigated in a nationwide Finnish case-control study of 690 IDDM children <15 yr of age. Each child was matched by date of birth and sex to a randomly selected population-based control child. Univariate analysis revealed that the risk of IDDM was increased by ∼1.5 in children for whom breast-feeding was terminated at <2 mo of age, doubled in those who were exclusively breast-fed for <2 mo, and doubled in those who were introduced to dairy products at <2 mo of age. In further multivariate analyses of these factors, it was found that introduction of dairy products at an early age was the most important risk factor, and the observed univariate effects of duration of breast-feeding variables were explained by their correlation with this factor. This is the first observational study to show that early introduction of dairy products is independently associated with an increased risk of IDDM. Adjustment for mother's education and age, child's birth order, or birth weight did not affect the results.
Mechanism of Awareness of Hypoglycemia: Perception of Neurogenic (Predominantly Cholinergic) Rather Than Neuroglycopenic Symptoms
Authors: Towler Dwight A , Havlin Carolyn E , Craft Suzanne , Cryer Philip
Published: September 2013

We sought 1) to determine which symptoms of hypoglycemia are reproducible, 2) to pharmacologically distinguish neurogenic (autonomic) from neuroglycopenic symptoms, and 3) to test the hypothesis that awareness of hypoglycemia is the result of perception of neurogenic rather than neuroglycopenic symptoms. Awareness of hypoglycemia and 19 symptoms were quantitated in 10 normal, young adults, each studied on four occasions in random sequence, during 1) clamped euglycemia (∼ 5 mM), 2) clamped hypoglycemia (∼ 2.5 mM), 3) clamped hypoglycemia with combined α- and β-adrenergic blockade (phentolamine and propranolol), and 4) clamped hypoglycemia with pan-autonomic blockade (phentolamine, propranolol and atropine). Significant (ANOVA, P < 0.001) treatment effects on the awareness of hypoglycemia (“blood sugar low”) were noted. No change occurred in the score for this during euglycemia, but the mean ± SE increase was 2.1 ± 0.4 during hypoglycemia. This increase was not reduced significantly by adrenergic blockade (1.6 ± 0.5), but was reduced significantly and substantially (∼ 70%) by pan-autonomic blockade (0.6 ± 0.3). Significant neurogenic symptoms included shaky/tremulous (P < 0.001), heart pounding (P < 0.001), and nervous/anxious (P = 0.002), all adrenergic; and sweaty (P < 0.001), hungry (P < 0.001), and tingling (P = 0.009), all cholinergic. Significant neuroglycopenic symptoms, those produced by hypoglycemia but not reduced by pan-autonomic blockade, included warm (P < 0.001), weak (P = 0.011), difficulty thinking/confused (P = 0.004), and tired/drowsy (P = 0.003). We conclude that muscarinic cholinergic mechanisms mediate an important and previously uncharacterized component of the neurogenic symptoms of hypoglycemia and awareness of hypoglycemia. Awareness of hypoglycemia is largely, perhaps exclusively, the result of perception of neurogenic rather than neuroglycopenic symptoms.
Differential Expression of GAD65 and GAD67 in Human, Rat, and Mouse Pancreatic Islets
Authors: Kim John , Richter Wiltrud , Aanstoot Henk-Jan , Shi Yuguang , Fu Qin , Rajotte Ray , Warnock Garth , Baekkeskov Steinunn
Published: September 2013

The smaller form of the autoantigen glutamic acid decarboxylase, GAD65 (formerly the 64,000 Mr autoantigen), is a major target of humoral autoimmunity in type I diabetes. Human autoantisera have been used extensively to characterize the GAD65 antigen in both rat and human islets, but the protein has escaped detection in mouse islets. We have now analyzed the expression of GAD65 and GAD67, the larger glutamic acid decarboxylase protein, in human, rat, and mouse islets of Langerhans and brain, using human monoclonal islet cell autoantibodies, human autoantisera, and experimentally raised antibodies to glutamic acid decarboxylase. Human monoclonal autoantibodies and experimentally raised antibodies reacted with mouse GAD65 produced in a baculovirus expression system by Western blotting and immunoprecipitation and with GAD65 in mouse brain by immunohistochemistry but failed to detect GAD65 in mouse islets by the latter two methods. However, analysis of mouse islets by Western blotting technique, using the most sensitive experimentally raised antibody, showed that mouse islets express both GAD65 and GAD67 but at levels that are severalfold lower than those in mouse brain or in human and rat islets. Furthermore, both human and rat islets predominantly express GAD65, whereas GAD67 is the major glutamic acid decarboxylase protein in mouse islets. Human islets are significantly distinct from mouse and rat islets and from brain because they only express GAD65, which is consistent with the predominant role of this form as a target of autoantibodies associated with β-cell destruction in humans. Human as well as rat islet GAD65 are found in both membrane-bound and soluble forms. The low level of glutamic acid decarboxylase expression in mouse islets compared with human and rat islets is likely to have implications for both the development of tolerance to glutamic acid decarboxylase as well as the homing of glutamic acid decarboxylase-specific lymphocytes to the mouse β-cell. In this context, the results suggest 1) that the mouse is ideal for studies of the consequences of an expression of high levels of glutamic acid decarboxylase in the β-cell from a transgene and 2) that the rat may be better suited than the mouse for development of nontransgenic animal models of glutamic acid decarboxylase autoimmunity by immunization.
The Effect of Glucose on Proteoglycans Produced by Cultured Mesangial Cells
Authors: Silbiger Silibiger , Schlondorff Detlef , Crowley Susan , Rosenberg Lawrence , Choi Haing , Hatcher Victor , Gordon Portia
Published: September 2013

Altered proteoglycan metabolism may play a role in the development of diabetic glomerulopathy. This study was conducted to examine the effects of glucose on the production and physical characteristics of proteoglycans generated by rat mesangial cells in culture. Rat mesangial cells were exposed to elevated glucose media (500 mg/dl) or standard glucose media (200 mg/dl) for 8–10 days, and proteoglycan synthesis was determined using 35S-labeling in conjunction with anion exchange and sizing chromatography. Rat mesangial cells generated predominantly chondroitin/dermatan sulfate proteoglycans, with small amounts of heparan sulfate proteoglycans. High glucose did not alter the number of rat mesangial cells after 24 h or after 8–10 days, compared with cells grown under standard glucose conditions. The total amount of glycosaminoglycan generated and the sizes of the major proteoglycans were not different between cultures grown in standard and elevated glucose medium. Levels of mRNA for the proteoglycan, biglycan (as assessed by Northern blot analysis), also were comparable between the standard and elevated glucose conditions. Exposure to media high in glucose did not change the rate of secretion of proteoglycans from the cell layer to the medium, but did result in a greater quantity of radiolabeled proteoglycan deposited in the extracellular matrix. The cell, extracellular matrix and medium proteoglycans isolated from the elevated glucose cultures, consistently eluted from the anion exchange column at a lower [NaCl] compared with those generated under standard glucose conditions, indicating a loss of anionic charges. We conclude that exposure of rat mesangial cells to media high in glucose results in a greater amount of labeled proteoglycan deposited in the extracellular matrix. These proteoglycans are the same size but have a lower charge density than those generated under standard glucose conditions.
Different Sensitivity of Glucose and Amino Acid Metabolism to Insulin in NIDDM
Authors: Luzi Livo , Petrides Alexander S , De Fronzo Ralph A
Published: September 2013

NIDDM subjects are characterized by impaired glucose tolerance and insulin resistance with respect to glucose metabolism. To examine whether the defect in glucose utilization extends to amino acid metabolism, 6 NIDDM subjects (64 ± 4 yr of age; ideal body weight of 107 ± 3%) and 7 control subjects (58 ± 4 yr of age; ideal body weight of 105 ± 2%) were studied with the euglycemic insulin clamp technique, in combination with [1-14C]leucine and indirect calorimetry. All subjects participated in two studies. In study 1, after 3 h of tracer equilibration, a 3-h insulin clamp (40 mU · m−2 · min−1) was performed to define the effect of insulin on leucine kinetics and glucose metabolism. In study 2, subjects received a repeat 3-h insulin clamp, and a balanced amino acid solution was infused to increase the plasma amino acid concentrations ∼ 2-fold to examine the effect of combined physiological hyperinsulinemia-hyperaminoacidemia on the rate of leucine and glucose disposal. Insulin-mediated total body glucose uptake was significantly reduced in NIDDM during both study 1 (5.6 ± 0.4 vs. 6.9 ± 0.6 mg · kg−1 · min−1 P < 0.01) and study 2 (5.2 ± 0.4 vs. 6.8 ± 0.6, P < 0.01). Basal plasma leucine (120 ± 10 vs. 123 ± 11 μM) and α-ketoisocaproic acid concentrations (28 ± 3 vs. 25 ± 2 μM) were similar in NIDDM and control subjects, respectively. In contrast, the basal plasma glucose concentration (8.9 ± 0.8 vs. 4.7 ± 0.2 μM) and the HbA1c (8.5 ± 0.2 vs. 5.7 ± 0.2%) were significantly increased in NIDDM (P < 0.01). In the postabsorptive state, endogenous leucine flux, leucine oxidation, and nonoxidative leucine disposal were similar in NIDDM and control subjects. When insulin was infused without amino acids (study 1), the decrement in plasma leucine (53 ± 5 vs. 48 ± 4 μM), endogenous leucine flux (13 ± 2 vs. 11 ± 1 μmol · m−2 · min−1), leucine oxidation (1.6 ± 0.2 vs. 1.3 ± 0.1 μmol · m−2 · min−1), and nonoxidative leucine disposal (10 ± 1 vs. 8 ± 1 μmol · m−2 · min−1) was comparable in both groups. During combined insulin and amino acid infusion (study 2), plasma leucine concentration (185 ± 20 vs. 190 ± 15 μM) rose similarly in NIDDM and control subjects. In NIDDM, the increment in leucine oxidation (9.0 ± 0.7 vs. 8.5 ± 0.6 μmol · m−2 · min−1) and nonoxidative leucine disposal (9.3 ± 0.7 vs. 10.5 ± 0.9 μmol · m−2 · min−1) was similar to that observed in control subjects; the decrement in endogenous leucine flux (22.3 ± 2.1 vs. 20.2 ±1.9 μmol · m−2 · min−1) was comparable in both groups. We conclude that 1) insulin-mediated glucose disposal is significantly impaired in NIDDM and 2) the effect of insulin on endogenous leucine flux (protein degradation), nonoxidative leucine disposal (protein synthesis), and leucine oxidation is similar in NIDDM and control subjects. These results indicate a clear-cut dissociation between the effect of insulin on glucose and protein metabolism in NIDDM.
Insulin Resistance in Aging Is Related to Abdominal Obesity
Authors: Kohrt Wendy M , Kirwan John P , Staten Myrlene A , Bourey Raymond E , King Douglas S , Holloszy John O
Published: September 2013

Studies have shown that insulin resistance increases with age, independent of changes in total adiposity. However, there is growing evidence that the development of insulin resistance may be more closely related to abdominal adiposity. To evaluate the independent effects of aging and regional and total adiposity on insulin resistance, we performed hyperinsulinemic euglycemic clamps on 17 young (21–33 yr) and 67 older (60–72 yr) men and women. We assessed FFM and total and regional adiposity by hydrodensitometry and anthropometry. Insulin-stimulated GORs at a plasma insulin concentration of ∼450 pM averaged 45.6 ± 3.3 μmol · kg FFM−1 · min−1 (mean ± SE) in the young subjects, 45.6 ± 10.0 μmol · kg FFM−1 · min−1 in 24 older subjects who were insulin sensitive, and 23.9 ± 11.7 μmol · kg FFM−1 · min−1 in 43 older subjects who were insulin resistant. Few significant differences were apparent in skin-fold and circumference measurements between young and insulin-sensitive older subjects, but measurements at most central body sites were significantly larger in the insulin-resistant older subjects. Waist girth accounted for >40% of the variance in insulin action, whereas age explained only 10–20% of the total variance and <2% of the variance when the effects of waist circumference were statistically controlled. These results suggest that insulin resistance is more closely associated with abdominal adiposity than with age.
Early Exposure to Cow's Milk and Solid Foods in Infancy, Genetic Predisposition, and Risk of IDDM
Authors: Kostraba Jill N , Cruickshanks Karen J , Lawler-Heavner Janet , Jobim Luiz F , Rewers Marian J , Gay Elizabeth C , Chase H Peter , Klingensmith Georgeanna , Hamman Richard F
Published: September 2013

Using a case-control study design, we examined the hypothesis that early exposure to cow's milk and solid foods increased the risk of IDDM. An infant diet history was collected from 164 IDDM subjects from the Colorado IDDM Registry with a mean birth year of 1973, and 145 nondiabetic population control subjects who were frequency matched to diabetic subjects on age, sex, and ethnicity. Early exposure was defined as exposure occurring before 3 mo of age. After controlling for ethnicity, birth order, and family income, more diabetic subjects were exposed early to cow's milk (OR 4.5, 95% Cl 0.9–21.4) and solid foods (OR 2.5, Cl 1.4–4.3) than control subjects. To examine this association while accounting for the genetic susceptibility to IDDM, we defined individuals as high and low risk by an HLA-DQB1 molecular marker. Early exposure to cow's milk was not associated with elevated risk for IDDM in low-risk individuals. Relative to unexposed low-risk individuals, early exposure to cow's milk was strongly associated in individuals with a high risk marker (OR 11.3, Cl 1.2–102.0). Similar findings were observed for early exposure to solid foods. These data indicate that early exposure to cow's milk and solid foods may be associated with increased risk of IDDM. The inclusion of HLA-encoded risk in the analyses demonstrates the combined effect of genetic and environmental factors.
Effects of Ponalrestat, an Aldose Reductase Inhibitor, on Neutrophil Killing of Escherichia Coli and Autonomic Function in Patients With Diabetes Mellitus
Authors: Boland Oliver M , Blackwell Caroline C , Clarke Basil F , Ewing David J
Published: September 2013

In diabetic subjects, polyol pathway activity might inhibit neutrophil function and cause nerve damage. The effects of ponalrestat, an aldose reductase inhibitor, were assessed on neutrophil intracellular killing of Escherichia coli and on autonomic function in diabetic subjects in a randomized double-blind, placebo-controlled, crossover trial. We studied 31 diabetic subjects with autonomic dysfunction and 21 age- and sex-matched control subjects. During two 12-wk treatment periods, the diabetic subjects took either 600 mg of ponalrestat or matching placebo once daily. Neutrophil killing of E. coli was measured by a microbiological assay technique. Kmax by neutrophils from the diabetic subjects was lower than in the control group (Kmax of diabetic subjects 54.5 ± 26.4 vs. control subjects 67.3 ± 16.3, P = 0.045). Ponalrestat significantly increased bacterial killing in the diabetic subjects (Kmax of ponalrestat 75.1 ± 16.5 vs. placebo 58.2 ± 20.8, P = 0.003) so that there was no longer any significant difference in Kmax between the control subjects and the diabetic subjects on active treatment. Ponalrestat had no significant effect on a range of standard cardiovascular autonomic nerve function tests. We conclude that neutrophil killing of E. coli is impaired in diabetic subjects with autonomic dysfunction. This is restored to normal by ponalrestat.
Opposite Effects of Hyperglycemia and Insulin Deficiency on Liver Glycogen Synthase Phosphatase Activity in the Diabetic Rat
Authors: Lavoie Louis , Dimitrakoudis Dimitrios , Marette Andre , Annabi Borhane , Klip Amira , Vranic Mladen , Werve Gerald Van De
Published: September 2013

The specific effect of hyperglycemia on the reported decrease in liver glycogen synthase phosphatase activity was studied in STZ-induced diabetic rats with normal fasting insulinemia. Four groups of animals were investigated: control (nondiabetic), diabetic hyperglycemic (STZ), diabetic normoglycemic (STZ followed by 3-day phloridzin treatment), and a diabetic normoglycemic group injected with glucose to reinstate hyperglycemia. None of the treatments significantly altered fasting plasma insulin and glucagon concentrations. We found that hepatic synthase phosphatase activity decreased in STZ-induced diabetic rats and was further markedly reduced when glycemia was normalized in the diabetic animals. This additional decrease in phosphatase activity was almost fully reversed when hyperglycemia was restored by acute glucose infusion of the normoglycemic diabetic rats. In parallel, the levels of liver G6P and F6P were markedly reduced in the diabetic normoglycemic rats and restored with reinstatement of hyperglycemia. In contrast, liver microsomal glucose-6-phosphatase activity was enhanced and glucokinase activity was lowered in all diabetic groups, regardless of glycemia. Our results indicate that hyperglycemia per se counteracts part of the loss of hepatic synthase phosphatase in diabetic animals and provokes the stable conversion of synthase phosphatase from a less active to a more active form.
Impact of Obesity on Insulin Action in NIDDM
Authors: Campbell Peter J , Carlson Michael G
Published: September 2013

Although a strong clinical association exists between obesity and NIDDM, previous studies have failed to confirm a significant relationship between the degree of obesity and the insulin resistance of NIDDM. We examined the impact of obesity on insulin-mediated glucose metabolism in 25 patients with NIDDM, whose BMI ranged from 20.8 to 36.9 kg/m2, during insulin infusions of 0.4, 1.0, and 10 μU/kg min using the glucose clamp technique. Rates of glucose disposal and HGP were compared in 14 of the diabetic patients with 19 nondiabetic control subjects of similar sex, age, and weight distribution. We found an inverse correlation between BMI and the glucose infusion rate necessary to maintain euglycemia during the first two of three insulin infusions (r = −0.63, P < 0.001, r = −0.57, P < 0.01, and r = −0.36, P = 0.08). Glucose disposal was correlated with BMI during the 1.0 mU/kg min insulin infusion (r = −0.57, P < 0.05), and glucose production was correlated at the 0.4 mU/kg min infusion (r = 0.69, P < 0.01). We concluded that obesity diminishes the insulin sensitivity of both hepatic and peripheral tissues in patients with NIDDM. The exacerbation of the insulin resistance of NIDDM by obesity indicates why weight reduction is an effective treatment for the obese NIDDM patient.
Diabetes and Embryonic Malformations: Role of Substrate-Induced Free-Oxygen Radical Production for Dysmorphogenesis in Cultured Rat Embryos
Authors: Eriksson Ulf J , Borg L A HÅkan
Published: September 2013

The aim of this study was to evaluate the role of free-oxygen radicals in the embryonic maldevelopment of diabetic pregnancy. Rat embryos cultured in vitro during early organogenesis showed growth retardation and severe malformations after exposure to 50 mM glucose, 3 mM PYR, 10 mM HBT, or 3 mM KIC. Combinations of 25 mM glucose, 2.5 mM HBT, and 1 mM KIC also elicited embryonic growth retardation and malformations. The deleterious effects on embryonic development by all agents were alleviated by addition of SOD to the culture media, which yielded increased enzyme activity in the embryos and their membranes. The endogenous SOD activity also increased in embryos subjected to a high concentration of glucose or PYR in the culture medium. Addition of the mitochondrial PYR transport inhibitor CHC to the culture media blocked the dysmorphogenesis caused by glucose and PYR, but was without effect on the teratogenic actions of HBT and KIC. These findings implicate the embryonic mitochondria as a likely site for enhanced substrate-induced production of free-oxygen radicals mediating the teratogenic effect of a diabetic environment. In particular, the teratogenic process in diabetic pregnancy may depend on an increased production of free-oxygen radicals in immature embryonic mitochondria in response to a metabolic overload. This notion implies that every oxidative substrate entering the mitochondrial metabolism in excess may induce embryonic malformations and emphasizes the need for an extended metabolic surveillance of pregnant diabetic women. Consequently, optimal metabolic control should aim at normalizing the maternal serum concentrations of all possible oxidative substrates.
Prevention of High- and Low-Dose STZ-Induced Diabetes With D-Glucose and 5-Thio-D-Glucose
Authors: Wang Zhiyong , Dohle Claudia , Friemann Johannes , Green Bernard S , Gleichmann Helga
Published: September 2013

To induce hyperglycemia in mice by administration of STZ, two experimental protocols that involve different pathogenic pathways are being used. First, the intraperitoneal injection of a single high dose (HD-STZ) exerts direct toxicity on β-cells, which results in necrosis within 48–72 h and overt permanent hyperglycemia. Second, injections of multiple low doses of STZ (LD-STZ), administered intraperitoneally on 5 consecutive days, induce both β-cytotoxic effects and STZ-specific T-cell-dependent immune reactions. In LD-STZ models, only a combination of toxic and immunological effects result in gradually increasing hyperglycemia, provided male mice of susceptible strains are being used. In this study, we found that 5-T-G, a glucose analogue that has sulfur for oxygen in the pyranose ring, prevented, in a dose-dependent way, both HD-STZ- and LD-STZ-induced hyperglycemia and that D-G, which was only tested in the LD-STZ system, was also protective, albeit somewhat less so than 5-T-G. This protective effect was achieved by intraperitoneally injecting 5-T-G and D-G, respectively, right before each STZ injection. Protection against hyperglycemia was already achieved with a total of 3 injections of 5-T-G, 1 injection each given before the first 3 of 5 LD-STZ injections. By means of OGTT, it was determined that pretreatment with 5-T-G afforded protection from substantial β-cell damage in vivo. In addition, significant (P < 0.005) in vitro β-cell protection with 5-T-G was documented by assaying for basal and glucose-stimulated IRI release in bulk cultures of islets, isolated from LD-STZ-injected and 5-T-G-pretreated donors. Interestingly, pretreatment with 5-T-G failed to prevent STZ-induced inflammatory reactions, as assessed by two different methods. First, by PLN assay, it was shown that the T-cell-dependent immune reactions to STZ as antigen were not affected by pretreatment with 5-T-G. Second, histological examinations indicated that the mononuclear cell infiltrates of pancreatic islets were not changed by pretreatment with 5-T-G. In conclusion, our data indicate that 5-T-G and D-G protected against hyperglycemia by preventing the β-cell toxic effects of STZ, not by preventing its inflammatory effect in the LD-STZ model.
Differential Expression of Glutamic Acid Decarboxylase in Rat and Human Islets
Authors: Petersen Jacob S , Russel Steven , Marshall Michael O , Kofod Hans , Buschard Karsten , Cambon Natalie , Karlsen Allan E , Boel Esper , Hagopian William A , Hejnæs Kim R , Lernmark ÅKe , Madsen Ole D , Michelsen Birgitte K
Published: September 2013

The GABA synthesizing enzyme GAD is a prominent islet cell autoantigen in type I diabetes. The two forms of GAD (GAD64 and GAD67) are encoded by different genes in both rats and humans. By in situ hybridization analysis of rat and human pancreases, expression of both genes was detected in rat islets, whereas only GAD64 mRNA was detected in human islets. Immunocytochemical analysis of rat and human pancreatic sections or isolated islets with antibodies to GAD64 and GAD67 in combination with antibodies to insulin, glucagon, or SRIF confirmed that a GAD64 and GAD67 expression were β-cell specific in rat islets. In contrast, only GAD64 was detected in human islets and was, in addition to β-cells, also surprisingly localized to some α-cells, δ-cells, and PP-cells. In long-term (4 wk) monolayer cultures of newborn rat islet cells, GAD64 expression remained β-cell specific as observed in vivo, whereas GAD67 was localized not only to the β-cells but also in the α-cells and δ-cells. A small but distinct fraction of GAD positive cells in these monolayer cultures did not accumulate GABA immunoreactivity, which may indicate cellular heterogeneity with respect to GABA catabolism or GAD enzyme activity. In a rat insulinoma cell line (NHI-6F) producing both glucagon and insulin depending on the culture conditions, GAD64 expression was detected only in cultures in which the insulin producing phenotype dominated. In conclusion, these data demonstrate that the two GAD isoforms are differentially expressed in rat and human islets but also that the expression differs according to culture conditions. These findings emphasize the need to consider both the species and culture conditions of islets.
Islet Cell DNA Is a Target of Inflammatory Attack by Nitric Oxide
Authors: Fehsel Karin , Jalowy Andreas , Qi Sun , Burkart Volker , Hartmann Bettina , Kolb Hubert
Published: September 2013

NO has been identified recently as the prime islet-toxic product of inflammatory macrophages. The adverse effects of IL-1 on isolated islets also have been reported to involve NO. We now show that exposure of an islet cell suspension to the NO donor nitroprusside or to activated macrophages leads to DNA strand breaks. Macrophages did not induce DNA damage in the presence of the NO synthase inhibitor NG-methyl-L-arginine. DNA strand breaks were demonstrated at the level of single cells by a modified nick-translation procedure and confirmed by analysis of DNA fragmentation by gel electrophoresis. DNA strand breaks occurred within 1 h and preceded islet cell lysis. DNA damage could not be prevented by inhibitors of endogenous endonucleases. We conclude that islet cell DNA is an early target of NO action.
Differences in the Ratio of RNA Encoding Two Isoforms of the Insulin Receptor Between Control and NIDDM Patients: The RNA Variant Without Exon 11 Predominates in Both Groups
Authors: Norgren Svante , Zierath Juleen , Galuska Dana , Wallberg-Henriksson Harriet , Luthman Holger
Published: September 2013

Two alternative forms of the insulin receptor with different affinities for insulin are expressed as a result of alternative splicing of RNA corresponding to exon 11 of the IR gene. The percentage of IR-RNA molecules without exon 11, encoding the high-affinity isoform, was determined by cDNA-mediated PCR amplification of RNA extracts from the quadriceps femoris muscle of healthy control subjects (n = 9) and NIDDM patients (n = 7). In both patients and control individuals, a majority of the IR-RNA molecules contained exon 11. In addition, the proportion of IR-RNA molecules without exon 11 was decreased in patients (21 ± 1%) compared with control subjects (31 ± 3%) (P = 0.018). Careful investigation of the kinetics of the PCR-based assay system, as well as the conditions for separation of the PCR products, allowed us to suggest a possible explanation of the discrepant results concerning the alternative splicing presented in previous reports. The diabetic subjects as a group had higher fasting insulin levels and lower insulin-mediated glucose uptake during a euglycemic-hyperinsulinemic clamp (P = 0.042). However, identification of the regulatory pathways leading to the splicing alteration in NIDDM patients requires further investigation.