bluuebunny/crossref_metadata_2025 huggingface dataset:
GADIA2-combi determination as first-line screening for improved prediction of type 1 diabetes in relatives.
Authors: Dittler J , Seidel D , Schenker M , Ziegler A G
Published: March 2007

A new radiobinding assay for the simultaneous detection of antibodies to GAD and the tyrosine phosphatase IA2 has been recently described in patients with newly diagnosed type 1 diabetes. Here we assessed sensitivity and predictive value of this GADIA2-combi test in first-degree relatives of type 1 diabetic patients compared with islet cell antibody (ICA) and insulin autoantibody (IAA) screening. Of 1,606 relatives, 77 (4.8%) had elevated GADIA2-combi titers above the 99th percentile of 105 nondiabetic control subjects, and results were confirmed by testing these samples for GAD antibody (GADA) and tyrosine phosphatase IA2 antibody (IA2A) in the single antibody test (29 GADA+/IA2A+, 44 GADA+/IA2A-, and 4 IA2A+/GADA-). A further 9 of 1,606 relatives had detectable ICA (1) or IAA (8), but they were negative in the GADIA2-combi assay as well as in the single test for GADA or IA2A. Twenty-four relatives progressed to IDDM within a median follow-up time of 5.6 years (range 0.5-8.2). The sensitivity of antibody determination in relatives with progression to IDDM was 92% for the GADIA2-combi assay, 96% for the combined testing of IAA and GADIA2-combi antibodies, and 83, 67, 67, and 79%, respectively, for GADA, IA2A, IAA, or ICA testing alone. The cumulative life-table risk of antibody-positive relatives was related to GADIA2-combi titers (5-year risk: >50 U, 51% [95% CI 30-73]; >10 to 50 U, 12% [1-24]; <10 U, 0.17% [0-0.5]; P=0.0001) and on the presence of IA2A in addition to GADA (5-year risk: GADA+/IA2A+, 47% [25-68]; GADA+/IA2A-, 15% [2-28]; P=0.006). In those with detectable antibodies, risk was not associated with age (<15 vs. >15 years) or relation to proband (offspring, sibling, parent). Relatives with GADIA2-combi antibodies >10 U and the additional presence of IAA had a slightly higher diabetes risk than relatives without IAA (5-year: IAA+, 46% [23-68]; IAA-, 19% [6-32]; P=0.07). Furthermore, low first-phase insulin release after intravenous glucose tolerance test was associated with risk in relatives with GADIA2-combi antibodies (P=0.01). These results indicate that the GADIA2-combi test is a valuable marker for first-line screening and risk assessment of type 1 diabetes in relatives. It can be used for venous as well as capillary blood samples.
Altered expression and subcellular localization of diacylglycerol-sensitive protein kinase C isoforms in diabetic rat glomerular cells.
Authors: Babazono T , Kapor-Drezgic J , Dlugosz J A , Whiteside C
Published: March 2007

Protein kinase C (PKC) is implicated in the pathogenesis of diabetic nephropathy. This study was designed to identify the expression of diacylglycerol (DAG)-sensitive PKC-alpha, -betaII, -delta, and -epsilon isoforms in normal and diabetic rat glomerular cells and to determine the effects of high glucose and insulin on PKC isoform cellular compartmentalization and PKC activity. Diabetic rats treated with or without insulin and normal rats were examined 2 and 4 weeks after streptozotocin/vehicle injection. Renal cortical tissue immunogold-labeled with anti-PKC-alpha, -betaII, -delta, or -epsilon antibody was visualized by electron microscopy. From isolated glomeruli, total cell lysate and cytosol and membrane fractions were immunoblotted with the same anti-PKC isoform antibodies. PKC activity in isolated glomeruli was measured by 32P-phosphorylation of the epidermal growth factor (EGF)-receptor substrate. Immunogold labeling revealed expression of the four PKC isoforms by glomerular visceral epithelial, endothelial, and mesangial cells of both normal and diabetic rats. Immunoblot analysis of the diabetic rat glomeruli at 2 weeks demonstrated a significant increase in membrane-associated PKC-alpha, -delta, and -epsilon and a significant decrease in membrane PKC-betaII content compared with normal, which were similar at 4 weeks. Insulin treatment normalized membrane PKC isoform contents and caused a significant decrease in the cytosol content of PKC-alpha, -betaII, and -delta and total cellular PKC-alpha compared with normal. Although PKC activity in the cells of diabetic rat glomeruli was increased by 20% compared with normal, the difference did not reach statistical significance. In insulin-treated diabetic rat glomeruli, PKC activity was significantly decreased compared with non-insulin-treated diabetic rat glomeruli. In conclusion, DAG-sensitive PKC-alpha, -betaII, -delta, and -epsilon isoforms are all found in the three major glomerular cell types in rats, and the expression, compartmentalization, and activity are modulated independently by high glucose and insulin.
Antioxidants diminish developmental damage induced by high glucose and cyclooxygenase inhibitors in rat embryos in vitro.
Authors: Wentzel P , Eriksson U J
Published: March 2007

Previous studies have suggested that the metabolism of arachidonic acid and radical oxygen species (ROS) are altered in diabetes and that these disturbances may induce severe embryonic dysmorphogenesis in diabetic pregnancy. We tested this hypothesis by studying whether an inhibition of the rate-limiting enzyme of prostaglandin biosynthesis, cyclooxygenase (COX), caused developmental disturbances analogous to those seen in embryos exposed to high glucose concentration. Whether antioxidants could prevent such developmental alterations was also investigated. Whole embryo culture was used in which day-9 embryos were exposed to high concentrations of glucose, arachidonic acid, prostaglandin (PG)E2, COX inhibitors, and antioxidants for 48 h. Increased glucose concentration (from 10 to 30 mmol/l) caused embryonic dysmorphogenesis, and addition of either 60 pmol/l arachidonic acid or 280 nmol/l PGE2 largely protected the embryo from this maldevelopment. Furthermore, exposure to the COX inhibitors indomethacin (200 micromol/l) or acetylsalicylic acid (700 micromol/l) in 10 mmol/l glucose concentration yielded embryonic dysmorphogenesis similar to that caused by 30 mmol/l glucose. Supplementation of either arachidonic acid or PGE2 to the culture medium with COX inhibitors in low glucose rectified the embryonic development, and PGE2 supplementation also normalized the development of embryos cultured with COX inhibitors in high glucose concentration. Interestingly, the antioxidants superoxide dismutase (SOD) and N-acetylcysteine (NAC) were each able to diminish the dysmorphogenesis induced by the COX inhibitors, at doses previously shown to diminish glucose-induced embryonic damage in the same in vitro culture system. In conclusion, the present study shows that a high glucose concentration disturbs embryonic development and that this disturbance may be partly mediated via altered metabolism of arachidonic acid and ROS in the embryo.
Short-term exposure to tumor necrosis factor-alpha does not affect insulin-stimulated glucose uptake in skeletal muscle.
Authors: Nolte L A , Hansen P A , Chen M M , Schluter J M , Gulve E A , Holloszy J O
Published: March 2007

It has been hypothesized that increased production of tumor necrosis factor-alpha (TNF-alpha) plays a role in causing the insulin resistance associated with obesity. Obesity with insulin resistance is associated with increased production of TNF-alpha by fat cells. Exposure of 3T3-L1 adipocytes to TNF-alpha for 3-4 days makes them insulin resistant. TNF-alpha has also been reported to rapidly (15-60 min) cause insulin resistance, with a decrease in insulin-stimulated tyrosine phosphorylation, in a number of cultured cell lines. Because skeletal muscle is the major tissue responsible for insulin-stimulated glucose disposal, we performed the present study to determine if acute exposure to TNF-alpha causes insulin resistance in muscle. We found that exposure of soleus muscles to 6 nmol/l TNF-alpha for 45 min in vitro had no inhibitory effect on insulin-stimulated tyrosine phosphorylation of the insulin receptor or insulin receptor substrate 1 (IRS-1) or on phosphatidylinositol 3-kinase association with IRS-1. Incubation of epitrochlearis and soleus muscles with 6 nmol/l TNF-alpha for 45 min or 4 h had no effect on insulin-stimulated 2-deoxyglucose (2-DG) uptake. Treatment of epitrochlearis muscles with 2 nmol/l TNF-alpha for 8 h also had no effect on insulin-stimulated 2-DG uptake. We conclude that in contrast to Fao hepatoma cells and 3T3-L1 fibroblasts, skeletal muscle does not become insulin resistant in response to short-term exposure to TNF-alpha.
Human islets of Langerhans express Fas ligand and undergo apoptosis in response to interleukin-1beta and Fas ligation.
Authors: Loweth A C , Williams G T , James R F , Scarpello J H , Morgan N G
Published: March 2007

IDDM results from a progressive loss of pancreatic beta-cells that, in humans, may be triggered by a combination of genetic and environmental factors. Recently, attention has been focused on the hypothesis that the loss of beta-cells is initiated by inappropriate induction of apoptosis. We now demonstrate that human islets of Langerhans undergo apoptosis upon exposure to interleukin-1beta. The cytokine also sharply increases the number of cells that enter apoptosis on treatment with a stimulatory anti-Fas antibody. Western blotting and immunocytochemistry clearly show for the first time that human pancreatic beta-cells normally express Fas ligand. The results suggest that human islet cells are primed to undergo apoptosis by interleukin-1beta and that this involves the close association between cell-surface Fas and its ligand.
Islet amyloid-associated diabetes in obese A(vy)/a mice expressing human islet amyloid polypeptide.
Authors: Soeller W C , Janson J , Hart S E , Parker J C , Carty M D , Stevenson R W , Kreutter D K , Butler P C
Published: March 2007

We have previously shown that hemizygous transgenic mice expressing human islet amyloid polypeptide (hIAPP) in pancreatic beta-cells have no diabetic phenotype, whereas in the homozygous state, they developed severe, early-onset hyperglycemia associated with impaired insulin secretion and beta-cell death. We investigated the possibility that when the hemizygous mice are crossed onto an obese, insulin-resistant strain such as agouti viable yellow (A(vy)/a), they would exhibit a phenotype more akin to human type 2 diabetes. The hIAPP-expressing A(vy) males (TG-Y) displayed fasting hyperglycemia at 90 days of age and by 1 year progressed to severe hyperglycemia relative to their nontransgenic counterparts. Plasma insulin concentrations and pancreatic insulin content dropped 10- to 20-fold, suggesting severe impairment of beta-cell function. Histopathological findings revealed beta-cell degeneration and loss consistent with the drop in the plasma insulin concentration. In addition, large deposits of IAPP amyloid were present in TG-Y islets. We conclude that in transgenic mice expressing hIAPP, insulin resistance can induce overt, slow-onset diabetes associated with islet amyloid and decreased beta-cell mass.
Development of insulin resistance in the JCR:LA-cp rat: role of triacylglycerols and effects of MEDICA 16.
Authors: Russell J C , Shillabeer G , Bar-Tana J , Lau D C , Richardson M , Wenzel L M , Graham S E , Dolphin P J
Published: March 2007

The JCR:LA-cp rat develops an extreme obese/insulin-resistant syndrome such that by 12 weeks of age, there is no longer any insulin-mediated glucose turnover. At 4 weeks of age, obese and lean rats have essentially identical basal and insulin-mediated glucose uptake in skeletal muscle. By 8 weeks of age, however, the obese rats no longer exhibit such intake. Plasma insulin concentrations in the normal fed state show only small increases up to 4 weeks, with a rapid rise to a marked hyperinsulinemia thereafter, with an age at half-development of 5.5 weeks. Plasma triacylglycerol concentrations in fed obese rats are elevated at 3 weeks and rise rapidly thereafter. The triacylglycerol content of skeletal muscle is significantly elevated in the obese rats at 4 weeks of age. Histological examination of Oil Red O-stained muscle tissue and transmission electron microscopy shows the presence of intracellular lipid droplets. Treatment with the potent triacylglycerol-lowering agent MEDICA 16 (beta,beta'-tetramethylhexadecanedioic acid) from 6 weeks of age reduces plasma lipids markedly, but it reduces body weight and insulin resistance only modestly. In contrast, treatment with MEDICA 16 from the time of weaning at 3 weeks of age results in the normalization of food intake and body weight to over 8 weeks of age. The development of hyperinsulinemia is also delayed until 8.5 weeks of age, and insulin levels remain strongly reduced. Plasma triacylglycerol concentrations remain at the same level as in lean rats, and neither an elevated muscle triacylglycerol content nor intracellular lipid droplets are found at 4 weeks of age. The results indicate that insulin resistance develops in the young animals and is not directly due to a genetically determined defect in insulin metabolism. The mechanism of induction instead appears to be related to an exaggerated triacylglycerol metabolism.
Glial reactivity and impaired glutamate metabolism in short-term experimental diabetic retinopathy. Penn State Retina Research Group.
Authors: Lieth E , Barber A J , Xu B , Dice C , Ratz M J , Tanase D , Strother J M
Published: March 2007

The early pathophysiology of diabetic retinopathy and the involvement of neural and vascular malfunction are poorly understood. Glial cells provide structural and metabolic support for retinal neurons and blood vessels, and the cells become reactive in certain injury states. We therefore used the streptozotocin rat model of short-term diabetic retinopathy to study glial reactivity and other glial functions in the retina in the first months after onset of diabetes. With a two-site enzyme-linked immunosorbent assay, we measured the expression of the intermediate filament glial fibrillary acidic protein (GFAP). After 1 month, GFAP was largely unchanged, but within 3 months of the beginning of diabetes, it was markedly induced, by fivefold (P < 0.04). Immunohistochemical staining showed that the GFAP induction occurred both in astrocytes and in Müller cells. Consistent with a glial cell malfunction, the ability of retinas to convert glutamate into glutamine, assayed chromatographically with an isotopic method, was reduced in diabetic rats to 65% of controls (P < 0.01). Furthermore, retinal glutamate, as determined by luminometry, increased by 1.6-fold (P < 0.04) after 3 months of diabetes. Taken together, these findings indicate that glial reactivity and altered glial glutamate metabolism are early pathogenic events that may lead to elevated retinal glutamate during diabetes. These data are the first demonstration of a specific defect in glial cell metabolism in the retina during diabetes. These findings suggest a novel understanding of the mechanism of neural degeneration in the retina during diabetes, involving early and possibly persistent glutamate excitotoxicity.
Sib-pair linkage analysis for susceptibility genes for microvascular complications among Pima Indians with type 2 diabetes. Pima Diabetes Genes Group.
Authors: Imperatore G , Hanson R L , Pettitt D J , Kobes S , Bennett P H , Knowler W C
Published: March 2007

The aim of this study was to identify loci influencing susceptibility to microvascular complications (nephropathy and retinopathy) in Pima Indians with type 2 diabetes. Affected sib-pair linkage analyses were performed on 98 diabetic sibling pairs with nephropathy in both members and on 103 sibling pairs with retinopathy in both members. Four chromosomal regions with some evidence of linkage (P < 0.01; logarithm of odds [LOD] >1.18) with nephropathy were identified. The strongest evidence for linkage with nephropathy was on chromosome 7, where two adjacent markers, D7S500 and D7S1804, were linked both by two-point analysis (LOD = 2.73 and LOD = 2.28; respectively) and by multipoint analysis (LOD = 2.04). Additional loci potentially linked to nephropathy were found on chromosome 3, near D3S3053 (multipoint LOD = 1.48); on chromosome 9, near D9S910 (multipoint LOD = 1.12) and D9S302 (two-point LOD = 1.28); and on chromosome 20, near D20S115 (multipoint LOD = 1.83) and GATA65E01 (two-point LOD = 1.89). Multipoint analyses showed two regions with some evidence for linkage to retinopathy: chromosome 3 between D3S3053 and D3S2427 (LOD = 1.36), and chromosome 9 between D9S1120 and D9S910 (LOD = 1.46). These linkage analyses suggest that a genetic element on chromosome 7 and possibly one on chromosome 20 influence susceptibility to diabetic nephropathy but not retinopathy. Genetic elements on chromosome 3 and 9 may determine susceptibility to both these complications. These loci could presumably influence susceptibility to the complications by influencing the microvasculature directly, by influencing the severity of hyperglycemia, or by other unknown mechanisms.
A functional analysis of the role of IGF2 in IDDM2-encoded susceptibility to type 1 diabetes.
Authors: Vafiadis P , Grabs R , Goodyer C G , Colle E , Polychronakos C
Published: March 2007

Genetic studies have identified a number of loci demonstrating linkage to type 1 diabetes. One of the largest single contributors to genetic susceptibility, after the major histocompatability complex, is the IDDM2 locus, which maps to a nontranscribed variable number of tandem repeats (VNTR) minisatellite upstream of the insulin (INS) and insulin-like growth factor 2 (IGF2) genes. In a progression from population to functional studies, recent reports have shown that VNTR susceptibility alleles (class I) have different transcriptional effects on INS than protective VNTR alleles (class III) in thymus and pancreas, two tissues important in the pathogenesis of the disease. Similar VNTR transcriptional effects on IGF2 have also been proposed as a mechanism by which the IDDM2 locus confers susceptibility in addition to, or instead of, effects on INS. We evaluated this hypothesis by comparing IGF2 expression levels from chromosomes with the protective class III alleles to those with class I alleles in tissues relevant to type 1 diabetes pathogenesis. In thymus, class III alleles were associated with an IGF2 mRNA level of 4.7 +/- 0.9 (mean +/- SE, arbitrary units, n = 12) compared with 4.7 +/- 1.3 for class I alleles (n = 17). The same absence of a significant difference was found in pancreas, where class III alleles were associated with a level of 28.4 +/- 4.2 (n = 7) and class I alleles with a level of 29.5 +/- 5.2 (n = 6). There was a significant correlation between fetal age and IGF2 in both tissues, but fetal ages were not different in the genotype groups compared. We therefore did not detect any significant difference in IGF2 mRNA levels associated with the protective class of VNTR alleles as compared with the predisposing class. This is evidence against the hypotheses that have suggested IGF2 is a mediator of IDDM2-encoded susceptibility and corroborates previous studies suggesting insulin is the gene involved.
Development and regulation of glucose-6-phosphatase gene expression in rat liver, intestine, and kidney: in vivo and in vitro studies in cultured fetal hepatocytes.
Authors: Chatelain F , Pégorier J P , Minassian C , Bruni N , Tarpin S , Girard J , Mithieux G
Published: March 2007

The mRNA and the activity of glucose-6-phosphatase (Glc-6-Pase) were present in the liver, kidney, and small intestine of 15-day-old suckling rats, but were absent from the stomach, colon, lung, white and brown adipose tissues, muscle, heart, brain, and spleen. The mRNA encoding Glc-6-Pase was present in the liver of 21-day-old fetal rats and increased markedly immediately after birth. From 5 days after birth to the end of the suckling period, it returned to 50% of the level found in the liver of 48-h starved adult rats. When rats were weaned at 21 days onto a high-carbohydrate, low-fat (HCLF) diet, the concentration of liver Glc-6-Pase mRNA was markedly increased. In the fetal rat jejunum, the activity and mRNA of Glc-6-Pase were very low. It increased during the 5 days after birth and then declined to reach very low levels. Neither mRNA nor activity of Glc-6-Pase was present in the fetal kidney. They appeared and increased slowly during the suckling period to reach maximal levels 15 days after birth and then remained constant. Weaning onto the HCLF diet did not change the Glc-6-Pase gene expression, neither in the jejunum nor in the kidney. The regulation of Glc-6-Pase gene expression by hormones and nutrients was studied in cultured hepatocytes from 20-day-old rat fetuses. Bt2cAMP stimulated the Glc-6-Pase gene expression in a dose-dependent manner. This probably resulted from an increased gene transcription since the half-life of the transcript was not affected by dibutyryl cAMP (Bt2cAMP). The Bt2cAMP-induced Glc-6-Pase mRNA accumulation was antagonized by insulin in a dose-dependent manner. Long-chain fatty acids (LCFAs), but not medium-chain fatty acids, induced the accumulation of Glc-6-Pase mRNA and the stabilization of the transcript. The peroxisome proliferator, clofibrate, induced a threefold increase in Glc-6-Pase mRNA concentration. Both stimulation of Glc-6-Pase mRNA by LCFAs and clofibrate were inhibited by insulin. Increasing concentrations of glucose (from 0 to 20 mmol/l) did not affect the Bt2cAMP-induced Glc-6-Pase gene expression. By contrast, high glucose concentration (25 mmol/l) markedly induced the Glc-6-Pase gene expression in fed adult rat hepatocytes. The difference in the response to glucose between fetal and adult rat hepatocytes is discussed. We conclude that the rapid increase in hepatic Glc-6-Pase mRNA levels that accompanies the fetal-to-neonatal transition in the rat is triggered by the reciprocal change in circulating insulin and LCFA concentrations, coupled to the rise in liver cAMP concentration.
Induction of GAD65-specific regulatory T-cells inhibits ongoing autoimmune diabetes in nonobese diabetic mice.
Authors: Tisch R , Liblau R S , Yang X D , Liblau P , McDevitt H O
Published: March 2007

IDDM is a T-cell-mediated autoimmune disease in which the insulin-producing beta-cells are destroyed. The disease process is complex, involving the recognition of several beta-cell autoantigens. One of these, GAD65, appears to have a critical and not fully defined role in IDDM in humans and in the NOD mouse. We provide evidence that an ongoing diabetogenic response in NOD mice can be suppressed after intravenous administration of GAD65, but not by other beta-cell autoantigens. Furthermore, suppression of the diabetogenic response is mediated by the induction of GAD65-specific CD4+ regulatory T-cells. Finally, cytokine analysis indicates that these CD4+ regulatory T-cells have a T-helper 2 phenotype.
Atypical protein kinase C isozyme zeta mediates carbachol-stimulated insulin secretion in RINm5F cells.
Authors: Tang S H , Sharp G W
Published: March 2007

Carbachol-stimulated insulin release in the RINm5F cell is associated with elevation of the cytosolic Ca2+ concentration ([Ca2+]i) through mobilization of Ca2+ from thapsigargin-sensitive intracellular stores and with the generation of diacylglycerol (DAG). Thus carbachol activates phospholipase C, and this was thought to be the means by which it stimulates insulin secretion. However, when the elevation of [Ca2+]i was blocked by thapsigargin, the effect of carbachol to stimulate insulin release was unchanged. Thus the effect of carbachol to increase [Ca2+]i was dissociated from the stimulation of release. When the role of protein kinase C (PKC) was examined, carbachol-stimulated insulin release was found to be unaffected by phorbol ester-induced downregulation of PKC, using 12-O-tetradecanoylphorbol-13-acetate (TPA), and by the PKC inhibitors staurosporine, bisindolylmaleimide, and 1-O-hexadecyl-2-O-methylglycerol (AMG-C16). These treatments abolished the stimulation of release by TPA. Thus the carbachol activation of PKC appeared also to be dissociated from the stimulation of insulin release. However, when the activation of several different PKC isozymes was studied, an atypical PKC isozyme, zeta, was found to be translocated by carbachol. By Western blotting analysis, carbachol selectively translocated the conventional PKC isozymes alpha and beta (the activation of which is dependent on Ca2+ and DAG) from the cytosol to the membrane. Carbachol also translocated the atypical PKC isozyme zeta, which is insensitive to Ca2+, DAG, and phorbol esters. The PKC inhibitors staurosporine, bisindolylmaleimide, and AMG-C16 blocked the stimulated translocation of PKC-alpha and -beta, but not that of PKC-zeta. Prolonged treatment of the cells with TPA downregulated PKC-alpha and -beta, but not PKC-zeta. Under all these conditions, carbachol-stimulated insulin release was unaffected. However, a pseudosubstrate peptide inhibitor specific for PKC-zeta inhibited the translocation of PKC-zeta and 70% of the carbachol-stimulated insulin secretion. The data indicate that carbachol-stimulated insulin release in RINm5F cells is mediated to a large degree by the activation of the atypical PKC isozyme zeta.
Arteriolar reactivity in conscious diabetic rats: influence of aminoguanidine treatment.
Authors: Crijns F R , Struijker Boudier H A , Wolffenbuttel B H
Published: March 2007

The effect of 6 weeks' streptozotocin (STZ)-induced (70 mg/kg) diabetes and aminoguanidine (AG) treatment (50 mg/kg s.c. or 250-750 mg/l given in drinking water) on arteriolar reactivity to vasoactive substances was investigated in conscious rats. Studies were performed in untreated control rats (n = 13), STZ-induced diabetic rats (n = 11), AG-treated control rats (n = 12), and AG-treated diabetic rats (n = 12). Rats were provided with a dorsal microcirculatory chamber that allowed intravital microscopy of striated muscle arterioles of varying diameter (A1, large; A2, intermediate; and A3, small arterioles) in conscious animals. The mean arterial pressure (MAP) and arteriolar diameter responses to intravenous infusion of the following drugs were examined: the endothelium-dependent vasodilator acetylcholine (ACh; 3, 10, and 30 microg x kg(-1) x min(-1)), the potassium-channel opener levcromakalim (LC; 30 microg/kg), and the vasoconstrictor agents ANG II (0.1 and 0.3 microg x kg(-1) x min(-1)) and norepinephrine (NE; 0.2, 0.6, and 2.0 microg x kg(-1) x min(-1)). Baseline MAP was lower in both diabetic groups versus the nondiabetic groups (P < 0.05). AG treatment had no influence on baseline MAP. The absolute change in MAP after drug infusion tended to be lower in the diabetic rats than in their nondiabetic littermates. Arteriolar vasodilatory responses to ACh and LC were attenuated in the diabetic animals (1 +/- 7 vs. 19 +/- 7% [P < 0.05] and 7 +/- 3 vs. 34 +/- 8% [P < 0.01] in A2, respectively). AG treatment of diabetic animals did not prevent the development of this disturbance. Vasoconstrictor responses were not influenced by the diabetic state. In the intermediate arterioles of AG-treated control rats, a hyperresponse was observed after ANG II infusion (-10 +/- 2 vs. -2 +/- 2%; P < 0.05) and a hyporesponse was observed after ACh and LC infusion (2 +/- 3 and 15 +/- 6%, respectively; P < 0.05 vs. untreated control rats). These data indicate that 6 weeks of experimental diabetes is associated with a decreased endothelium-dependent and -independent vasodilatation. AG treatment had no beneficial effect on this disturbance.
Insulin increases endothelin-1-evoked intracellular free calcium responses by increased ET(A) receptor expression in rat aortic smooth muscle cells.
Authors: Hopfner R L , Hasnadka R V , Wilson T W , McNeill J R , Gopalakrishnan V
Published: March 2007

While insulin is known to promote vascular smooth muscle (VSM) relaxation, it also enhances endothelin-1 (ET-1) secretion and action in conditions such as NIDDM and hypertension. We examined the effect of insulin pretreatment on intracellular free calcium ([Ca2+]i) responses to ET-1 in cultured aortic smooth muscle cells (ASMCs) isolated from Sprague-Dawley (SD) rats and measured ET(A) receptor characteristics and ET-1-evoked tension responses in aorta obtained from insulin-resistant, hyperinsulinemic Zucker-obese (ZO) and control Zucker-lean (ZL) rats. Pretreatment of rat ASMCs with insulin (10 nmol/l for 24 h) failed to affect basal [Ca2+]i levels but led to a significant increase in peak [Ca2+]i response (1.7-fold; P < 0.01) to ET-1. The responses to IRL-1620 (an ET(B) selective agonist), ANG II, and vasopressin remained unaffected. ET-1-evoked peak [Ca2+]i responses were significantly attenuated by the inclusion of the ET(A) antagonist, BQ123, in both groups. The ET(B) antagonist, BQ788, abolished [Ca2+]i responses to IRL-1620 but failed to affect the exaggerated [Ca2+]i responses to ET-1. Saturation binding studies revealed a twofold increase (P < 0.01) in maximal number of binding sites labeled by 125I-labeled ET-1 in insulin-pretreated cells and no significant differences in sites labeled by 125I-labeled IRL-1620 between control and treatment groups. Northern blot analysis revealed an increase in ET(A) mRNA levels after insulin pretreatment for 20 h, an effect that was blocked by genistein, actinomycin D, and cycloheximide. Maximal tension development to ET-1 was significantly greater (P < 0.01), and microsomal binding studies using [3H]BQ-123 revealed a twofold higher number of ET(A) specific binding sites (P < 0.01) in aorta from ZO rats compared with that of ZL rats. These data suggest that insulin exaggerates ET-1-evoked peak [Ca2+]i responses via increased vascular ET(A) receptor expression, which may contribute to enhanced vasoconstriction observed in hyperinsulinemic states.
Measurements of oxygen tension in native and transplanted rat pancreatic islets.
Authors: Carlsson P O , Liss P , Andersson A , Jansson L
Published: March 2007

This study was performed to measure the oxygen tension before and after revascularization of pancreatic islets transplanted beneath the renal capsule and to investigate to what extent this was affected by acute and chronic hyperglycemia. In addition, the oxygen tension in islets within the pancreas was determined. PO2 was measured with a modified Clark electrode (tip 2-6 microm o.d.). Within native pancreatic islets, the mean PO2 was higher (31-37 mmHg) than within the exocrine pancreas (20-23 mmHg). The mean oxygen tension in the transplanted islets the day after implantation was half of that recorded in native islets (14-19 mmHg) and did not differ between normoglycemic and diabetic recipients. At 1 month after transplantation, when revascularization had occurred, the mean PO2 in the islet grafts was 9-15 mmHgf in normoglycemic animals but was lower (6-8 mmHg) in diabetic animals, whereas the blood perfusion of the transplants, as measured with laser-Doppler flowmetry (probe diameter 0.45 mm), was similar in both groups. The mean oxygen tension in the superficial renal cortex surrounding the implanted islets was similar in all groups and remained stable at 13-21 mmHg. Intravenous administration of D-glucose (1 g/kg) did not affect the oxygen tension in any of the investigated tissues. We conclude that the mean PO2 in islets implanted under the renal capsule is markedly lower than in native islets, not only in the immediate posttransplantation period but also 1 month after implantation, i.e., when revascularization has occurred. Furthermore, persistent hyperglycemia in the recipient leads to a further decrease in graft oxygen tension. To what extent this may contribute to islet graft failure is at present unknown.
Sex difference in resistance to dexamethasone-induced apoptosis in NOD mice: treatment with 1,25(OH)2D3 restores defect.
Authors: Casteels K M , Gysemans C A , Waer M , Bouillon R , Laureys J M , Depovere J , Mathieu C
Published: March 2007

The NOD mouse, a model for type 1 diabetes, is characterized by resistance to apoptosis in immunocytes. The aim of this study was to investigate a link between apoptosis in NOD thymocytes and autoimmunity. First, we demonstrated that the sexual dimorphism in diabetes incidence in NOD mice (females are more diabetes-prone than males) is reflected by differences in apoptosis. Apoptosis in NOD thymocytes, 24 h after dexamethasone, was decreased in both sexes compared with C57B1/6, but it was lower in female mice (26 +/- 2%) than in male mice (50 +/- 3%, P < 0.001). Further, we demonstrated that sex hormones themselves play a central role in this difference, since castration of NOD male mice, which increases diabetes incidence, decreased apoptosis levels (32 +/- 2%), while treatment of NOD female mice with dihydrotestosterone, which protects against diabetes, restored apoptosis to male levels (42 +/- 1.5%). Finally, we demonstrated that 1,25-dihydroxyvitamin D3, a steroid hormone that prevents diabetes in NOD mice, restored apoptosis levels to C57B1/6 reference levels. This improved apoptosis was seen in male (68 +/- 1 vs. 50 +/- 3% in untreated NOD mice, P < 0.001) but especially in female NOD mice (51 +/- 5 vs. 26 +/- 2% in untreated NOD mice, P < 0.001). Fluorescence-activated cell sorter analysis of thymocyte subsets revealed marked differences, especially in CD4+CD8+ and CD4+ cells. We conclude that the sexual dimorphism in diabetes incidence in NOD mice is paralleled by a dimorphism in resistance to apoptotic signals in NOD thymocytes. This resistance to apoptosis is driven by sex hormones and is corrected by 1,25-dihydroxyvitamin D3.
Peptones stimulate both the secretion of the incretin hormone glucagon-like peptide 1 and the transcription of the proglucagon gene.
Authors: Cordier-Bussat M , Bernard C , Levenez F , Klages N , Laser-Ritz B , Philippe J , Chayvialle J A , Cuber J C
Published: March 2007

Truncated glucagon-like peptide (GLP)-1 is a potent incretin. Its synthesis and secretion are modulated by food, but the influence of individual nutrients remains to be established. The hypothesis that protein hydrolysates (peptones) can directly regulate both GLP-1 secretion and proglucagon (PG) gene transcription was tested in this study, ex vivo in the isolated vascularly perfused rat intestine and in vitro in the murine enteroendocrine cell line STC-1. Peptones were albumin egg hydrolysate (AEH) and meat hydrolysate (MH). We demonstrate in these two models that peptones dose-dependently stimulate GLP-1 release, whereas isocaloric quantities of bovine serum albumin or of an amino acid mixture had no stimulatory effect. A strong and rapid increase of PG RNA level was observed in STC-1 cells treated with peptones (14-fold and 7-fold increase after 4 h of incubation with 3% wt/vol MH and AEH, respectively). Peptones also increased the PG RNA level in the colonic PG-expressing cell line GLUTag. In contrast, peptones did not modify the PG RNA level in two pancreatic glucagon-producing cell lines, namely, the RINm5F and INR1G9 cells. The peptone effect in STC-1 cells was completely abolished by blocking transcription before MH treatment. The stability of proglugacon transcripts was not modified by MH treatment, but nascent transcripts were more abundant in STC-1 cells preincubated with MH. Finally, MH treatment strongly stimulated (15-fold stimulation) the transcriptional activity of two PG gene promoter fragments (-1100 and -350 base pair) linked to the CAT reporter gene transiently transfected in STC-1 cells. Overall, peptones evoke an as yet undescribed release of GLP-1 when brought into contact with native intestinal L-cells or with STC-1 enteroendocrine cells. The increased transcription of the glucagon gene in the latter system suggests an important role of protein hydrolysates in the control of not only the secretion but also the synthesis of the incretin hormone.
Glucose augmentation of mastoparan-stimulated insulin secretion in rat and human pancreatic islets.
Authors: Straub S G , James R F , Dunne M J , Sharp G W
Published: March 2007

Mastoparan, a tetradecapeptide component of wasp venom, activates heterotrimeric G-proteins and stimulates exocytosis in several cell types, including the pancreatic beta-cell. In this study, its effects on insulin secretion were assessed in both rat and human pancreatic islets, along with the ability of glucose and alpha-ketoisocaproate (alpha-KIC) to augment mastoparan-stimulated release. In Ca2+-free Krebs-Ringer bicarbonate buffer containing 2.8 mmol/l glucose, 20 micromol/l mastoparan stimulated insulin secretion 12- and 14-fold in rat and human islets, respectively. The inactive analog mastoparan-17 had no effect on release. Under the same Ca2+-free conditions, 11.1 mmol/l glucose had no effect on insulin release alone, but augmented mastoparan-stimulated release by 74% in both rat and human islets. Stimulation of release by mastoparan and augmentation of release by glucose were unaffected by treatment with pertussis toxin. The effect of cellular GTP depletion on the mastoparan stimulation of release and augmentation by alpha-KIC was studied by culturing rat islets in the presence of 25 microg/ml mycophenolic acid for 20 h. In the control islets, alpha-KIC augmented mastoparan-stimulated insulin release by 80%. In the GTP-depleted rat islets, mastoparan-stimulated insulin release was not changed, while the augmentation by alpha-KIC was eliminated. Mannoheptulose completely blocked the augmentation by glucose. In conclusion, mastoparan stimulates insulin release by activation of a signal transduction pathway that can be augmented by nutrients such as glucose and alpha-KIC. Nutrient augmentation of this pathway is heavily dependent on GTP.
Long-term exposure of beta-INS cells to high glucose concentrations increases anaplerosis, lipogenesis, and lipogenic gene expression.
Authors: Roche E , Farfari S , Witters L A , Assimacopoulos-Jeannet F , Thumelin S , Brun T , Corkey B E , Saha A K , Prentki M
Published: March 2007

Chronic exposure of pancreatic beta-cells to high glucose has pleiotropic action on beta-cell function. In particular, it induces key glycolytic genes, promotes glycogen deposition, and causes beta-cell proliferation and altered insulin secretion characterized by sensitization to low glucose. Postglycolytic events, in particular, anaplerosis and lipid signaling, are thought to be implicated in beta-cell activation by glucose. To understand the biochemical nature of the beta-cell adaptive process to hyperglycemia, we studied the regulation by glucose of lipogenic genes in the beta-cell line INS-1. A 3-day exposure of cells to elevated glucose (5-25 mmol/l) increased the enzymatic activities of fatty acid synthase 3-fold, acetyl-CoA carboxylase 30-fold, and malic enzyme 1.3-fold. Pyruvate carboxylase and citrate lyase expression remained constant. Similar observations were made at the protein and mRNA levels except for malic enzyme mRNA, which did not vary. Metabolic gene expression changes were associated with chronically elevated levels of citrate, malate, malonyl-CoA, and conversion of glucose carbon into lipids, even in cells that were subsequently exposed to low glucose. Similarly, fatty acid oxidation was suppressed and phospholipid and triglyceride synthesis was enhanced independently of the external glucose concentration in cells preexposed to high glucose. The results suggest that a coordinated induction of glycolytic and lipogenic genes in conjunction with glycogen and triglyceride deposition, as well as increased anaplerosis and altered lipid partitioning, contribute to the adaptive process to hyperglycemia and glucose sensitization of the beta-cell.
Regulation of skeletal muscle hexokinase II by insulin in nondiabetic and NIDDM subjects.
Authors: Kruszynska Y T , Mulford M I , Baloga J , Yu J G , Olefsky J M
Published: March 2007

Impaired muscle glucose phosphorylation to glucose-6-phosphate by hexokinases (HKs)-I and -II may contribute to insulin resistance in NIDDM and obesity. HK-II expression is regulated by insulin. We tested the hypothesis that basal and insulin-stimulated expression of HK-II is decreased in NIDDM and obese subjects. Skeletal muscle HK-I and HK-II activities were measured in seven lean and six obese normal subjects and eight patients with NIDDM before and at 3 and 5 h of a hyperinsulinemic (80 mU x m(-2) x min(-1)) euglycemic clamp. To assess whether changes in HK-II expression seen during a glucose clamp are likely to be physiologically relevant, we also measured HK-I and HK-II activity in 10 lean normal subjects before and after a high-carbohydrate meal. After an overnight fast, total HK, HK-I, and HK-II activities were similar in lean and obese control subjects; but HK-II was lower in NIDDM patients than in lean subjects (1.42 +/- 0.16 [SE] vs. 2.33 +/- 0.24 nmol x min(-1) x mg(-1) molecular weight, P < 0.05) and accounted for a lower proportion of total HK (33 +/- 3 vs. 47 +/- 3%, P < 0.025). HK-II (but not HK-I) activity increased during the clamp in lean and obese subjects by 34 and 36% after 3 h and by 14 and 22% after 5 h of hyperinsulinemia; no increase was found in the NIDDM patients. In the lean subjects, muscle HK-II activity also increased by 15% 4 h after the meal, from 2.47 +/- 0.19 basally to 2.86 +/- 0.28 nmol x min(-1) x mg(-1) protein (P < 0.05). During the clamps, muscle HK-II activity correlated with muscle citrate synthase activity in the normal subjects (r = 0.58, P < 0.05) but not in the NIDDM patients. A weak relationship was noted between muscle HK-II activity and glucose disposal rate at the end of the clamp when all three groups were combined (r = 0.49, P < 0.05). In summary, NIDDM patients have lower muscle HK-II activity basally and do not increase the activity of this enzyme in response to a 5-h insulin stimulus. This defect may contribute to their insulin resistance. In nondiabetic obese subjects, muscle HK-II expression and its regulation by insulin are normal.
Aminoguanidine inhibits reactive oxygen species formation, lipid peroxidation, and oxidant-induced apoptosis.
Authors: Giardino I , Fard A K , Hatchell D L , Brownlee M
Published: March 2007

Aminoguanidine (AG) treatment, like nerve growth factor (NGF) treatment, prevents diabetes-induced apoptosis of retinal Müller cells in the rat eye, but the mechanism involved is unknown. In this study, the effects of preincubation with AG on oxidant-induced apoptosis, oxidant-induced intracellular reactive oxygen species (ROS) production, and lipid peroxidation were determined in rat retinal Müller cells and compared with the effects of NGF, a protein that protects neuronal cells from oxidative stress. The effect of AG on rabbit vitreous lipid peroxide levels was also determined. After exposure to increasing concentrations of H2O2, there was a corresponding increase in the percentage of apoptotic Müller cells. Preincubation with AG for 48 h completely inhibited oxidant-induced apoptosis in response to 10 micromol/l H2O2 (+AG 0 vs. 10 micromol/l, NS), and reduced the percentage of apoptotic cells in response to 50 micromol/l H2O2 by 50% (+AG vs. -AG, P < 0.01). Longer preincubation did not increase the antiapoptotic effect of AG. The effect of AG was dose-dependent. Similar results were obtained after preincubation with NGF. Both AG and NGF preincubation prevented the twofold increase in oxidant-induced lipid peroxides. The fivefold increase in oxidant-induced ROS production was decreased 100% by NGF, but only 61% by AG preincubation. The twofold increase in vitreous lipid peroxide level in diabetic rabbits was completely prevented by AG treatment. AG reduced H2O2-induced benzoate hydroxylation in a dose-dependent manner. Intracellular glutathione content was unchanged. These data demonstrate that AG can act as an antioxidant in vivo, quenching hydroxyl radicals and lipid peroxidation in cells and tissues and preventing oxidant-induced apoptosis.
Diabetic patients produce an increase in coronary sinus endothelin 1 after coronary artery bypass grafting.
Authors: Fogelson B G , Nawas S I , Vigneswaran W T , Ferguson J L , Law W R , Sharma A C
Published: March 2007

Diabetes is associated with altered vascular responses, and diabetic patients demonstrate increased morbidity and mortality after coronary artery bypass grafting (CABG). We tested whether endothelin (ET)-1 levels in this patient population differed from those in nondiabetic subjects after CABG. Of 14 consecutive patients who underwent CABG by the same surgeon, 7 had type 2 diabetes and 7 were nondiabetic. The two groups did not differ significantly in preoperative ejection fraction, number of vessels bypassed, cross-clamp time, or Parsonnet's score. Coronary sinus blood samples were obtained before cardioplegic arrest and then obtained at 1 and 15 min after each of two reperfusion periods: reperfusion A (native coronary perfusion plus the left internal mammary artery), reperfusion B (saphenous vein graft perfusion). ET-1 was significantly increased at all reperfusion time points in diabetic patients compared with nondiabetic patients. In diabetic patients, reperfusion after CABG can trigger the release of ET-1, which may be a contributing factor in the increased cardiac morbidity seen in this patient population.
Major susceptibility locus for nephropathy in type 1 diabetes on chromosome 3q: results of novel discordant sib-pair analysis.
Authors: Moczulski D K , Rogus J J , Antonellis A , Warram J H , Krolewski A S
Published: March 2007

Diabetic nephropathy (DN) clusters in families with type 1 diabetes and the degree of clustering suggests that a major gene having a common disease allele may be responsible. To investigate the chromosomal regions containing genes for the renin-angiotensin system, we performed a linkage study using pairs of siblings with type 1 diabetes who were discordant for DN. Theoretical considerations supported by simulation studies indicated that such discordant pairs, rather than the usual concordant pairs, would be more effective in detecting a major susceptibility gene for DN. We applied this novel strategy to test for linkage between DN and chromosomal regions containing genes for the ACE, angiotensinogen (AGT), and angiotensin II type 1 receptor (AT1). Two polymorphic markers were genotyped in the vicinity of each of the three loci in 66 discordant sib pairs and were analyzed with multipoint methods. The regions containing ACE and AGT loci were not linked with DN, while the region containing the AT1 locus showed linkage with DN. As a result of these positive findings, eight additional polymorphic markers spanning a 63-cM region around AT1 locus were genotyped. Linkage was demonstrated between DN and a 20-cM region that includes AT1 (P = 7.7 x 10(-5)), an obvious candidate gene for DN. To investigate whether AT1 could account for the observed linkage, we sequenced all exons, splicing junctions, and the promoter region and examined the identified polymorphisms/mutations for association with DN using the transmission disequilibrium test. Four new polymorphisms in the gene were found, but neither these nor previously described polymorphisms were associated with DN. Thus, while our study does not implicate AT1 itself in the etiology of DN, it provides very strong evidence that a 20-cM region around AT1 contains a major locus for susceptibility to DN.
Dominance of cyclooxygenase-2 in the regulation of pancreatic islet prostaglandin synthesis.
Authors: Robertson R P
Published: March 2007

Dramatic, scientifically important discoveries in prostaglandin (PG) pharmacology and physiology have taken place over the past decade. Chief among these discoveries is the identification of two separate forms of cyclooxygenase (COX), a constitutive and an inducible form, both of which exist in most tissues. The pancreatic islet is an exception to this rule because it continually and dominantly expresses the inducible form, COX-2. It has also been learned that nonsteroidal anti-inflammatory drugs affect the two forms of COX with different potencies, a finding with far-reaching clinical implications. An equally important finding is that PGE2, which is known to negatively modulate glucose-induced insulin secretion, has at least four different subtypes of receptors with different mechanisms of action and metabolic consequences. These recent changes in our understanding of the molecular regulation of PG synthesis call for a reconsideration of previous hypotheses involving PGE2 as a regulator of beta-cell function in physiological and pathophysiological states.