bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of Expression of Insulin Resistance and Hyperglycemia by Different Genetic Factors in Diabetic C57BL/6J Mice
Authors: Surwit Richard S , Seldin Michael F , Kuhn Cynthia M , Cochrane Christina , Feinglos Mark N
Published: September 2013

The inheritance of the tendency to develop diet-induced non-insulin-dependent (type II) diabetes was analyzed in crosses between diabetes-prone C57BL/6J (BL/6) mice and diabetes-resistant A/J mice. The effects of a diabetogenic diet on blood glucose and insulin levels, insulin sensitivity, and weight were evaluated in F1 and both (BL/6 × A/J) F1 × BL/6 and (BL/6 × A/J) F1 × A/J backcross mice. These results suggest that diet-induced hyperglycemia is largely determined by a recessive gene and diet-induced insulin resistance by a dominant gene. Analyses of both backcrosses indicated that insulin sensitivity and blood glucose levels were unrelated, suggesting that they are controlled by different genetic factors. This conclusion was supported by data from nine recombinant inbred BXA strains in which no correlation was observed between these variables. Furthermore, insulin sensitivity and body weight correlated differently in the two backcross groups, suggesting that insulin resistance is not simply a function of obesity. The number of genes that predominantly influence diabetic traits was estimated by comparing the variance observed in (BL/6 × A/J) F1 × BL/6 backcross mice with that observed in parental mice. The data suggest that relatively few genes predominantly affect the diabetic phenotype in this murine model.
Evidence for Impaired Coupling of Receptors to Gi Protein in Adipocytes From Streptozocin-Induced Diabetic Rats
Authors: Green Allan , Johnson Jeff L
Published: September 2013

Adenosine and prostaglandins of the E series inhibit lipolysis in adipocytes by binding to cell surface receptors. This inhibition is mediated via Gi. It has been reported that Gi is almost absent in livers from diabetic rats. Therefore, we have evaluated the sensitivity of adipocytes from diabetic rats to the adenosine analogue N6-phenylisopropyl adenosine (PIA) and to prostaglandin E1 (PGE1). Diabetes was induced with streptozocin (65 mg/kg i.v.), and after 7 days, adipocytes were isolated. Lipolysis (measured in the presence of adenosine deaminase) was inhibited by PIA and PGE, in both control and diabetic cells. However, the dose-response curves were markedly shifted to the right in the cells from diabetic rats. The IC50 for PIA was 0.30 ± 0.02 nM in controls and 0.83 ± 0.08 in diabetic rats (P < 0.001), and the IC50 for PGE, was 3.16 ± 0.18 nM in controls and 5.26 ± 0.57 nM in diabetic rats (P < 0.02). These findings indicate decreased sensitivity to both adenosine and PGE1. Adipocyte membranes were isolated from control and diabetic rats. Adenosine receptors (measured by binding of 125I-labeled hydroxy-PIA) were not altered in cells from diabetic rats. However, the ability of Gpp(NH)p (a nonhydrolyzable GTP analogue) to inhibit adenosine-receptor binding was markedly decreased in membranes from diabetic rats, suggesting a change at the level of G1. The α-subunits of G11, G12, G13, and Gs were quantitated on Western blots with a series of recently characterized anti-peptide antisera. This revealed that the amounts of each of these G proteins were normal in membranes from the diabetic rats. These findings suggest that there is a functional but not quantitative abnormality in G1 in adipocytes from diabetic rats that results in abnormal coupling of both A1-adenosine receptors and prostaglandin receptors and would explain the decreased sensitivity to PIA and PGE1.
Insulin, Prostaglandins, and the Pathogenesis of Hypertension
Authors: Axelrod Lloyd
Published: September 2013

Hypertension is associated with hyperinsulinemia in the presence or absence of obesity or glucose intolerance. Physiological concentrations of insulin decrease the catecholamine-induced production of prostaglandin I2 (PGI2; prostacyclin) and PGE2, two potent vasodilators, in adipose tissue, one of the largest organs in the body. This finding suggests that hyperinsulinemia increases peripheral vascular resistance and blood pressure by inhibiting the stimulatory effect of adrenergic agonists (and perhaps other agonists) on the production of PGI2 and PGE2 in adipose tissue (and perhaps other tissues). This concept is supported by evidence that PGI2 and PGE2 modulate vascular reactivity in states of health and disease. For example, during insulin deficiency, i.e., in diabetic ketoacidosis, PGI2 and PGE2 production by adipose tissue are increased, and peripheral vascular resistance and blood pressure are decreased. This hypothesis is also supported by evidence that blood flow through rat and human adipose tissue is decreased in obesity and that insulin decreases the blood flow through adipose tissue in nonobese rats. Thus, insulin may regulate PGI2 and PGE2 production by adipose tissue (and possibly other tissues) through a wide range of concentrations with important physiological and clinical consequences.
Inhibition by AICA Riboside of Gluconeogenesis in Isolated Rat Hepatocytes
Authors: Vincent M Françoise , Marangos Paul J , Gruber Harry E , van den Berghe Georges
Published: September 2013

5-Amino-4-imidazolecarboxamide (AICA) riboside, the nucleoside corresponding to AICA ribotide (AICAR or ZMP), an intermediate of the de novo pathway of purine biosynthesis, was found to exert a dose-dependent inhibition on gluconeogenesis in isolated rat hepatocytes. Production of glucose from lactate-pyruvate mixtures was half-maximally inhibited by ∼100 μM and completely suppressed by 500 μM AICA riboside. AICA riboside also inhibited the production of glucose from all other gluconeogenic precursors investigated, i.e., fructose, dihydroxyacetone, and L-proline. Measurements of intermediates of the glycolytic-gluconeogenic pathway showed that AICA riboside provoked elevations of triose phosphates and fructose-1,6-bisphosphate and decreases in fructose-6-phosphate and glucose-6-phosphate. The effects of AICA riboside persisted when the cells were washed 10 min after its addition but were suppressed by 5-iodotubercidin, an inhibitor of adenosine kinase. AICA riboside provoked a dose-dependent buildup of normally undetectable Z nucleotides. After 20 min of incubation with 500 μM AICA riboside, ZMP, ZTP, and ZDP reached 3, 0.3, and 0.1 μmol/g cells, respectively. Concentrations of ATP were not significantly modified by addition of up to 500 μM AICA riboside when the cells were incubated with lactate-pyruvate but decreased with fructose or dihydroxyacetone. The activity of rat liver fructose-1,6-bisphosphatase was inhibited by ZMP with an apparent Ki of 370 μM. It is concluded that AICA riboside exerts a suppressive effect on gluconeogenesis because it provokes an accumulation of ZMP, which inhibits fructose-1,6-bisphosphatase. Because gluconeogenesis is increased in diabetes, our observations warrant a search for related inhibitors of f ructose-1,6-bisphosphatase, which may have therapeutic potential in this disorder.
Differential Regulation of Glycosylated Phosphatidylinositol Subtypes by Insulin
Authors: Gaulton Glen N
Published: September 2013

Glycosylated phosphatidylinositol (gly-Pl) molecules have been implicated as precursors for insulin-sensitive second messengers (1–4) and lipid-anchored membrane proteins (5–9). The relationship between the diverse functions of these lipids and their predicted structural heterogeneity within gly-Pl subtypes was examined in human T lymphocytes. Four subtypes of gly-Pl molecules were identified in T lymphocytes after separation over high-performance thin-layer chromatography by sensitivity to Pl-specific phospholipase C and nitrous acid. Antibody probes of the glycan domain of gly-Pl were developed and used to assess the partial sensitivity of gly-Pl to insulin action. This analysis showed that the effects of insulin are linked to differential utilization of only two of the four gly-Pl subtypes in T lymphocytes. Polar fragments of this reaction were identified in extracellular supernatants from insulin-treated cells. The biological significance of insulin-dependent gly-Pl hydrolysis was demonstrated by insulin and inositol phosphoglycan regulation of glucose metabolism in intact lymphocytes. These results support the hypothesis that multifunctional roles of gly-Pl are served by discrete gly-Pl populations and that metabolites of gly-Pl subsets participate as signaling elements in insulin action.
Sorbitol, myo-Inositol, and Rod Outer Segment Phagocytosis in Cultured hRPE Cells Exposed to Glucose: In Vitro Model of myo-Inositol Depletion Hypothesis of Diabetic Complications
Authors: Monte Monte A Del , Rabbani Ramin , Diaz Tarcisio C , Lattimer Sarah A , Nakamura Jiro , Brennan Mary C , Greene Douglas A
Published: September 2013

The “myo-inositol depletion hypothesis” remains a leading but still controversial contender among proposed pathogenetic mechanisms for the chronic complications of diabetes. The multifaceted interrelationships among altered tissue myo-inositol content and metabolism and tissue function have been difficult to elucidate in diabetic animal models due in part to the complex, heterogeneous nature of tissues prone to diabetic complications. The retinal pigment epithelium consists of a homogenous cell monolayer that exhibits related alterations in myo-inositol metabolism and function in diabetic animals. Nontransformed human retinal pigment epithelial (hRPE) cells, which retain their general phenotypic and morphological characteristics during monolayer culture in vitro, were examined for parallel alterations in myo-inositol metabolism and cell function when grown under carefully controlled conditions in medium containing hyperglycemic concentrations of glucose. Exposure of hRPE cells to 20–40 mM glucose produced time- and dose-dependent increases in sorbitol content and decreases in myo-inositol content that were partially blocked by the aldose reductase inhibitor sorbinil. myo-Inositol was taken up by two Na-dependent transport systems, at least one of which was competitively inhibited by glucose. Exposure to 20 mM glucose impaired the ability of hRPE cells to take up human retinal rod outer segments, an important physiological function of these cells. The impairment of rod outer segment uptake by high glucose levels was prevented by an aldose reductase inhibitor or elevated medium myo-inositol that corrected the fall in myo-inositol content. Thus, hRPE cells provide a new in vitro model in which to examine the biochemical-functional interrelationships of the myo-inositol depletion hypothesis.
Effects of IGF-I and Glucose on Protein and Proteoglycan Synthesis by Human Fetal Mesangial Cells in Culture
Authors: Moran Antoinette , Brown David M , Kim Youngki , Klein David J
Published: September 2013

Abnormalities in proteoglycan metabolism have been implicated in the pathogenesis of diabetic nephropathy. Whether hyperglycemia plays a direct role in these events is unknown. To evaluate the effects of high glucose concentrations and insulinlike growth factor I (IGF-I) on kidney proteoglycan and protein metabolism, we incubated quiescent, subconfluent human fetal mesangial cells for 24 h in serum-free media containing either physiological (5.6-mM) or elevated (25-mM) glucose concentrations with or without 1.3 × 10−9 M IGF-I. In the presence of physiological glucose concentrations, IGF-I stimulated incorporation of [3H]leucine into protein and [35S]sulfate or [3H]glucosamine into proteoglycans. High glucose concentrations significantly amplified IGF-I–mediated stimulation of protein synthesis but totally abolished IGF-I–induced proteoglycan synthesis. The hydrodynamic size and proportions of heparan-35SO4 and chondroitin/dermatan-35SO4 proteoglycans in all experimental media were the same. However, high glucose concentrations decreased the iduronic acid content of dermatan-35SO4. In separate experiments, quiescent cells were cultured for 7 days in media supplemented with 2% fetal calf serum. IGF-I had no effect on mesangial cell proliferation, but as cells reached confluence, high glucose concentrations significantly inhibited cell proliferation. This inhibition was not mimicked by isosmolar concentrations of mannitol. After 7 days, uptake of radioactive precursors into proteoglycans and proteins over 24 h was similar under all culture conditions. However, IGF-I decreased the ratio of [35S]sulfate to [3H]glucosamine in proteoglycans and their glycosaminoglycan side chains. This difference persisted in disaccharides derived by chondroitin ABC lyase digestion of dermatan-35SO4. A higher specific activity of [3H]glucosamine without a change in [35S]sulfate uptake or proteoglycan structure indicated that IGF-I treatment of confluent mesangial cells altered the glucosamine pool size. Thus, in subconfluent human mesangial cells, high concentrations of glucose amplified IGF-I–stimulated protein synthesis but abrogated IGF-I–stimulated proteoglycan synthesis, resulting in a relatively proteoglycan-depleted state. This effect was not seen in cells that had been confluent for several days.
Effect of Insulin and Statil on Aldose Reductase Expression in Diabetic Rats
Authors: Ghahary Aziz , Chakrabarti Subrata , Sima Anders A F , Murphy Liam J
Published: September 2013

Tissue accumulation of sorbitol secondary to enhanced polyol-pathway activity is believed to play an important role in the development of diabetic complications. We previously demonstrated sorbitol accumulation, due in part to enhanced expression of aldose reductase (AR) in the diabetic kidney. In this study, we quantitated AR enzyme activity, immunoreactivity, and mRNA in various tissues from nondiabetic and diabetic BB/Wor rats 3 mo after onset of diabetes. In addition, the effects of intensive insulin treatment (3–6 U/day) and the effects of the AR inhibitor Statil (25 mg · kg−1 · day−1) on AR expression were determined. Of 13 tissues examined,AR activity was significantly increased in the lens, kidney, sciatic nerve, skeletal muscle, retina, and spinal cord from diabetic rats compared with age-matched nondiabetic control rats. In most tissues, AR immunoreactivity and AR mRNA were proportionately elevated. Intensive insulin treatment, which normalized blood glucose and glycosylated hemoglobin, significantly reduced AR activity and immunoreactivity. AR mRNA abundance was also reduced in tissues from insulin-treated diabetic rats. Statil treatment had no significant effect on AR immunoreactivity or AR mRNA abundance, although AR activity in tissues from Statil-treated diabetic rats was significantly reduced compared with untreated diabetic rats. These studies demonstrate that the expression of the AR gene is upregulated inmost tissues of the diabetic rat, that insulin treatment reverses this phenomenon, and that AR inhibition has no effect on AR gene expression.
Chronic Undernutrition Accentuates Insulin Deficiency in Rats With Mild Streptozocin-Induced Diabetes
Authors: Rao R Harsha
Published: September 2013

The effect of chronic undernutrition on coexisting diabetes mellitus was studied in pair-fed littermate rats with mild streptozocin-induced diabetes. They were either fed adlibitum or 50% food restricted for 9 wk. Undernourished diabetic rats, in which weight gain was reduced by 40%, had significantly higher glucose intolerance (mean ± SE, fractional rate of glucose disappearance during glucose tolerance test [Kgtt] 1.77 ± 0.16) than diabetic littermates fed ad libitum(2.33 ± 0.21, P < 0.05) or nondiabetic controls (3.8 ± 0.7, P < 0.01). The total area under the insulin response curve was significantly reduced in both groups of diabetic rats, but the undernourished group showed a 45% greater reduction in overall secretion than normally nourished diabetic littermates (21.3 ± 2.7 vs. 39.4 ± 6.3 nM · min in the diabetic group, P < 0.01, and 65.7 ± 6.1 nM · min in controls). There was also a marked reduction in first-phase insulin secretion in the undernourished rats (4.75 ± 0.24 vs. 9.84 ± 1.36 nM, P < 0.05, and 14.3 ± 1.8 nM, P < 0.01, respectively, in normally nourished diabetic littermates and controls). After refeeding, a significant improvement occurred in Kgtt (to 2.67 ± 0.24, P < 0.01) and first-phase insulin secretion (to 9.69 ± 1.65 nM, P < 0.05). The postrefeeding values were not different from those in the normally nourished diabetic littermates, indicating that the effect wasfully reversible and solely attributable to undernutrition rather than to enhanced β-cell cytotoxicity from streptozocin. It is concluded that chronic malnutrition accentuates β-cell dysfunction in rats with mild streptozocin-induced diabetes, indicating that its effects can amplify the deficits induced by another diabetogenic influence. This may be important in elderly diabetic individuals who are often undernourished. It may also contribute to the insulinopenia observed in malnutrition-related diabetes mellitus.
Inhibition of N-Acetylheparosan Deacetylase in Diabetic Rats
Authors: Kofoed-Enevoldsen Allan , Eriksson Ulf J
Published: September 2013

The effects on N-acetylheparosan deacetylase (N-deacetylase) activity exerted by poorly and well-regulated diabetes and variation of genetic background were investigated in insulin-treated streptozocin-induced diabetic rats of two different strains (H and U). N-deacetylase plays a key role in heparan sulfate biosynthesis, because N-deacetylation is a prerequisite for N- and further O-sulfation. Specific activity of the enzyme was reduced by 50% in poorly regulated diabetic rats compared with nondiabetic rats (P< 0.001). The decrease in specific activity was accompanied by a reduction in theestimated KM from 34 ± 3 to 27 ± 4 mg/L (P < 0.001). Optimal insulin treatment, leading to near normalization of blood glucose, preventedreduction in N-deacetylase activity. In rat strain U, however, a 20% reduction was found despite optimal insulin treatment (P = 0.01), and the nondiabetic animals of this strain had reduced N-deacetylaseactivity compared with nondiabetic rats from the H strain. This might suggest a genetic difference between the rat strains in the regulation of the enzyme activity. The diabetes-induced inhibition of N-deacetylase may have an important role in the pathogenesis of nephropathy and vascular complications in human diabetes mellitus.
Sustained Pulsatile Insulin Secretion From Adenomatous Human β-Cells: Synchronous Cycling of Insulin, C-Peptide, and Proinsulin
Authors: Chou Hsu-Fang , Ipp Eli , Bowsher Ronald R , Berman Nancy , Ezrin Calvin , Griffiths Stephanie
Published: September 2013

The endocrine pancreas secretes insulin in a pulsatile fashion. This rhythm is generated at a site within the pancreas, although its precise location has not been determined. With an in vitro system, we tested the possibility that β-cells might generate spontaneous pulsatile insulin secretion in the absence of any external influence. Human insulinoma tissue from five patients was perifused for 7–10 h with RPMI-1640 medium and constant concentrations of glucose (5.5 mM). Insulin, C-peptide, and proinsulin were measured in the effluent collected at 3.3-min intervals. All three peptides demonstrated pulsatility of secretion in a similar, synchronous fashion that was sustained throughout each study. The Clifton cycle detection program demonstrated cycling in all five tumors, with an average period for all tumors of 28, 29, and 26 min for insulin, C-peptide, and proinsulin, respectively. Spectral analysis confirmed the regularity and consistency of the hormonal secretory patterns. Mean hormone concentrations secreted by different tumors varied, but insulin and C-peptide were secreted in a nearly 1:1 ratio. This study demonstrates 1) that β-cells are able to generate spontaneous pulsatile insulin secretory activity, which is independent of innervation or the presenceof other islet cells, and 2) proinsulin secretion from the β-cell also has an inherent pulsatility. The synchrony observed in the cycles of proinsulin andits peptide products confirms their common secretory pathway in the β-cell. We conclude that the β-cell may be the originator of insulin cycling.
Effects of Insulin and myo-Inositol on Embryo Growth and Development During Early Organogenesis in Streptozocin-Induced Diabetic Rats
Authors: Akashi Masaharu , Akazawa Shoichi , Akazawa Mihoko , Trocino Romulo , Hashimoto Masumi , Maeda Yasuo , Yamamoto Hidefumi , Kawasaki Eiji , Takino Hirofumi , Yokota Atsushi , Nagataki Shigenobu
Published: September 2013

We have previously shown that myo-inositol depletion in the embryonic tissue at a critical stage of organogenesis has a crucial role in hyperglycemia-induced embryopathy. This study tested whether myo-inositol depletion in early organogenesis contributes to the pathogenesis of streptozocin-induced diabetic embryopathy. Rats were made diabetic by streptozocin administration before conception, and the diabetic rats were treated with diet supplemented by 2% myo-inositol or insulin from 6 to 11 gestational days during the period of maximum teratological susceptibility. In each group on the 11th gestational day, growth retardation and incidence of malformations were recorded, and myo-inositol and sorbitol content in the embryonic and extraembryonic tissues were examined. In diabetic rats, the myo-inositol content of the embryos was decreased by 36% (P < 0.01) compared with control rats, and there was growth retardation (crown-rump length 3.37 ± 0.04 vs. 3.87 ± 0.03 mm, P < 0.01; somite no. 27.5 ± 0.2 vs. 29.1 ± 0.2, P < 0.01) and a significantly increased incidence of the neural lesions (17.6 vs. 1.9%, P < 0.01). Insulin treatment resulted in near normalization of maternal serum glucose and complete restoration of myo-inositol content in the embryos with significant improvement of the growth retardation (crown-rump length 3.55 ± 0.06 vs. 3.37 ± 0.04 mm, P < 0.05; somite no. 28.2 ± 0.13 vs. 27.5 ± 0.2, P < 0.05) and a significantly lowered incidence of neural lesions (2.5 vs. 17.6%, P < 0.01) compared with those of the untreated diabetic rats. Despite complete restoration of myo-inositol content in the embryos by oral supplementation with myo-inositol of the diabetic rats, the embryos showed no improvement of growth retardation and had partially lowered incidence of neural lesions (9.6 vs. 17.6%, P < 0.05) but still had higher incidence of neural lesions than controls (9.6 vs. 1.9%, P < 0.05). We demonstrated that metabolic improvement of maternal diabetes by insulin treatment could prevent early growth retardation and dysmorphogenesis, whereas correction of only myo-inositol depletion did not. These data suggest that diabetes-induced growth retardation and dysmorphogenesis during early organogenesis may be caused by multifactorial factors and cannot be explained solely by the myo-inositol depletion hypothesis.
Intestinal and Hepatic Cholesterogenesis in Hypercholesterolemic Dyslipidemia of Experimental Diabetes in Dogs
Authors: Kwong Linda K , Feingold Kenneth R , Peric-Golia Ludvic , Le Thanh , Karkas John D , Alberts Alfred W , Wilson Dana E
Published: September 2013

We previously reported that dog diabetes results in hypercholesterolemia and the accumulation of a high-density lipoprotein (HDL) subclass, HDL1. Hypercholesterolemic diabetic rodents exhibit hyperphagia, intestinal hypertrophy, and increased intestinal cholesterol synthesis and absorption; intestinal 3-hydroxy-3-methylglutaryl (HMG) CoA reductase activity is increased, whereas hepatic activity is unchanged or reduced. To determine whether similar mechanisms operate in the hypercholesterolemic diabetic dog, we measured hepatic and intestinal cholesterologenesis. Streptozocin-alloxan–induced diabetic dogs allowed access to food ad libitum were hyperphagic and hypercholesterolemic (10.1 vs. 4.47 mM) but normotriglyceridemic. Plasma HDL1 concentrations were markedly increased. Differences in renal and hepatic function were not statistically significant, except serum alkaline phosphatase, which was elevated 4-fold (P = 0.0003). Urinary mevalonate, an index of whole-body cholesterol synthesis, was increased 6-fold. Intestinal and hepatic weights were both increased, and direct measurements showed crypt and villus thickening. The activity of HMG CoA reductase per gram organ weight was increased 1.7-fold in liver and 2.1-fold in intestine. Calculated whole-organ activity in intestine was nearly twice that in liver. These observations provide strong evidence that intestinal cholesterogenesis is involved in the pathogenesis of hypercholesterolemia in dog diabetes and support the conclusion that increased cholesterol synthesis plays a role in the hypercholesterolemia of diabetes.
HLA-DQB1 Alleles and Absence of Asp 57 as Susceptibility Factors of IDDM in Finland
Authors: Reijonen Helena , Ilonen Jorma , Knip Mikael , Åkerblom Hans K
Published: September 2013

It has been proposed that negatively charged aspartic acid at position 57 of the HLA-DQ β-chain determines resistance to development of insulin-dependent diabetes mellitus (IDDM), whereas genetic susceptibility to IDDM correlates with a neutral amino acid residue. The disease rate is very low in Oriental populations with high frequencies of Asp 57. This raises a question whether the high incidence of IDDM in Finland could be explained by the distribution of this disease marker. In this study, the polymerase chain reaction products of 86 diabetic patients and 115 nondiabetic control subjects were analyzed with seven sequence-specific oligonucleotide probes. Only 25.5% of the diabetic subjects were phenotyped as Asp 57+ compared to 82% of control subjects, which suggests that Asp 57 negativity is a definite risk marker for developing IDDM in Finnish patients. However, the susceptibility conferred by various non-Asp and Asp haplotypes was not equally strong: DQw8 was the most important risk marker and DQw6 the most protective one. The frequency of Asp 57+ DQw4 was similar in diabetic patients and control subjects. The highest genotype-associated relative risk was defined by DQw2/DQw8 heterozygosity (RR 91), whereas it was 13 for non-Asp homozygosity. In the control subjects, the frequency of Asp 57+ phenotypes was higher than in several white populations with lower IDDM incidence figures. We conclude that the disease risk in Finland appears to be most strongly related to specific Asp 57− alleles, although other HLA- or non-HLA–associated genes may also contribute to IDDM susceptibility in this population.
Charge Selectivity of Proteinuria in Type I Diabetes Explored by Ig Subclass Clearance
Authors: Pietravalle Patrizia , Morano Susanna , Cristina Giuseppe , De Rossi Maria Grazia , Mariani Guido , Cotroneo Patrizia , Ghirlanda Giovanni , Clementi Aldo , Andreani Domenico , Di Mario Umberto
Published: September 2013

To investigate the role of protein charge in early diabetic proteinuria, the clearance of proteins differing in charge and/or size (anionic and cationic Igs, albumin) was evaluated in 98 insulin-dependent (type I) diabetic patients selected as a representative sample of the 418 patients attending our clinics. Of the patients, 12.9% were microalbuminuric and 4.8% were macroalbuminuric. Anionic and total IgG clearances were significantly increased in 30.6 and 12.2% of patients and were correlated with duration of disease. Anionic IgG4 clearances were increased in patients (9.2%) with normal IgG excretion, suggesting that charge-selectivity impairment is responsible for protein loss. Anionic Ig clearances were also higher in some patients (14.3%) with normal albumin clearance, probably as a result of different glomerular filtration and/or tubular reabsorption. The anionic-cationic IgG clearance ratio tended to increase in parallel with albumin clearance, but once above macroalbuminuric levels, it tended to fall again, indicating the concomitant presence of size-selectivity loss. The anionic IgG clearance and the anionic-cationic IgG ratio, in addition to albumin excretion, may be valuable in assessing early kidney protein charge-selectivity impairment and better characterizing normoalbuminuric patients and those in the preclinical stage of diabetic nephropathy.
Skeletal Muscle Insulin-Receptor Kinase: Effects of Substrate Inhibition and Diabetes
Authors: Block Nancy E , Komori Katsutoshi , Dutton Sandra L , Robinson Katherine A , Buse Maria G
Published: September 2013

Insulin receptor tyrosine kinase activity solubilized from hind limb muscle of control and streptozocin-induced diabetic (STZ-D) rats (2–3 wk) was studied with the substrates histone H2B and poly glutamic acid–tyrosine (glu-tyr) (4:1). Basal and insulin-stimulated kinase activities were inhibited when high concentrations of either substrate were added before initiation of phosphorylation with ATP. Under these conditions, insulin-stimulated activities of diabetic- and control-derived receptor kinase toward H2B were similar at 0.008 mg/ml H2B. However, higher concentrations of H2B (0.04–1 mg/ml) progressively reduced the ratios of diabetic-derived to control-derived receptor kinase activities to ∼0.5. When inhibition of receptor kinase activities was prevented by allowing maximal autophosphorylation of insulin receptors before addition of H2B, kinase activity of diabetic- and control-derived receptors was similar at all H2B concentrations. Diabetic-derived insulin-receptor tyrosine kinase activity toward poly glu-tyr (4:1) was not significantly different from that of control rats. Under conditions of substrate inhibition (0.4 mg/ml H2B), insulin receptor H2B kinase activity from muscles of rats with severe diabetes (85 mg/kg STZ, 7 days) was significantly decreased, whereas the same activity from rats with moderate diabetes (50 mg/kg STZ, 7 days) was not significantly different from control rats. Insulin receptor α,β dimers were not detectable in muscle preparations from control or diabetic rats. The data suggest that the impairment of muscle-derived insulin-receptor tyrosine kinase activity associated with insulinopenic diabetes reflects, in part, enhanced inhibition by some substrates. If solubilized insulin receptors and the exogenous substrates studied model in vivo events, impaired signaling of the muscle insulin receptor in insulinopenic diabetes may depend on the type and concentration of intracellular tyrosine kinase substrates and the severity of the metabolic derangements.
Radioreceptor Assay for Advanced Glycosylation End Products
Authors: Radoff Steven , Makita Zenji , Vlassara Helen
Published: September 2013

Previous assays for nonenzymatic advanced glycosylation end products (AGEs) formed in tissues and/or circulating in blood are unsatisfactory. Based on our earlier identification of AGE-specific receptors on the macrophagelike tumor cell line RAW 264.7, a new assay system for AGEs has been devised. RAW 264.7 cells were used in competitive radioreceptor assays (RRA) after a 3-day culture in 96-well plates with 1 μCi/ml [3H]glycine. Bovine serum albumin (BSA), modified extensively by incubation with glucose-6-phosphate in vitro to form AGE-BSA, was labeled with 125I and was used as a model ligand at a concn of 10 μg/ml. One unit of AGE was defined as the amount of test protein required to inhibit 50% of the specific binding of [125I]-labeled AGE-BSA to the AGE-receptors of intact RAW 264.7 cells. Nonlabeled AGE-BSA was used as a specific competitor to construct standard curves. The reproducibility of the assay was assessed at AGE levels equivalent to mean, maximum, and minimum levels of sensitivity for assays run on a single day and over an extended period, and the RRA had a reproducibility (coefficient of variation) between 5.9 and 14.7%. Protease hydrolysis of in vitro glycosylated proteins before assay increases the competitive ability of these proteins in proportion to their glycosylation. Little or no AGE cross-reactivity was detected in native BSA, Amadori-BSA, maleylated BSA, formaldehyde-treated BSA, palmitic acid–BSA, and acetylated low-density lipoproteins (acetyl-LDL). Polyanions such as heparin or fucoidan strongly interfere with this receptor binding assay. In addition, with this RRA, we quantitated AGE formed on proteins other than albumin, such as collagen, LDL, and RNase, modified by glucose in vitro.
Comparative Modulations of Insulin Secretion, Pancreatic Insulin Content, and Proinsulin mRNA in Rats: Effects of 50% Pancreatectomy and Dexamethasone Administration
Authors: Orland Matthew J , Permutt M Alan
Published: September 2013

These studies compared measurements of in vivo insulin secretion, insulin stores, and insulin synthesis. Rats were studied at 24 wk of age, either 1 or 20 wk after a sham operation (Sham) or 50% pancreatectomy (Px), reducing β-cell number. By 20 wk after surgery, an adaptation to pancreatectomy was apparent from results of serial glucose tolerance tests, done in a preliminary protocol. Some of the rats also received dexamethasone (ShamDex and PxDex, respectively), imposing insulin resistance. Insulin secretion was assessed with the acute insulin response to arginine under basal (AIRbas) and maximum glucose-potentiated (AIRmax) states. Pancreatic insulin was measured, and insulin synthesis was estimated by measurement of proinsulin mRNA. At 1 wk after surgery, there was no difference among Sham and Px rats in AIRbas, but in the Px rats, expected reductions of AIRmax, pancreatic insulin, and proinsulin mRNA were found. ShamDex rats had a markedly augmented AIRbas and increased AIRmax and proinsulin mRNA. However, pancreatic insulin was reduced both in ShamDex and PxDex rats. At 20 wk after surgery, the predicted adaptation to Px was substantiated by AIRmax and proinsulin mRNA in Px rats not different from those in Sham rats, but pancreatic insulin in the Px rats remained low. AIRbas and proinsulin mRNA were augmented in ShamDex and PxDex rats, but pancreatic insulin was again reduced, and in PxDex rats, low AIRmax and fed hyperglycemia were seen. Linear correlations of AIRbas and AIRmax with proinsulin mRNA were observed over a roughly fourfold range of secretion and synthesis. Greater augmentation of secretion than of synthesis, occurring when conditions stressful to β-cells were imposed, may have produced the reduction of insulin stores seen among ShamDex and PxDex rats and, as a result of the depleted stores, the unexpectedly low AIRmax in PxDex rats seen at 20 wk after surgery. These findings may be pertinent to investigations of insulin secretion and insulin stores accompanying spontaneous diabetes in animals and humans.
Insulin-Receptor cDNA Sequence in NIDDM Patient Homozygous for Insulin-Receptor Gene RFLP
Authors: Kusari Jyotirmoy , Olefsky Jerrold M , Strahl Cathy , McClain Donald A
Published: September 2013

Resistance to insulin action is a well-established feature of non-insulin-dependent diabetes mellitus (NIDDM) and is believed to contribute to the etiology of this condition. A strong genetic contribution to the etiology of NIDDM exists, and we previously identified an insulin-receptor gene restriction-fragment–length polymorphism (RFLP) associated with the NIDDM phenotype. In an attempt to elucidate whether structural defects in the insulin receptor could be a primary cause of insulin resistance in NIDDM, we analyzed the insulin-receptor cDNA sequence in a subject with NIDDM who is also homozygous for this RFLP. The insulin-receptor cDNA was sequenced with the polymerase chain reaction (PCR). mRNA from transformed lymphocytes was reverse transcribed and amplified with five overlapping sets of primers that span the coding sequence of both α- and β-subunits. No difference was found in the predicted amino acid sequence of the subject's insulin receptor compared with the normal insulin receptor. At nucleotide positions 831 and 2247, the subject is heterozygous for silent nucleotide polymorphisms that do not affect the amino acid sequence. Exon 11 encodes a 12–amino acid insert in the α-subunit, which, due to alternate splicing, is not expressed in lymphocyte insulin-receptor mRNA. Consequently, exon 11 was amplified from genomic DNA by PCR; the sequence of exon 11 was found to be normal. In addition, when this patient's transformed lymphocytes were maintained in culture, no abnormalities in insulin binding were observed. We conclude that the insulin resistance seen in this NIDDM subject is not due to a structural alteration in the insulin receptor itself. Consequently, the RFLP for which this patient is homozygous is not associated with a coding-sequence abnormality in the insulin-receptor gene.
Studies on Autoimmunity for Initiation of β-Cell Destruction: VII. Evidence for Antigenic Changes on β-Cells Leading to Autoimmune Destruction of β-Cells in BB Rats
Authors: Ihm Sung-Hee , Lee Ki-Up , Yoon Ji-Won
Published: September 2013

The diabetic syndrome in BioBreeding (BB) rats is believed to result from the destruction of β-cells by autoimmune responses. However, the initial events that cause the autoimmune destruction of β-cells remain largely unknown. This investigation was initiated to see whether there are any antigenic changes on the β-cells from neonatal to adult BB rats that may lead to the autoimmune destruction of β-cells. Pancreatic grafts from neonatal BB rats remained largely intact without insulitis when transplanted into the renal subcapsular space of acutely diabetic BB rats. Similarly transplanted islet grafts from neonatal BB rats were also not subject to autoimmune destruction. In contrast, islet grafts obtained from adult BB rats, which had been treated with silica to prevent insulitis, were rapidly destroyed in diabetic recipients. These results indicate that β-cells from neonatal BB rats are different from β-cells from adult BB rats, at least regarding their recognition by immunologic effectors. Considering our observations and previous information on the initial role of macrophages/dendritic cells in the development of insulitis in BB rats, we suggest that β-cell–specific antigenic changes that precede insulitis may result in the autoimmune destruction of β-cells in BB rats.
Preconception Counseling and Contraception After Gestational Diabetes
Authors: Mølsted-Pedersen Lars , Skouby Sven O , Damm Peter
Published: September 2013

Women with gestational diabetes mellitus (GDM) diagnosed in the period 1978–1984 were followed for on average 6 yr after the index pregnancy. Thirty percent had diabetes mellitus at the follow-up examination, and preliminary results indicate that at least another third will develop diabetes during a subsequent pregnancy. Therefore, family planning and contraceptive guidance should follow the lines for women with pregestational diabetes. When low-dose hormonal contraceptives containing ethinyl estradiol and levonorgestrel were given to women with previous GDM, glucose tolerance and lipoprotein levels remained unchanged during a 6-mo treatment. However, insulin response to oral glucose increased significantly after hormonal intake for 6 mo. A triphasic preparation resulted in a significantly lower insulin response than a low-dose monophasic preparation. However, the results indicate that low-dose oral-contraceptive compounds appear to be safe for women with previous GDM when administered for limited periods. At the follow-up examination, we found no increased risk of developing diabetes in women with previous GDM who used oral contraception. We consider the intrauterine contraceptives (IUD) a safe and effective alternative for women with previous GDM. Of 154 women with GDM, 33% chose IUD, 22% a combination-type oral contraceptive, and 16% barrier methods as their first choice of contraception 2 mo postpartum. We conclude that family planning and qualified contraceptive advice are important in women with previous GDM.
Impact of Maternal Fuels and Nutritional State on Fetal Growth
Authors: Kalkhoff Ronald K
Published: September 2013

Several maternal plasma fuel abnormalities have been described in gestational diabetes mellitus (GDM), and all may contribute to the development of fetal macrosomia, generally because of the surfeit of calories they provide. Elevated maternal plasma glucose and amino acid concentrations represent key disturbances, because they are also well-known fetal pancreatic β-cell secretagogues. Fetal hyperinsulinemia contributes to macrosomia in a special way by selectively accelerating fuel utilization and storage in insulin-sensitive fetal tissues. Maternal obesity intensifies the insulin resistance already present in late pregnancy and probably exaggerates the metabolic abnormalities attending GDM that impact on fetal growth and development. However, the means by which maternal obesity per se promotes the development of heavy babies in nondiabetic pregnancies remains poorly defined. Significant correlations exist between newborn birth weight and the levels of maternal plasma glucose, amino acids, free fatty acids, and triglycerides in diabetic pregnancies. However, the relative influence of each disturbance on fetal birth weight remains controversial and requires more detailed investigation.
Sonographic Measurement of Fetal Humeral Soft Tissue Thickness in Pregnancy Complicated by GDM
Authors: Landon Mark B , Sonek Jiri , Foy Pamela , Hamilton Lisa , Gabbe Steven G
Published: September 2013

Sonographic measurement of fetal humeral soft tissue thickness (STT) was performed in 93 women with gestational diabetes mellitus during the third trimester. STT measurements revealed accelerated growth in large for gestational age infants at 31 wk gestation. This new measurement proved to be the most accurate predictor of excessive fetal size compared with other standard ultrasound parameters (sensitivity 82%, specificity 95%, positive predictive value 90%). Asymmetrical growth was more evident in infants with large STT measurements in utero. Humeral STT measurement may distinguish large fetuses with truncal obesity from those that are symmetrically large, thereby allowing prediction of risk for birth trauma before delivery.
Peptide-Based Radioimmunoassay Specific for GLUT1 Glucose Transporter
Authors: Tsukuda Katsunori , Asano Tomoichiro , Lin Jiann-Liang , Katagiri Hideki , Ishihara Hisamitsu , Takaku Fumimaro , Oka Yoshitomo
Published: September 2013

A radioimmunoassay for the GLUT1 glucose transporter was developed with a synthesized peptide based on the sequence of the cDNA for GLUT1. A peptide corresponding to the COOH-terminal domain of the GLUT1 glucose transporter (Thr-Pro-Glu-Glu-Leu-Phe-His-Pro-Leu-Gly-Ala-Asp-Ser-Gln-Val) was synthesized and conjugated to keyhole limpet hemocyanin through the NH2-terminal of the peptide. An antibody was raised against this complex and affinity purified with the immobilized peptide. A second peptide, with tyrosine residue added to the NH2-terminal of the above peptide, was synthesized and used as a standard and iodinated for preparation of the radioactive ligand. The assay is highly reproducible, sensitive, and specific for the COOH-terminal domain of the GLUT1 glucose transporter. It has no cross-reactivity with the other glucose-transporter isoforms GLUT2 and GLUT4. Furthermore, the results obtained with this radioimmunoassay on the number of glucose transporters in human erythrocytes were in good agreement with previous studies based on cytochalasin B binding, suggesting that this radioimmunoassay is able to quantify the number of glucose transporters. The assay is completed within 4 h and can be used for simultaneous measurement of GLUT1 in many samples. In addition, it can be applied to the measurement of GLUT1 in several types of tissue.
Frequency and Effects of Apolipoprotein E Polymorphism in Mexican-American NIDDM Subjects
Authors: Shriver Mark D , Boerwinkle Eric , Hewett-Emmett David , Hanis Craig L
Published: September 2013

We typed 254 non-insulin-dependent diabetic (NIDDM) Mexican Americans living in Starr County, Texas, for the three common apolipoprotein E (apoE) alleles. Typing was performed via DNA amplification and Hha I restriction. The allele frequencies (ϵ2 = 0.041, ϵ3 = 0.860, ϵ4 = 0.099) were in Hardy-Weinberg equilibrium (χ2 = 0.60, df = 3) and did not differ from a random sample from the same population (χ2 = 0.16, df = 2). Analysis of variance was used to test for mean differences in lipid, lipoprotein, and glucose levels among apoE types. Significant differences among types were detected for low-density lipoprotein cholesterol (LDL-chol; P = 0.042, R2 = 2.6) and β-lipoprotein cholesterol (P = 0.019, R2 = 3.3) levels. Mean LDL-chol in E2/3 individuals was 2.69 mM, E3/3 was 3.26 mM, and E4/3 was 3.36 mM. Mean β-lipoprotein cholesterol in E2/3 individuals was 3.05 mM, E3/3 was 3.64 mM, and E4/3 was 3.67 mM. Based on these results, we conclude that the effects of the apoE polymorphism on lipid profiles and glucose levels are the same in NIDDM subjects as in nondiabetic Mexican Americans and other populations. Other studies investigating the role of apoE polymorphism in diabetic subjects have found increased triglyceride levels in individuals possessing an ϵ2-allele and an increased frequency of the ϵ2-allele in hyperlipidemic diabetic subjects. We found no significant difference in mean triglyceride levels among genotypes. Possible reasons for this discrepancy are discussed, including DNA- versus protein-typing methods.
Lack of Evidence for Improvement in Long-Term Glycemic Control by Pulsatile Insulin Infusion in Streptozocin-Induced Diabetic Baboon
Authors: Weigle David S , Rumbaoa Astor V , Goodner Charles J
Published: September 2013

To assess the potential therapeutic use of pulsatile intravenous insulin delivery, five streptozocin-induced diabetic baboons were treated with alternate 3- to 6-wk periods of pulsatile and continuous insulin infusion. Time-averaged insulin concentrations were matched during two pulsatile administration periods (P1 and P2) and an intervening period of continuous insulin administration (C). There were no significant differences among the overall means of four daily glucose determinations performed during the three periods (P1, 5.7 ± 1 mM; C, 5.6 ± 0.9 mM; P2, 5.3 ± 0.9 mM); the mean M value, a measure of the stability of glycemic control (P1, 4 ± 1.7; C, 3.9 ± 1.8; P2, 3.6 ± 1.5); the percentage of glucose values <2.8 mM (P1, 13 ± 8.5%; C, 14 ± 12%; P2, 13 ± 9.1%); or the glycosylated hemoglobin levels determined at the end of the P1 and C (7.5 ± 3.4 and 6.5 ± 1.8%, respectively [all values are means ± SD]). Fasting hepatic glucose production was suppressed to a similar degree during pulsatile and continuous insulin infusion (P1, 23 ± 3 μmol · kg−1 · min−1; C, 24 ± 8 μmol · kg−1 · min−1). Arterial glucagon levels were similar during pulsatile and continuous insulin infusion, both in the fasting state (84 ± 29 and 84 ± 31 ng/L, respectively) and postprandially (30 ± 14 and 27 ± 12 ng/L, respectively). Pulsatile insulin infusion failed to entrain a corresponding glucagon secretory rhythm. These data suggest that the metabolic consequences of long-term pulsatile and continuous insulin infusion in an animal model of human non-insulin-dependent diabetes are comparable.