bluuebunny/crossref_metadata_2025 huggingface dataset:
Brain-derived neurotrophic factor regulates glucose metabolism by modulating energy balance in diabetic mice.
Authors: Nakagawa T , Tsuchida A , Itakura Y , Nonomura T , Ono M , Hirota F , Inoue T , Nakayama C , Taiji M , Noguchi H
Published: March 2007

We previously reported that brain-derived neurotrophic factor (BDNF) regulates both food intake and blood glucose metabolism in rodent obese diabetic models such as C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice. To elucidate the effect of BDNF on glucose metabolism, we designed a novel pellet pair-feeding apparatus to eliminate the effect of appetite alteration on glucose metabolism. The apparatus was used to synchronize food intake precisely between BDNF-treated and vehicle-treated db/db mice. It was shown using this pellet pair-feeding apparatus that BDNF administered daily (20 mg x kg(-1) x day(-1)) to db/db mice significantly lowered blood glucose compared with pellet pair-fed db/db mice. To evaluate the effect of BDNF on insulin action, we used streptozotocin-induced type 1 diabetic mice. In this case, BDNF did not lower blood glucose concentration but rather enhanced the hypoglycemic action of insulin. In hyperglycemic db/db mice, pancreatic insulin content was reduced and glucagon content was increased compared with normoglycemic db/m mice. BDNF administered to db/db mice significantly restored both pancreatic insulin and glucagon content. Histological observations of aldehyde-fuchsin staining and immunostaining with anti-insulin indicated that insulin-positive pancreatic beta-cells were extensively regranulated by BDNF administration. We also studied the effect of BDNF on KK mice, normoglycemic animals with impaired glucose tolerance. In these mice, BDNF administration improved insulin resistance in the oral glucose tolerance test. To elucidate how blood glucose was metabolized in BDNF-treated animals, we investigated the effect of BDNF on the energy metabolism of db/db mice. Body temperature and oxygen consumption of the pellet pair-fed vehicle-treated mice were remarkably lower than the ad libitum-fed vehicle-treated mice. Daily BDNF administration for 3 weeks completely ameliorated both of the reductions. Finally, to clarify its action mechanism, the effect of intracerebroventricular administration of BDNF on db/db mice was examined. Here, a small dose of BDNF was found to be effective in lowering blood glucose concentration. This indicates that BDNF regulates glucose metabolism by acting directly on the brain.
Increased levels of soluble vascular cell adhesion molecule 1 are associated with risk of cardiovascular mortality in type 2 diabetes: the Hoorn study.
Authors: Jager A , van Hinsbergh V W , Kostense P J , Emeis J J , Nijpels G , Dekker J M , Heine R J , Bouter L M , Stehouwer C D
Published: March 2007

Membrane-bound vascular cell adhesion molecule 1 (VCAM-1) allows the tethering and rolling of monocytes and lymphocytes as well as firm attachment and transendothelial migration of leukocytes. Soluble forms of VCAM (sVCAM-1) may serve as monitors of increased expression of membrane-bound VCAM-1 and thus may reflect progressive formation of atherosclerotic lesions. Levels of sVCAM-1 have been found to be increased among type 2 diabetic as compared with nondiabetic subjects. To study the association of plasma sVCAM-1 concentration and risk of cardiovascular and all-cause mortality among nondiabetic and diabetic subjects, we investigated an age-, sex-, and glucose-tolerance-stratified sample (n = 631) of a population-based cohort aged 50-75 years that was followed prospectively. Plasma levels of sVCAM-1 were determined in frozen -70 degrees C baseline samples. After 7.4 years (mean) of follow-up, 107 (17%) subjects had died (42 of cardiovascular causes). In the entire group, increased sVCAM-1 levels were significantly associated with increased risk of cardiovascular mortality (relative risks [RRs] per 100 ng/ml sVCAM-1 increase, 1.10 [1.05-1.15] after adjustment for age, sex, and glucose tolerance status). This RR was somewhat diminished by further adjustment for the presence of hypertension and cardiovascular disease; levels of total, HDL, and LDL cholesterol and homocysteine; the presence of microalbuminuria (a putative marker of endothelial dysfunction); levels of von Willebrand factor (a marker of endothelial dysfunction) and C-reactive protein (a marker of low-grade inflammation); and estimates of glomerular filtration rate. However, the RR remained statistically significant. The RR among type 2 diabetic subjects was 1.13 (1.07-1.20) per 100 ng/ml sVCAM-1 increase after adjustment for age and sex, which was somewhat higher but not significantly different from the RR in nondiabetic subjects (P value for interaction term, 0.12). Further adjustment for other risk factors gave similar results. In conclusion, levels of sVCAM-1 are independently associated with the risk of cardiovascular mortality in type 2 diabetic subjects and therefore might be useful for identifying subjects at increased cardiovascular risk. Increased plasma sVCAM-1 levels may reflect progressive formation of atherosclerotic lesions, or sVCAM-1 itself may have bioactive properties related to cardiovascular risk. Our data, however, argue against the hypotheses of sVCAM-1 levels simply being a marker of endothelial dysfunction, of low-grade inflammation, or of an impaired renal function.
Genetic and physical mapping of a type 1 diabetes susceptibility gene (IDDM12) to a 100-kb phagemid artificial chromosome clone containing D2S72-CTLA4-D2S105 on chromosome 2q33.
Authors: Marron M P , Zeidler A , Raffel L J , Eckenrode S E , Yang J J , Hopkins D I , Garchon H J , Jacob C O , Serrano-Rios M , Martinez Larrad M T , Park Y , Bach J F , Rotter J I , Yang M C , She J X
Published: March 2007

Polymorphic markers within the CTLA4 gene on chromosome 2q33 have been shown to be associated with type 1 diabetes. Therefore, a gene responsible for the disease (IDDM12) most likely lies within a region of <1-2 cM of CTLA4. To define more precisely the IDDM12 interval, we genotyped a multiethnic (U.S. Caucasian, Mexican-American, French, Spanish, Korean, and Chinese) collection of 178 simplex and 350 multiplex families for 10 polymorphic markers within a genomic interval of approximately 300 kb, which contains the candidate genes CTLA4 and CD28. The order of these markers (D2S346, CD28, GGAA19E07, D2S307, D2S72, CTLA4, D2S105, and GATA52A04) was determined by sequence tagged site content mapping of bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) clones. The transmission disequilibrium test (TDT) analyses of our data revealed significant association/linkage with three markers within CTLA4 and two immediate flanking markers (D2S72 and D2S105) on each side of CTLA4 but not with more distant markers including the candidate gene CD28. Tsp analyses revealed significant association only with the three polymorphic markers within the CTLA4 gene. The markers linked and associated with type 1 diabetes are contained within a phagemid artificial chromosome clone of 100 kb, suggesting that the IDDM12 locus is either CTLA4 or an unknown gene in very close proximity.
Sex-determining region Y-related protein SOX13 is a diabetes autoantigen expressed in pancreatic islets.
Authors: Kasimiotis H , Myers M A , Argentaro A , Mertin S , Fida S , Ferraro T , Olsson J , Rowley M J , Harley V R
Published: March 2007

The SOX (sex-determining region [SRY]-type high mobility group [HMG] box) family of transcription factors play key roles in determining cell fate during organ development. In this study, we have identified a new human SOX gene, SOX13, as encoding the type 1 diabetes autoantigen, islet cell antigen 12 (ICA12). Sequence analysis showed that SOX13 belongs to the class D subgroup of SOX transcription factors, which contain a leucine zipper motif and a region rich in glutamine. SOX13 autoantibodies occurred at a significantly higher frequency among 188 people with type 1 diabetes (18%) than among 88 with type 2 diabetes (6%) or 175 healthy control subjects (4%). Deletion mapping of the antibody epitopes showed that the autoantibodies were primarily directed against an epitope requiring the majority of the protein. SOX13 RNA was detected in most human tissues, with the highest levels in the pancreas, placenta, and kidney. Immunohistochemistry on sections of human pancreas identified SOX13 in the islets of Langerhans, where staining was mostly cytoplasmic. In mouse pancreas, Sox13 was present in the nucleus and cytoplasm of beta-cells as well as other islet cell types. Recombinant SOX13 protein bound to the SOX consensus DNA motif AACAAT, and binding was inhibited by homodimer formation. These observations-along with the known molecular interactions of the closely related protein, rainbow trout Sox23-suggest that SOX13 may be activated for nuclear import and DNA binding through heterodimer formation. In conclusion, we have identified ICA12 as the putative transcription factor SOX13 and demonstrated an increased frequency of autoantibody reactivity in sera from type 1 diabetic subjects compared with type 2 diabetic and healthy control subjects.
Selection of insulinoma cell lines with resistance to interleukin-1beta- and gamma-interferon-induced cytotoxicity.
Authors: Chen G , Hohmeier H E , Gasa R , Tran V V , Newgard C B
Published: March 2007

Engineered insulinoma cell lines may represent an alternative to isolated islets for transplantation therapy of type 1 diabetes. Success of this approach may require development of cell lines that can withstand cytokine-mediated damage. To this end, we have cultured INS-1 insulinoma cells in increasing concentrations of interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma), with approximate weekly iterations over an 8-week period. Based on the C,N diphenyl-N'-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium+ ++ bromide (MTT) viability assay, the selected cells, termed INS-1res, were 100% viable after 5 days of treatment with 10 ng/ml of IL-1beta. These cells were also 78 +/- 1.2% viable after 5 days of exposure to the combination of 10 ng/ml IL-1beta and 100 U/ml IFN-gamma, whereas parental INS-1 cells treated in the same manner were only 0.3 +/- 0.03% viable. INS-1res cells were also resistant to treatment with supernatants from activated rat peripheral blood mononuclear cells, whereas only 20% of parental INS-1 cells survived such treatment. The resistance to IL-1beta conferred by this procedure was stable, whereas the partial resistance to IFN-gamma was transient but reinducible by culture in the presence of cytokines. Stable transfection of INS-1res cells with a plasmid containing the human insulin cDNA and expansion of the transfected colonies in the absence of cytokines produced cell lines that were on average more resistant to IL-1beta + IFN-gamma (53 +/- 11%) than similarly transfected clones derived from parental INS-1 cells (15 +/- 7%). Importantly, several INS-1res-derived clones retained the capacity to secrete insulin in response to glucose concentrations over the normal physiological range. With regard to the mechanism by which selection was conferred, we found normal levels of IFN-gamma receptor mRNA, but a 60% reduction in expression of the IL-1 receptor type I (IL-1RI) in INS-1res cells compared with parental INS-1 cells. IL-1beta signaling through p38 MAP kinase was found to be normal in INS-1res cells, suggesting that their expression of IL-1RI is sufficient to maintain cytokine action. However, normal IL-1beta-mediated translocation of NF-kappaB and induction of inducible nitric oxide synthase expression and nitric oxide production was severely impaired in the INS-1res cell lines, suggesting a mechanism for the IL-1beta resistance. In sum, this study defines a strategy for isolation of cytokine-resistant beta-cell lines and provides a new system for studying the mechanisms by which such resistance can be achieved.
Decreased insulin receptor tyrosine kinase activity and plasma cell membrane glycoprotein-1 overexpression in skeletal muscle from obese women with gestational diabetes mellitus (GDM): evidence for increased serine/threonine phosphorylation in pregnancy and GDM.
Authors: Shao J , Catalano P M , Yamashita H , Ruyter I , Smith S , Youngren J , Friedman J E
Published: March 2007

The cellular mechanisms for the insulin resistance of pregnancy and gestational diabetes mellitus (GDM) are unknown. The membrane protein plasma cell membrane glycoprotein-1 (PC-1) has been identified as an inhibitor of insulin receptor tyrosine kinase (IRTK) activity. We investigated insulin receptor function and PC-1 levels in muscle from three groups of obese subjects: women with GDM, pregnant women with normal glucose tolerance, and nonpregnant control subjects. Subjects (n = 6 for each group) were similar in age and degree of obesity (body fat >30%). IRTK activity, insulin receptor tyrosine phosphorylation, and protein levels of membrane glycoprotein PC-1 were determined in rectus abdominus muscle biopsies obtained at the time of either elective cesarean section or gynecological surgery. No significant differences were evident in basal insulin receptor tyrosine phosphorylation or IRTK activity in the three groups. After maximal insulin (10(-7) mol/l) stimulation, IRTK activity measured with the artificial substrate poly(Glu,Tyr) increased in all subjects but was lower in women with GDM by 25% (P < 0.05) and 39% (P < 0.001) compared with pregnant and nonpregnant control subjects, respectively. Similarly, insulin receptor tyrosine phosphorylation was significantly decreased in subjects with GDM (P < 0.05) compared with pregnant and nonpregnant control subjects. Treatment of the insulin receptors with alkaline phosphatase to dephosphorylate serine/threonine residues increased insulin-stimulated IRTK activity significantly in pregnant control and GDM subjects (P < 0.05), but these rates were still lower compared with nonpregnant control subjects (P < 0.05). PC-1 content in muscle from GDM subjects was increased by 63% compared with pregnant control subjects (P < 0.05) and by 206% compared with nonpregnant control subjects (P < 0.001). PC-1 content was negatively correlated with insulin receptor phosphorylation (r = -0.55, P < 0.05) and IRTK activity (r = -0.66, P < 0.05). These results indicate that pregnant control and GDM subjects had increased PC-1 content and suggest excessive phosphorylation of serine/threonine residues in muscle insulin receptors and that both may contribute to decreased IRTK activity. These changes worsen in women with GDM when controlling for obesity. These postreceptor defects in insulin signaling may contribute to the pathogenesis of GDM and the increased risk for type 2 diabetes later in life.
Effect of glucagon-like peptide 1(7-36) amide on glucose effectiveness and insulin action in people with type 2 diabetes.
Authors: Vella A , Shah P , Basu R , Basu A , Holst J J , Rizza R A
Published: March 2007

Although it is well established that glucagon-like peptide 1(7-36) amide (GLP-1) is a potent stimulator of insulin secretion, its effects on insulin action and glucose effectiveness are less clear. To determine whether GLP-1 increases insulin action and glucose effectiveness, subjects with type 2 diabetes were studied on two occasions. Insulin was infused during the night on both occasions to ensure that baseline glucose concentrations were comparable. On the morning of study, either GLP-1 (1.2 pmol x kg(-1) x min(-1)) or saline were infused along with somatostatin and replacement amounts of glucagon. Glucose also was infused in a pattern mimicking that typically observed after a carbohydrate meal. Insulin concentrations were either kept constant at basal levels (n = 6) or varied so as to create a prandial insulin profile (n = 6). The increase in glucose concentration was virtually identical on the GLP-1 and saline study days during both the basal (1.21 +/- 0.15 vs. 1.32 +/- 0.19 mol/l per 6 h) and prandial (0.56 +/- 0.14 vs. 0.56 +/- 0.10 mol/l per 6 h) insulin infusions. During both the basal and prandial insulin infusions, glucose disappearance promptly increased after initiation of the glucose infusion to rates that did not differ on the GLP-1 and saline study days. Suppression of endogenous glucose production also was comparable on the GLP-1 and saline study days during both the basal (-2.7 +/- 0.3 vs. -3.1 +/- 0.2 micromol/kg) and prandial (-3.1 +/- 0.4 vs. -3.0 +/- 0.6 pmol/kg) insulin infusions. We conclude that when insulin and glucagon concentrations are matched, GLP-1 has negligible effects on either insulin action or glucose effectiveness in people with type 2 diabetes. These data strongly support the concept that GLP-1 improves glycemic control in people with type 2 diabetes by increasing insulin secretion, by inhibiting glucagon secretion, and by delaying gastric emptying rather than by altering extrapancreatic glucose metabolism.
Effects of troglitazone on blood concentrations of plasminogen activator inhibitor 1 in patients with type 2 diabetes and in lean and obese normal subjects.
Authors: Kruszynska Y T , Yu J G , Olefsky J M , Sobel B E
Published: March 2007

Low plasma fibrinolytic activity in association with increased plasma plasminogen activator inhibitor 1 (PAI-1) levels has been linked to an increased risk of atherosclerosis in obesity and type 2 diabetes. We tested the hypothesis that troglitazone, which improves insulin sensitivity and lowers plasma insulin levels in insulin-resistant obese subjects and patients with type 2 diabetes, would also lower circulating PAI-1 antigen concentrations and activity. We assessed insulin sensitivity (5-h, 80 mU x m(-2) x min(-1) hyperinsulinemic-euglycemic clamp) and measured plasma PAI-1 antigen and activities and tissue plasminogen activator (tPA) in 14 patients with type 2 diabetes and 20 normal control subjects (10 lean, 10 obese) before and after 3 months of treatment with troglitazone (600 mg/day). At baseline, plasma PAI-1 antigen levels after an overnight fast were significantly higher in the obese (33.5 +/- 4.7 microg/l) and type 2 diabetic subjects (54.9 +/- 6.3 microg/l) than in the lean control subjects (16.3 +/- 3.2 microg/l; P < 0.01 and P < 0.001, respectively). Troglitazone decreased plasma PAI-1 antigen concentrations in the diabetic patients (36.8 +/- 5.0 microg/l; P < 0.001 vs. baseline), but the reduction in the obese subjects did not reach statistical significance (baseline, 33.5 +/- 4.7; after troglitazone, 25.6 +/- 5.2 microg/l). Changes in plasma PAI-1 activity paralleled those of PAI-1 antigen. The extent of the reduction in plasma PAI-1 antigen concentrations in the diabetic patients after troglitazone correlated with the reductions in fasting plasma insulin (r = 0.60, P < 0.05), nonesterified fatty acid (r = 0.63, P < 0.02), and glucose concentrations (r = 0.64, P < 0.02) but not with the improvement in glucose disposal rates during the glucose clamps. Three nonresponders to troglitazone with respect to effects on insulin sensitivity and fasting glucose and insulin levels also had no reduction in circulating PAI-1. In conclusion, troglitazone enhances fibrinolytic system activity in insulin-resistant type 2 diabetic patients. This effect appears to be intimately linked to its potential to lower plasma insulin levels and improve glycemic control through its peripheral tissue insulin-sensitizing effects.
Use of a novel impermeable biotinylated photolabeling reagent to assess insulin- and hypoxia-stimulated cell surface GLUT4 content in skeletal muscle from type 2 diabetic patients.
Authors: Ryder J W , Yang J , Galuska D , Rincón J , Björnholm M , Krook A , Lund S , Pedersen O , Wallberg-Henriksson H , Zierath J R , Holman G D
Published: March 2007

Cell surface GLUT4 levels in skeletal muscle from nine type 2 diabetic subjects and nine healthy control subjects have been assessed by a new technique that involves the use of a biotinylated photo-affinity label. A profound impairment in GLUT4 translocation to the skeletal muscle cell surface in response to insulin was observed in type 2 diabetic patients. Levels of insulin-stimulated cell surface GLUT4 above basal in type 2 diabetic patients were only approximately 10% of those observed in healthy subjects. The magnitude of the defect in GLUT4 translocation in type 2 diabetic patients was greater than that observed for glucose transport activity, which was approximately 50% of that in healthy subjects. Reduced GLUT4 translocation is therefore a major contributor to the impaired glucose transport activity in skeletal muscle from type 2 diabetic subjects. When a marked impairment in GLUT4 translocation occurs, the contribution of other transporters to transport activity becomes apparent. In response to hypoxia, marked reductions in skeletal muscle cell surface GLUT4 levels were also observed in type 2 diabetic patients. Therefore, a defect in a common late stage in signal transduction and/or a direct impairment in the GLUT4 translocation process accounts for reduced glucose transport in type 2 diabetic patients.
Endothelin receptor blockade prevents augmented extracellular matrix component mRNA expression and capillary basement membrane thickening in the retina of diabetic and galactose-fed rats.
Authors: Evans T , Deng D X , Chen S , Chakrabarti S
Published: March 2007

Endothelins (ETs) are a family of vasoactive peptides that have mitogenic properties and have also been associated with altered long-term nuclear signaling. We have previously shown that mRNA levels for ET-1, ET-3, and their receptors are upregulated under hyperhexosemic conditions. In this study, an endothelin antagonist was used to assess the effects of endothelin blockage on the production of two basement membrane transcripts, fibronectin and collagen alpha1 (IV). The microvascular basement membranes were analyzed using ultrastructural morphometry. Streptozotocin-induced diabetic rats, galactose-fed rats (30% galactose in diet), and nondiabetic, non-galactose-fed control rats were studied after 1-month and 6-month follow-up. Simultaneously, similar animal groups were treated with a general ET receptor blocker (bosentan, 100 mg x kg(-1) x day(-1)) and investigated. Semiquantitative reverse transcription-polymerase chain reaction for fibronectin and collagen alpha1 (IV) was conducted after 1 month of follow-up with comparison to beta-actin housekeeping gene using slot blot hybridization and densitometry. Basement membrane thickness was assessed after 6 months of follow-up in diabetic rats, using the orthogonal intercept method. After 1 month of follow-up, increased fibronectin and collagen alpha1 (IV) mRNA were present in the retina of diabetic and galactosemic animals, and the bosentan-treated groups exhibited mRNA levels similar to the control animals. After 6 months of follow-up, diabetes and galactose feeding induced basement membrane thickening, which was partially prevented by bosentan treatment. The above findings indicate that increased production of extracellular matrix proteins leading to thickening of microvascular basement membrane, secondary to hyperhexosemia, may be mediated via augmented ET production.
Molecular basis and characterization of the hyperinsulinism/hyperammonemia syndrome: predominance of mutations in exons 11 and 12 of the glutamate dehydrogenase gene. HI/HA Contributing Investigators.
Authors: Stanley C A , Fang J , Kutyna K , Hsu B Y , Ming J E , Glaser B , Poncz M
Published: March 2007

Glutamate dehydrogenase (GDH) is allosterically activated by the amino acid leucine to mediate protein stimulation of insulin secretion. Children with the hyperinsulinism/hyperammonemia (HI/HA) syndrome have symptomatic hypoglycemia plus persistent elevations of plasma ammonium. We have reported that HI/HA may be caused by dominant mutations of GDH that lie in a unique allosteric domain that is encoded within GDH exons 11 and 12. To examine the frequency of mutations in this domain, we screened genomic DNA from 48 unrelated cases with the HI/HA syndrome for exon 11 and 12 mutations in GDH. Twenty-five (52%) had mutations in these exons; 74% of the mutations were sporadic. Clinical manifestations included normal birth weight, late onset of hypoglycemia, diazoxide responsiveness, and protein-sensitive hypoglycemia. Enzymatic studies of lymphoblast GDH in seven of the mutations showed that all had reduced sensitivity to inhibition with GTP, consistent with an increase in enzyme activity. Mutations had little or no effect on enzyme responses to positive allosteric effectors, such as ADP or leucine. Based on the three-dimensional structure of GDH, the mutations may function by impairing the binding of an inhibitory GTP to a domain responsible for the allosteric and cooperativity properties of GDH.
Fuel selection in human skeletal muscle in insulin resistance: a reexamination.
Authors: Kelley D E , Mandarino L J
Published: March 2007

For many years, the Randle glucose fatty acid cycle has been invoked to explain insulin resistance in skeletal muscle of patients with type 2 diabetes or obesity. Increased fat oxidation was hypothesized to reduce glucose metabolism. The results of a number of investigations have shown that artificially increasing fat oxidation by provision of excess lipid does decrease glucose oxidation in the whole body. However, results obtained with rodent or human systems that more directly examined muscle fuel selection have found that skeletal muscle in insulin resistance is accompanied by increased, rather than decreased, muscle glucose oxidation under basal conditions and decreased glucose oxidation under insulin-stimulated circumstances, producing a state of "metabolic inflexibility." Such a situation could contribute to the accumulation of triglyceride within the myocyte, as has been observed in insulin resistance. Recent knowledge of insulin receptor signaling indicates that the accumulation of lipid products in muscle can interfere with insulin signaling and produce insulin resistance. Therefore, although the Randle cycle is a valid physiological principle, it may not explain insulin resistance in skeletal muscle.
Effects of free fatty acid elevation on postabsorptive endogenous glucose production and gluconeogenesis in humans.
Authors: Roden M , Stingl H , Chandramouli V , Schumann W C , Hofer A , Landau B R , Nowotny P , Waldhäusl W , Shulman G I
Published: March 2007

Effects of free fatty acids (FFAs) on endogenous glucose production (EGP) and gluconeogenesis (GNG) were examined in healthy subjects (n = 6) during stepwise increased Intralipid/heparin infusion (plasma FFAs 0.8+/-0.1, 1.8+/-0.2, and 2.8+/-0.3 mmol/l) and during glycerol infusion (plasma FFAs approximately 0.5 mmol/l). Rates of EGP were determined with D-[6,6-2H2]glucose and contributions of GNG from 2H enrichments in carbons 2 and 5 of blood glucose after 2H2O ingestion. Plasma glucose concentrations decreased by approximately 10% (P < 0.01), whereas plasma insulin increased by approximately 47% (P = 0.02) after 9 h of lipid infusion. EGP declined from 9.3+/-0.5 (lipid) and 9.0+/-0.8 pmol x kg(-1) x min(-1) (glycerol) to 8.4+/-0.5 and 8.2+/-0.7 micromol x kg(-1) x min(-1), respectively (P < 0.01). Contribution of GNG similarly rose (P < 0.01) from 46+/-4 and 52+/-3% to 65+/-8 and 78+/-7%. To exclude interaction of FFAs with insulin secretion, the study was repeated at fasting plasma insulin (approximately 35 pmol/l) and glucagon (approximately 90 ng/ml) concentrations using somatostatin-insulin-glucagon clamps. Plasma glucose increased by approximately 50% (P < 0.005) during lipid but decreased by approximately 12% during glycerol infusion (P < 0.005). EGP remained unchanged over the 9-h period (9.9+/-1.2 vs. 9.0+/-1.1 micromol x kg(-1) x min(-1)). GNG accounted for 62+/-5 (lipid) and 60+/-6% (glycerol) of EGP at time 0 and rose to 74+/-3% during lipid infusion only (P < 0.05 vs. glycerol: 64+/-4%). In conclusion, high plasma FFA concentrations increase the percent contribution of GNG to EGP and may contribute to increased rates of GNG in patients with type 2 diabetes.
Acceleration of type 1 diabetes by a coxsackievirus infection requires a preexisting critical mass of autoreactive T-cells in pancreatic islets.
Authors: Serreze D V , Ottendorfer E W , Ellis T M , Gauntt C J , Atkinson M A
Published: March 2007

Coxsackievirus infections have been proposed as an environmental trigger for the development of T-cell-mediated autoimmune (type 1) diabetes by either providing a molecular mimic of the candidate pancreatic beta-cell autoantigen GAD or inducing bystander inflammation in the pancreas. In this study in the NOD mouse model, we found that infection with a pancreatrophic coxsackievirus isolate can accelerate type 1 diabetes development through the induction of a bystander activation effect, but only after a critical threshold level of insulitic beta-cell-autoreactive T-cells has accumulated. Thus, coxsackievirus infections do not appear to initiate beta-cell autoreactive immunity but can accelerate the process once it is underway. These findings indicate that the timing of a coxsackievirus infection, rather than its simple presence or absence, may have important etiological implications for the development of T-cell-mediated autoimmune type 1 diabetes in humans.
Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas.
Authors: Stoffers D A , Kieffer T J , Hussain M A , Drucker D J , Bonner-Weir S , Habener J F , Egan J M
Published: March 2007

Diabetes is caused by a failure of the pancreas to produce insulin in amounts sufficient to meet the body's needs. A hallmark of diabetes is an absolute (type 1) or relative (type 2) reduction in the mass of pancreatic beta-cells that produce insulin. Mature beta-cells have a lifespan of approximately 48-56 days (rat) and are replaced by the replication of preexisting beta-cells and by the differentiation and proliferation of new beta-cells (neogenesis) derived from the pancreatic ducts. Here, we show that the insulinotropic hormone glucagon-like peptide (GLP)-1, which is produced by the intestine, enhances the pancreatic expression of the homeodomain transcription factor IDX-1 that is critical for pancreas development and the transcriptional regulation of the insulin gene. Concomitantly, GLP-1 administered to diabetic mice stimulates insulin secretion and effectively lowers their blood sugar levels. GLP-1 also enhances beta-cell neogenesis and islet size. Thus, in addition to stimulating insulin secretion, GLP-1 stimulates the expression of the transcription factor IDX-1 while stimulating beta-cell neogenesis and may thereby be an effective treatment for diabetes.
Response of pancreatic beta-cells to improved insulin sensitivity in women at high risk for type 2 diabetes.
Authors: Buchanan T A , Xiang A H , Peters R K , Kjos S L , Berkowitz K , Marroquin A , Goico J , Ochoa C , Azen S P
Published: March 2007

The purpose of this study was to examine the response of pancreatic beta-cells to changes in insulin sensitivity in women at high risk for type 2 diabetes. Oral glucose tolerance tests (OGTTs) and frequently sampled intravenous glucose tolerance tests (FSIGTs) were conducted on Latino women with impaired glucose tolerance and a history of gestational diabetes before and after 12 weeks of treatment with 400 mg/day troglitazone (n = 13) or placebo (n = 12). Insulin sensitivity was assessed by minimal model analysis, and beta-cell insulin release was assessed as acute insulin responses to glucose (AIRg) and tolbutamide (AIRt) during FSIGTs and as the 30-min incremental insulin response (30-min dINS) during OGTTs. Beta-cell compensation for insulin resistance was assessed as the product (disposition index) of minimal model insulin sensitivity and each of the 3 measures of beta-cell insulin release. In the placebo group, there was no significant change in insulin sensitivity or in any measure of insulin release, beta-cell compensation for insulin resistance, or glucose tolerance. Troglitazone treatment resulted in a significant increase in insulin sensitivity, as reported previously. In response, AIRg did not change significantly, so that the disposition index for AIRg increased significantly from baseline (P = 0.004) and compared with placebo (P = 0.02). AIRt (P = 0.001) and 30-min dINS (P = 0.02) fell with improved insulin sensitivity during troglitazone treatment, so that the disposition index for each of these measures of beta-cell function did not change significantly from baseline (P > 0.20) or compared with placebo (P > 0.3). Minimal model analysis revealed that 89% of the change from baseline in insulin sensitivity during troglitazone treatment was accounted for by lowered plasma insulin concentrations. Neither oral nor intravenous glucose tolerance changed significantly from baseline or compared with placebo during troglitazone treatment. The predominant response of beta-cells to improved insulin sensitivity in women at high risk for type 2 diabetes was a reduction in insulin release to maintain nearly constant glucose tolerance.
IGF-I treatment in adults with type 1 diabetes: effects on glucose and protein metabolism in the fasting state and during a hyperinsulinemic-euglycemic amino acid clamp.
Authors: Carroll P V , Christ E R , Umpleby A M , Gowrie I , Jackson N , Bowes S B , Hovorka R , Croos P , Sönksen P H , Russell-Jones D L
Published: March 2007

Type 1 diabetes is associated with abnormalities of the growth hormone (GH)-IGF-I axis. Such abnormalities include decreased circulating levels of IGF-I. We studied the effects of IGF-I therapy (40 microg x kg(-1) x day(-1)) on protein and glucose metabolism in adults with type 1 diabetes in a randomized placebo-controlled trial. A total of 12 subjects participated, and each subject was studied at baseline and after 7 days of treatment, both in the fasting state and during a hyperinsulinemic-euglycemic amino acid clamp. Protein and glucose metabolism were assessed using infusions of [1-13C]leucine and [6-6-2H2]glucose. IGF-I administration resulted in a 51% rise in circulating IGF-I levels (P < 0.005) and a 56% decrease in the mean overnight GH concentration (P < 0.05). After IGF-I treatment, a decrease in the overnight insulin requirement (0.26+/-0.07 vs. 0.17+/-0.06 U/kg, P < 0.05) and an increase in the glucose infusion requirement were observed during the hyperinsulinemic clamp (approximately 67%, P < 0.05). Basal glucose kinetics were unchanged, but an increase in insulin-stimulated peripheral glucose disposal was observed after IGF-I therapy (37+/-6 vs. 52+/-10 micromol x kg(-1) x min(-1), P < 0.05). IGF-I administration increased the basal metabolic clearance rate for leucine (approximately 28%, P < 0.05) and resulted in a net increase in leucine balance, both in the basal state and during the hyperinsulinemic amino acid clamp (-0.17+/-0.03 vs. -0.10+/-0.02, P < 0.01, and 0.25+/-0.08 vs. 0.40+/-0.06, P < 0.05, respectively). No changes in these variables were recorded in the subjects after administration of placebo. These findings demonstrated that IGF-I replacement resulted in significant alterations in glucose and protein metabolism in the basal and insulin-stimulated states. These effects were associated with increased insulin sensitivity, and they underline the major role of IGF-I in protein and glucose metabolism in type 1 diabetes.
Genotype/phenotype relationships in HNF-4alpha/MODY1: haploinsufficiency is associated with reduced apolipoprotein (AII), apolipoprotein (CIII), lipoprotein(a), and triglyceride levels.
Authors: Shih D Q , Dansky H M , Fleisher M , Assmann G , Fajans S S , Stoffel M
Published: March 2007

Hepatocyte nuclear factor (HNF)-4alpha is a transcription factor that plays an important role in regulation of gene expression in pancreatic beta-cells and in the liver. Heterozygous mutations in the HNF-4alpha gene are responsible for maturity-onset diabetes of the young 1 (MODY1), which is characterized by pancreatic beta-cell-deficient insulin secretion. HNF-4alpha is a major transcriptional regulator of many genes expressed in the liver. However, no liver defect has been identified in individuals with HNF-4alpha mutations. In this study, we have identified HNF-4alpha target genes that are mainly expressed in the liver, including alpha1-antitrypsin, alpha1-antichymotrypsin, alpha-fetal protein, ceruloplasmin, IGF binding protein 1, transferrin, apolipoprotein(AI) [apo(AI)], apo(AII), apo(B), and apo(CIII). Serum levels of these proteins and Lp(a) and triglycerides were measured in 24 members of the HNF-4alpha/MODY1 RW pedigree (Q268X mutation), including 12 diabetic patients with HNF-4alpha mutations (D-HNF4+/-), 6 nondiabetic subjects with HNF-4alpha mutations (N-HNF4+/-), 6 normal relatives (N-HNF4+/+), 6 unrelated normal matched control subjects (N-HNF4+/+), and 12 matched diabetic (non-MODY1-5) patients (D-HNF4+/+). Serum levels of apo(AII), apo(CIII), lipoprotein(a) [Lp(a)], and triglyceride were significantly reduced in HNF4+/- subjects (26.9, 19.8, 12.1, and 72.1 mg/dl, respectively) compared with HNF4+/+ subjects (37.4, 26.5, 45.2, and 124.2 mg/dl, respectively) (P = 0.00001, P = 0.01, P = 0.00006, and P = 0.000003, respectively). This reduction was not found when apo(AII), apo(CIII), Lp(a), and triglyceride levels were compared in D-HNF4+/- versus N-HNF4+/- or in D-HNF4+/+ versus N-HNF4+/+ subjects, which indicates that HNF-4alpha haploinsufficiency rather than hyperglycemia is the primary cause of decreased serum protein and triglyceride concentrations. Furthermore, we determined that genetic or environmental modifiers other than HNF-4alpha do not appear to contribute to the observed decrease of HNF-4alpha-regulated serum proteins. This study demonstrates that a heterozygous HNF-4alpha mutation leads to an HNF-4alpha-dependent hepatocyte secretory defect of liver-specific proteins.
Glucosamine activates the plasminogen activator inhibitor 1 gene promoter through Sp1 DNA binding sites in glomerular mesangial cells.
Authors: Goldberg H J , Scholey J , Fantus I G
Published: March 2007

Increased flux through the hexosamine biosynthetic pathway is associated with altered gene expression. To investigate the underlying mechanisms, we treated glomerular mesangial cells with glucosamine and studied the regulation of the plasminogen activator inhibitor (PAI)-1 gene. Incubating mesangial cells with 2 mmol/l glucosamine for 4 days resulted in a 3.1+/-0.4-fold increase in PAI-1 mRNA levels (P < 0.01) and a 33+/-9-fold increase in the activity of a transiently transfected PAI-1 promoter-luciferase reporter gene (P < 0.01). Cotransfection of an expression vector for a dominant-negative type II TGF-beta receptor with the PAI-1 promoter-reporter gene did not interfere with this effect of glucosamine. However, mutation of 2 putative Sp1 sites in the PAI-1 promoter, at -76 to -71 and -44 to -39, markedly reduced induction of PAI-1 luciferase activity by glucosamine, from 8.9+/-1.9-fold to 1.7+/-0.5-fold (P < 0.01). An electrophoretic mobility shift assay demonstrated that glucosamine increased Sp1 DNA binding by 31+/-11% (P < 0.05), implying that the effects of glucosamine were explained, in part, by changes in Sp1 DNA binding. High glucose (20 mmol/l) also activated the transiently transfected PAI-1 promoter (2.5+/-0.4-fold). This effect was diminished by mutation of both the PAI-1 promoter Sp1 sites (1.2+/-0.3-fold, P < 0.05). In addition, 6-diazo-5-oxo-L-norleucine, a glutamine:fructose-6-phosphate-amidotransferase inhibitor, blocked the induction by high glucose (4.7+/-0.8- to 0.9+/-0.1-fold, P < 0.01). These results indicate that stimulation of the PAI-1 promoter by both high glucose and glucosamine involves Sp1 and that the hexosamine pathway may be involved in the regulation of gene expression by high glucose in glomerular mesangial cells.
The CART gene and human obesity: mutational analysis and population genetics.
Authors: Challis B G , Yeo G S , Farooqi I S , Luan J , Aminian S , Halsall D J , Keogh J M , Wareham N J , O'Rahilly S
Published: March 2007

The cocaine- and amphetamine-regulated transcript (CART) peptide is a recently characterized neuropeptide implicated in the control of appetite. We hypothesized that genetic variation in CART may contribute to human obesity. The entire coding region of CART was determined by nucleotide sequencing in 91 unrelated subjects with severe early-onset obesity. A novel amino acid change, Ser66Thr, was found in 2 probands and in 0 of 100 control subjects but did not cosegregate with obesity in family studies. Two common polymorphisms were found in the 3'-untranslated region (A1475G and deltaA1457). An effect of these polymorphisms on body composition and intermediate phenotypes related to obesity was examined in a large Caucasian population in the U.K. Neither polymorphism showed any significant relationship with obesity; however, men heterozygous for the A1475G variant had significantly lower waist-to-hip ratio (WHR), fasting plasma insulin, and fasting triglycerides. Regression analysis indicated that the effects on insulin and triglycerides were likely to be secondary to the effects on WHR. Thus, we have conducted the first systematic study of the CART gene in human obesity, and although no clear association with obesity was found, the data suggest that genetic variation in the CART locus might influence fat distribution and variables related to syndrome X.
Serine residues 1177/78/82 of the insulin receptor are required for substrate phosphorylation but not autophosphorylation.
Authors: Bossenmaier B , Strack V , Stoyanov B , Krützfeldt J , Beck A , Lehmann R , Kellerer M , Klein H , Ullrich A , Lammers R , Häring H U
Published: March 2007

Serine residues of the human insulin receptor (HIR) may be phosphorylated and negatively regulate the insulin signal. We studied the impact of 16 serine residues in HIR by mutation to alanine and co-overexpression in human embryonic kidney (HEK) 293 cells together with the docking proteins insulin receptor substrate (IRS)-1, IRS-2, or (SHC) Src homologous and collagen-like. As a control, IRS-1 was also cotransfected with an HIR with a juxtamembrane deletion (HIR delta JM) and therefore not containing the domain required for interaction with IRS-1. Coexpression of HIR with IRS-1, IRS-2, and SHC strongly enhanced tyrosine phosphorylation of these proteins. A similar increase in tyrosine phosphorylation was observed in cells overexpressing IRS-1, IRS-2, or SHC together with all HIR mutants except HIR delta JM and a mutant carrying exchanges of serines 1177, 1178, and 1182 to alanine (HIR1177/78/82), although this mutant showed normal autophosphorylation. Analysis of total cell lysates with anti-phosphotyrosine antibodies showed that in addition to the overexpressed substrates, other cellular proteins displayed reduced levels of tyrosine phosphorylation in these cells. To study consequences for phosphatidylinositol 3-kinase (PI 3-kinase) activation, we established stable NIH3T3 fibroblast cell lines overexpressing wild-type HIR, HIR1177/78/82, and other HIR mutants as the control. Again, HIR1177/78/82 showed normal autophosphorylation but showed a clear decrease in tyrosine phosphorylation of endogenous IRS-1 and activation of PI 3-kinase. This decrease in kinase activity also occurred in an in vitro kinase assay towards recombinant IRS-1. Finally, we performed a separation of the phosphopeptides by high-performance liquid chromatography and could not detect any differences in the profiles of HIR and HIR1177/78/82. In conclusion, we have defined a region in HIR that is important for substrate phosphorylation but not autophosphorylation. Therefore, this mutant may provide new insights into the mechanism of kinase activation and substrate phosphorylation.
Adipocyte metabolism in adipocyte fatty acid binding protein knockout mice (aP2-/-) after short-term high-fat feeding: functional compensation by the keratinocyte [correction of keritinocyte] fatty acid binding protein.
Authors: Shaughnessy S , Smith E R , Kodukula S , Storch J , Fried S K
Published: March 2007

Mice null for adipocyte fatty acid binding protein (AFABP) compensate by increasing expression of keratinocyte fatty acid binding protein (KFABP) (Hotamisligil et al. Science 274:1377-1379, 1996). In the present study, AFABP knockout (KO) and wild-type (WT) mice became equally obese on a high-fat diet, as judged by fat pad weights, adipocyte size, and body composition analysis. High-fat feeding led to moderate insulin resistance in both WT and AFABP knockout mice, as indicated by an approximately 2-fold increase in plasma insulin. However, in the high fat-fed mice, plasma glucose levels were approximately 15% lower in the AFABP-KO mice. Adipocytes isolated from AFABP-KO and WT mice fed high- or low-fat diets exhibited similar rates of basal and norepinephrine-stimulated lipolysis and insulin-stimulated rates of glucose conversion to fatty acids and glyceride-glycerol. However, basal glucose conversion to fatty acids was higher in adipocytes of AFABP-KO mice. Adipocyte tumor necrosis factor-alpha release was similarly increased by high-fat diet-induced obesity in both WT and AFABP-KO mice. As assessed by Western blot analysis, the level of KFABP protein in AFABP-KOs was approximately 40% of the level of AFABP in WT controls. The binding affinities of KFABP for long-chain fatty acids were 2- to 4-fold higher than those of AFABP, but the relative affinities for different fatty acids were similar. As for AFABP, the rate of fatty acid transfer from KFABP to model phospholipid vesicles was increased with acceptor membrane concentration and by inclusion of acidic phospholipids, indicating a similar mechanism of transfer. We conclude KFABP can functionally compensate for the absence of AFABP, resulting in no major alterations in adipocyte metabolism or fat accumulation in response to short-term feeding of high-fat diets that result in moderate hyperinsulinemia.