bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypic Correction of Diabetic Mice by Adenovirus-Mediated Glucokinase Expression
Authors: Desai Urvi J. , Slosberg Eric D. , Boettcher Brian R. , Caplan Shari L. , Fanelli Barbara , Stephan Zouhair , Gunther Vicky J. , Kaleko Michael , Connelly Sheila
Published: March 2007

Hyperglycemia of diabetes is caused in part by perturbation of hepatic glucose metabolism. Hepatic glucokinase (GK) is an important regulator of glucose storage and disposal in the liver. GK levels are lowered in patients with maturity-onset diabetes of the young and in some diabetic animal models. Here, we explored the adenoviral vector–mediated overexpression of GK in a diet-induced murine model of type 2 diabetes as a treatment for diabetes. Diabetic mice were treated by intravenous administration with an E1/E2a/E3-deleted adenoviral vector encoding human hepatic GK (Av3hGK). Two weeks posttreatment, the Av3hGK-treated diabetic mice displayed normalized fasting blood glucose levels (95 ± 4.8 mg/dl; P < 0.001) when compared with Av3Null (135 ± 5.9 mg/dl), an analogous vector lacking a transgene, and vehicle-treated diabetic mice (134 ± 8 mg/dl). GK treatment also resulted in lowered insulin levels (632 ± 399 pg/ml; P < 0.01) compared with the control groups (Av3Null, 1,803 ± 291 pg/ml; vehicle, 1,861 ± 392 pg/ml), and the glucose tolerance of the Av3hGK-treated diabetic mice was normalized. No significant increase in plasma or hepatic triglycerides, or plasma free fatty acids was observed in the Av3hGK-treated mice. These data suggest that overexpression of GK may have a therapeutic potential for the treatment of type 2 diabetes.
Pharmacological Antilipolysis Restores Insulin Sensitivity During Growth Hormone Exposure
Authors: Nielsen Steen , Møller Niels , Christiansen Jens S. , Jørgensen Jens O.L.
Published: March 2007

Stimulation of lipolysis and the induction of resistance to insulin’s actions on glucose metabolism are well-recognized effects of growth hormone (GH). To evaluate whether these two features are causally linked, we studied the impact of pharmacologically induced antilipolysis in seven GH-deficient patients (mean [± SE] age 37 ± 4 years). Each subject was studied under four different conditions: during continuation of GH replacement alone (A), after discontinuation of GH replacement for 2 days (B), after GH replacement and short-term coadministration of acipimox (250 mg, p.o., b.i.d., for 2 days) (C), and after administration of acipimox alone (D). At the end of each study, total and regional substrate metabolisms were assessed in the basal state and after a 3-h hyperinsulinemic/euglycemic clamp. Serum levels of free fatty acids (FFAs) were elevated with GH alone (A) and suppressed with acipimox (C and D). Basal rates of lipid oxidation were highest with GH alone (A), and suppressed by 50% with acipimox (B versus D, P < 0.01; A versus C, P < 0.05). Basal glucose oxidation rates were lowest with GH alone (A) and highest with acipimox (C and D) (P = 0.01). Insulin-stimulated rates of total glucose turnover were significantly lower with GH alone as compared with all other conditions (P = 0.004). Insulin sensitivity as assessed by the M value (rate of glucose infusion) was reduced with GH alone as compared with all other conditions (M value in mg · kg−1 · min−1: GH alone [A], 2.55 ± 0.64; discontinuation of GH [B], 4.01 ± 0.70; GH plus acipimox [C], 3.96 ± 1.34; acipimox alone [D], 4.96 ± 0.91; P < 0.01). During pharmacological antilipolysis, GH did not significantly influence insulin sensitivity (C versus D; P = 0.19). From our results, we reached the following conclusions: 1) Our data strongly suggest that the insulin antagonistic actions of GH on glucose metabolism are causally linked to the concomitant activation of lipolysis. 2) In addition, GH may induce residual insulin resistance through non–FFA-dependent mechanisms. 3) The cellular and molecular mechanisms subserving the insulin antagonistic effects of GH remain to be elucidated.
Thiazolidinedione Treatment Prevents Free Fatty Acid–Induced Insulin Resistance in Male Wistar Rats
Authors: Hevener Andrea L. , Reichart Donna , Janez Andrej , Olefsky Jerrold
Published: March 2007

We sought to ascertain whether pretreatment with troglitazone (20 days) could prevent acute free fatty acid (FFA)–induced insulin resistance in male Wistar rats. Animals were divided into three groups: 1) control, 2) FFA infusion alone (FFA1), and 3) thiazolidinedione (TZD)-treated + FFA infusion (FFA1). Days before a hyperinsulinemic-euglycemic clamp, all animals were cannulated in the jugular vein (infusion) and carotid artery (sampling). Animals were allowed 5 days to recover from surgery and fasted 12 h before the experiment. Glucose (variable), insulin (40 mU · kg−1 · min−1), and Liposyn (heparinized 10% lipid emulsion) infusions were initiated simultaneously and continued from 0–120 min. Steady-state glucose, 8.3 ± 0.14 mmol/l, and insulin concentrations, 7.3 ± 2.45 nmol/l, were the same between groups. Interestingly, steady-state FFA levels were significantly lower in animals pretreated with TZD compared with FFA alone (1.83 ± 0.26 vs. 2.96 ± 0.25 mmol/l; P = 0.009), despite matched intralipid infusion rates. A second group of TZD-treated animals (TZD + FFA2) were infused with intralipid at a higher infusion rate (44%) to match the arterial concentrations of FFA1. The glucose infusion and insulin-stimulated glucose disposal rates (GDRs) were significantly decreased (40%) for untreated Liposyn infused (FFA1) compared with control rats. In addition, insulin receptor substrate-1 (IRS-1) phosphorylation and IRS-1–associated phosphatidylinositol (PI) 3-kinase activity was significantly reduced, 30–50%, in FFA1 rats. TZD pretreatment prevented the FFA-induced decrement in insulin signaling. Fatty acid translocase (FAT/CD36) also was significantly reduced (56%) in untreated FFA1 rats after the clamp but remained identical to control values for TZD-treated rats. In conclusion, acutely elevated FFA levels 1) induced a significant reduction in tracer-determined GDR paralleled by impaired tyrosine phosphorylation of IRS-1 and reduced IRS-1–associated PI 3-kinase activity and 2) induced a significant reduction in FAT/CD36 total protein. TZD pretreatment prevented FFA-induced decrements in insulin action and prevented the reduction in FAT/CD36 protein.
Caffeine Ingestion Decreases Glucose Disposal During a Hyperinsulinemic-Euglycemic Clamp in Sedentary Humans
Authors: Greer Felicia , Hudson Robert , Ross Robert , Graham Terry
Published: March 2007

The purpose of this investigation was to examine the effect of caffeine (an adenosine receptor antagonist) on whole-body insulin-mediated glucose disposal in resting humans. We hypothesized that glucose disposal would be lower after the administration of caffeine compared with placebo. Healthy, lean, sedentary (n = 9) men underwent two trial sessions, one after caffeine administration (5 mg/kg body wt) and one after placebo administration (dextrose) in a double-blind randomized design. Glucose disposal was assessed using a hyperinsulinemic-euglycemic clamp. Before the clamp, there were no differences in circulating levels of methylxanthines, catecholamines, or glucose. Euglycemia was maintained throughout the clamp with no difference in plasma glucose concentrations between trials. The insulin concentrations were also similar in the caffeine and placebo trials. After caffeine administration, glucose disposal was 6.38 ± 0.76 mg/kg body wt compared with 8.42 ± 0.63 mg/kg body wt after the placebo trial. This represents a significant (P < 0.05) decrease (24%) in glucose disposal after caffeine ingestion. In addition, carbohydrate storage was 35% lower (P < 0.05) in the caffeine trial than in the placebo trial. Furthermore, even when the difference in glucose disposal was normalized between the trials, there was a 23% difference in the amount of carbohydrate stored after caffeine administration compared with placebo administration. Caffeine ingestion also resulted in higher plasma epinephrine levels than placebo ingestion (P < 0.05). These data support our hypothesis that caffeine ingestion decreases glucose disposal and suggests that adenosine plays a role in regulating glucose disposal in resting humans.
High Glucose–Enhanced Mesangial Cell Extracellular Signal–Regulated Protein Kinase Activation and α1(IV) Collagen Expression in Response to Endothelin-1
Authors: Hua Hong , Goldberg Howard J. , Fantus I.G. , Whiteside Catharine I.
Published: March 2007

High glucose (HG) stimulates glomerular mesangial cell (MC) expression of extracellular matrix, a process involving protein kinase C (PKC) isozymes and enhanced signaling by autocrine peptides such as endothelin-1 (ET-1). The purpose of this study was to identify the specific PKC isozymes mediating the effects of HG on MC extracellular signal–regulated protein kinase (ERK1/2) signaling and α1(IV) collagen expression in response to ET-1. HG (30 mmol/l for 72 h) enhanced ET-1–stimulated α1(IV) collagen mRNA expression from 1.2 ± 0.1–fold to 1.9 ± 0.2–fold (P < 0.05 vs. normal glucose [NG] + ET-1), and the effect was significantly reduced by Calphostin C or the MEK (mitogen-activated protein kinase kinase) inhibitor PD98059. In transiently transfected MCs, dominant-negative (DN)–PKC-δ, -ε, or -ζ inhibited ET-1 activation of ERK1/2. Likewise, downstream of ERK1/2, ET-1 stimulated Elk-1–driven GAL4 luciferase activity to 11 ± 1–fold (P < 0.002 vs. NG + ET-1) in HG, and DN-PKC–δ, –ε, or –ζ attenuated this response to NG levels. HG enhanced ET-1–stimulated intracellular α1(IV) collagen protein expression, assessed by confocal immunofluorescence imaging, showed that individual DN–PKC-δ, -ε, -ζ, as well as DN–PKC-α and -β, attenuated the response. Thus, HG-enhanced ET-1 stimulation of α1(IV) collagen expression requires PKC-δ, -ε, and -ζ to act through an ERK1/2-dependent pathway and via PKC-α and -β, which are independent of ERK1/2.
A Major Gene Effect on Fasting Insulin and Insulin Sensitivity in Familial Combined Hyperlipidemia
Authors: Pihlajamäki Jussi , Austin Melissa , Edwards Karen , Laakso Markku
Published: March 2007

The most common inherited dyslipidemia, familial combined hyperlipidemia (FCHL), is associated with insulin resistance. Whether insulin sensitivity in these families is inherited is not known. Therefore, we investigated the inheritance of insulin sensitivity in 352 nondiabetic family members from 37 families with FCHL, 105 of whom had undergone testing using the hyperinsulinemic-euglycemic clamp technique for the measurement of insulin sensitivity. First, complex segregation analysis of fasting insulin levels (both unadjusted and age-, age2-, and BMI-adjusted) was used for modeling of the variance in fasting insulin levels. In these analyses, Mendelian codominant inheritance (P = 0.320 for unadjusted and P = 0.295 for adjusted insulin values) was not rejected over the most general model and fit the data significantly better than the sporadic model (P < 0.001). Polygenic and environmental models were rejected (P < 0.001). The Mendelian codominant model explained 44 and 45% of the variance in unadjusted and adjusted fasting insulin levels, respectively. The proposed genotypes of this locus, based on segregation analysis, were associated with directly measured insulin sensitivity in 105 FCHL family members who underwent the hyperinsulinemic-euglycemic clamp (P < 0.001). These results provide evidence for a major gene regulating insulin sensitivity in FCHL families. Possible pleiotropic effects of this insulin sensitivity locus on dyslipidemias in FCHL remain to be elucidated.
Characterization of the Annexin I Gene and Evaluation of Its Role in Type 2 Diabetes
Authors: Lindgren Cecilia M. , Nilsson Anita , Orho-Melander Marju , Almgren Peter , Groop Leif C.
Published: March 2007

In a previous study, we identified suggestive linkage between type 2 diabetes and a locus on chromosome 9p13-q21. This region contains the gene annexin I (ANXA1), encoding a protein suggested to be involved in both insulin secretion and insulin action. In this study, we sequenced the exon/intron boundaries of the human ANXA1 gene and performed mutation screening in 41 individuals from the initial linkage study. We identified five single nucleotide polymorphisms A58G, A401G, intronic variance sequence (IVS)8-28A/G, IVS11 +31A/G, and IVS12-11T/G, which were further tested for association to diabetes in 197 parent/offspring trios using the transmission disequilibrium test. No significant association with type 2 diabetes was observed, although the common A allele of the +58A/G variant gave a 22:12 transmission distortion (P = 0.12). This variant was further genotyped in 481 case and control subjects, but no difference in allele, genotype, or haplotype frequencies were observed between the groups. Further, a novel polymorphic (CA)15–25 repeat in intron 11 was genotyped in the subjects included in the initial linkage study. No improvement of the original finding was observed. We therefore concluded that the ANXA1 gene is unlikely to harbor variants that contribute to risk of type 2 diabetes.
A Common Hormone-Sensitive Lipase i6 Gene Polymorphism Is Associated With Decreased Human Adipocyte Lipolytic Function
Authors: Hoffstedt Johan , Arner Peter , Schalling Martin , Pedersen Nancy L. , Sengul Selim , Ahlberg Susanne , Iliadou Anastasia , Lavebratt Catharina
Published: March 2007

Hereditary factors may be involved in the pathogenesis of type 2 diabetes. A polymorphism in the hormone-sensitive lipase (HSL) gene (HSLi6) is associated with obesity and diabetes, although it is unknown whether the polymorphism is functional and thereby influences lipolysis. We genotyped 355 apparently healthy nonobese male and female subjects for the HSLi6 polymorphism. Allele 5 was found to be the most common allele (allele frequency 0.57). In 117 of the subjects, we measured abdominal subcutaneous fat cell lipolysis induced by drugs acting at various steps in the lipolytic cascade. The lipolysis rate induced by norepinephrine isoprenaline (acting on β-adrenoceptors), forskolin (acting on adenylyl cyclase), and dibutyryl cyclic AMP (acting on HSL) were all decreased by ∼50% in allele 5 homozygotes, as compared with noncarriers. Heterozygotes showed an intermediate lipolytic rate. The difference in lipolysis rate between genotypes was more pronounced in men than in women. We conclude that allele 5 of the HSLi6 polymorphism is associated with a marked decrease in the lipolytic rate of abdominal fat cells. This may in turn contribute to the development of obesity.
International Workshop on Lessons From Animal Models for Human Type 1 Diabetes
Authors: Bonifacio Ezio , Atkinson Mark , Eisenbarth George , Serreze David , Kay Thomas W.H. , Lee-Chan Edwin , Singh Bhagirath
Published: March 2007

Several self-antigens have been reported as targets of the autoimmune response in nonobese diabetic (NOD) mice. The aim of this workshop was to identify autoantibody assays that could provide useful markers of autoimmunity in this animal model for type 1 diabetes. More than 400 serum samples from NOD (4, 8, and 12 weeks of age and at diabetes onset), BALB/c, and B6 mice were collected from six separate animal facilities, coded, and distributed to five laboratories for autoantibody measurement. Insulin autoantibodies (IAA) were measured by radiobinding assay (RBA) by four laboratories and by enzyme-linked immunosorbent assay (ELISA) in one laboratory. Using the 99th percentile of BALB/c and B6 control mice as the threshold definition of positivity, IAA by RBA were detected in NOD mice at frequencies ranging from 10 to 30% at age 4 weeks, from 26 to 56% at 8 weeks, from 42 to 56% at 12 weeks, and from 15 to 75% at diabetes onset. With ELISA, IAA signals differed significantly between control mouse strains and increased with age in both control and NOD mice, with frequencies in NOD animals being 0% at 4 weeks, 14% at 8 weeks, 19% at 12 weeks, and 42% at diabetes onset. For IAA, the ELISA results were relatively discordant with those of RBA. GAD autoantibody (GADA) and IA-2 autoantibody (IA-2A) signals obtained by RBA were low (maximum 2.5% of total) but were increased in NOD mice compared with control mice at diabetes onset (GADA 29–50%; IA-2A 36–47%). ELISA also detected GADA (42%) and IA-2A (50%) at diabetes onset, with results concordant with those of RBA. Remarkably, GADA and IA-2A frequencies varied significantly with respect to the source colony of NOD mice. Furthermore, whereas neither GADA nor IA-2A correlated with IAA, there was strong concordance between GADA and IA-2A in individual mice. Sera with increased binding to GAD and IA-2 also had increased binding to the unrelated antigen myelin oligodendrocyte glycoprotein, and binding to GAD could not be inhibited with excess unlabeled antigen, suggesting nonspecific interactions. In sum, this workshop demonstrated that IAA measured by sensitive RBA are a marker of autoimmunity in NOD mice and draw into question the true nature of GADA and IA-2A in this animal model.
Report From the 1st International NOD Mouse T-Cell Workshop and the Follow-Up Mini-Workshop
Authors: Kaufman Daniel L. , Tisch Roland , Sarvetnick Nora , Chatenoud Lucienne , Harrison Leonard C. , Haskins Kathryn , Quinn Anthony , Sercarz Eli , Singh Bhagi , von Herrath Matthias , Wegmann Dale , Wen Li , Zekzer Dan
Published: March 2007

A workshop on autoreactive T-cell responses in NOD mice was held to optimize autoreactive T-cell detection methodologies. Using different proliferation assay protocols, 1 of the 11 participating laboratories detected spontaneous T-cell responses to GAD(524-543) and insulin(9-23) in their NOD mice. Two other laboratories were able to detect autoreactive responses when using enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) analysis of cytokines in culture supernatants, suggesting that these assays provided greater sensitivity. To address the divergent findings, a follow-up mini-workshop tested NOD mice from four different colonies side-by-side for T-cell proliferative responses to an expanded panel of autoantigens, using the protocol that had enabled detection of responses in the 1st International NOD Mouse T-Cell Workshop. Under these assay conditions, 16 of 16 NOD mice displayed proliferative responses to whole GAD65, 13 of 16 to GAD(524-543), 9 of 16 to GAD(217-236), 7 of 16 to insulin(9-23), and 5 of 16 to HSP277. Thus, spontaneous proliferative T-cell responses can be consistently detected to some β-cell autoantigens and peptides thereof. Overall, the results suggest that more sensitive assays (e.g., ELISPOT, ELISA analysis of cytokines in supernatants, or tetramer staining) may be preferred for the detection of autoreactive T-cells.
Autoantibody Response to Islet Transplantation in Type 1 Diabetes
Authors: Bosi Emanuele , Braghi Simona , Maffi Paola , Scirpoli Miriam , Bertuzzi Federico , Pozza Guido , Secchi Antonio , Bonifacio Ezio
Published: March 2007

Islet allotransplantation into patients with autoimmune type 1 diabetes represents a reexposure to autoantigen. Here, measurement of antibodies to GAD and IA-2 autoantigens before and after islet transplantation in 36 patients (33 receiving islet plus kidney grafts with cyclosporin and steroid-based immunosuppression, and 3 receiving solitary islet transplants with mycophenolate but cyclosporin-free immunosuppression) demonstrated marked rises in GAD antibodies within 7 days posttransplantation in 5 patients (3 receiving islet after kidney transplants, and 2 receiving solitary islet transplants) and within 30 days in the third patient receiving solitary islet transplantation. GAD antibodies were of the IgG1 subclass, against major autoantigenic epitopes, and in cases of islet after kidney transplants, the responses were short-lived and not accompanied by HLA antibodies. Two of these patients had subsequent marked rises of IA-2 antibodies, and an additional patient had a marked rise in IgM-GAD antibodies 3 years after transplantation. Insulin independence was not achieved in patients with autoantibody elevations and was significantly less frequent in these patients. These data are consistent with a reactivation of autoimmunity that may be dependent on immunosuppression therapy and is associated with impaired graft function.
Antecedent Hyperglycemia, Not Hyperlipidemia, Is Associated With Increased Islet Triacylglycerol Content and Decreased Insulin Gene mRNA Level in Zucker Diabetic Fatty Rats
Authors: Harmon Jamie S. , Gleason Catherine E. , Tanaka Yoshito , Poitout Vincent , Robertson R. Paul
Published: March 2007

Type 2 diabetes is caused by a combination of β-cell dysfunction and insulin resistance. Over time, hyperglycemia worsens, a phenomenon that has been attributed to deleterious effects of chronic hyperglycemia (glucotoxicity) or chronic hyperlipidemia (lipotoxicity) on β-cell function and is often accompanied by increased islet triacylglycerol (TAG) content and decreased insulin gene expression. To examine these two potentially pathogenic forces, we studied Zucker rats (leptin receptor wild type, +/+; heterozygous, +/−; and mutant, −/−). First, +/+ and +/− Zucker rats were compared metabolically. At 6 weeks of age, the +/− rats had a lower level of islet insulin mRNA compared with +/+. At 12 weeks of age, differences were found in body weight and islet TAG content; however, levels of insulin mRNA were equivalent. Second, we examined whether worsening of the diabetic state in the homozygous mutant (−/−) Zucker diabetic fatty (ZDF) rat is related more to chronic hyperglycemia or to hyperlipidemia. The ZDF rats were treated for 6 weeks with either bezafibrate, a lipid-lowering drug that does not affect plasma glucose levels, or phlorizin, a drug that reduces plasma glucose without lowering lipid levels. Bezafibrate treatment lessened the rise in plasma TAG observed in nontreated rats (239 ± 16 vs. 388 ± 36 mg/dl, treated versus nontreated; P < 0.0001) but did not prevent the rise in fasting plasma glucose. Despite lowering plasma TAG, bezafibrate was not effective in preventing an increased islet TAG content and did not prevent the associated decrease in insulin mRNA levels. Phlorizin treatment prevented hyperglycemia (61 ± 2 vs. 145 ± 7 mg/dl, treated versus nontreated; P < 0.0001) and lowered islet TAG content (32.7 ± 0.7 vs. 47.8 ± 2.7 ng/islet, treated versus nontreated; P < 0.0001) and preserved insulin mRNA levels without preventing hypertriglyceridemia. Plasma free fatty acid level did not correlate with changes in islet TAG or insulin mRNA levels. We conclude that antecedent elevated plasma glucose levels, not plasma lipid levels, are associated with elevated islet TAG content and decreased insulin mRNA levels in ZDF animals.
The Newly Inbred Cohen Diabetic Rat
Authors: Weksler-Zangen Sara , Yagil Chana , Zangen David H. , Ornoy Asher , Jacob Howard J. , Yagil Yoram
Published: March 2007

The newly inbred Cohen diabetic rat is an exceptional experimental model of diet-induced type 2 diabetes mellitus that is the result of secondary inbreeding nearly 30 years after it originally had been established. Animals from the original colony were selectively inbred by stringent criteria for 10 additional generations, bringing overall inbreeding to >50 generations. The metabolic phenotypes of the resulting contrasting strains, designated as the Cohen diabetic-sensitive (CDs) and -resistant (CDr) rats, were characterized. The phenotype of the CDs strain that was fed a regular diet consisted of fasting normoglycemia, normal glucose tolerance to intraperitoneal glucose loading, normal fasting insulin levels, and a normal insulin response to glucose loading. In contrast, CDs rats that were fed a custom-prepared high-sucrose low-copper diabetogenic diet became overtly diabetic: fasting glucose levels were normal or elevated, and the blood glucose insulin response to glucose loading was markedly abnormal. CDr rats that were fed a regular or diabetogenic diet did not develop diabetes and maintained normal glucose tolerance and insulin secretion. A striking sex difference was observed in CDs rats that were fed a diabetogenic diet: males had a lower growth rate and a more severe glucose intolerance pattern than females. Gonadectomy shortly after weaning did not prevent the development of the diabetic phenotype in its early phase in either sex but markedly attenuated its expression in males at a later phase, abolishing the sex differences. Alternate-day feeding, as opposed to daily feeding, also attenuated the metabolic phenotype in males. The development of the diabetic phenotype in CDs rats that were fed a diabetogenic diet was not accompanied by obesity or hyperlipidemia. The genetic profile of the strains was established using 550 microsatellite markers evenly distributed throughout the rat genome. The rate of homozygosity within strain was ≥96%. The rate of polymorphism between the contrasting strains was 43%. We conclude that the metabolic phenotypes of the rebred colony of CDs and CDr rats and their genetic makeup render the Cohen diabetic rat a useful experimental model that is highly suitable for studying the interaction between nutritional-metabolic environmental factors and genetic susceptibility (sensitivity and resistance) for the development of type 2 diabetes. The model is also distinctively useful for investigating the effect of sex on the expression of the diabetic phenotype.
Mutation of the RIIβ Subunit of Protein Kinase A Prevents Diet-Induced Insulin Resistance and Dyslipidemia in Mice
Authors: Schreyer Sandra A. , Cummings David E. , McKnight G. Stanley , LeBoeuf Renée C.
Published: March 2007

The mechanisms by which obesity contributes to diabetic phenotypes remain unclear. We evaluated the role of protein kinase A (PKA) signaling events in mediating diabetes associated with obesity. PKA comprises two regulatory subunits and two catalytic subunits and is activated by cAMP. The RIIβ regulatory subunit is abundantly expressed in adipose tissue and brain. Knockout mice lacking this subunit are lean and display remarkable resistance to diet-induced obesity. We investigated whether these mice were also resistant to diet-induced diabetes and whether this effect was dependent on reduced adiposity. Mice were fed a high-fat, high-carbohydrate diet and weight gain and diabetes phenotypes were examined. RIIβ−/− mice displayed decreased body weights, reduced insulin levels, improved insulin sensitivity, and improved total-body glucose disposal as compared with wild-type controls. Plasma levels of VLDL and LDL cholesterol were also reduced in high fat–fed RIIβ−/− mice compared with wild-type mice. Taken together, these data demonstrate that loss of RIIβ protects mice from diet-induced obesity, insulin resistance, and dyslipidemia.
Effects of Intravenous and Dietary Lipid Challenge on Intramyocellular Lipid Content and the Relation With Insulin Sensitivity in Humans
Authors: Bachmann Oliver P. , Dahl Dominik B. , Brechtel Klaus , Machann Jürgen , Haap Michael , Maier Thomas , Loviscach Mattias , Stumvoll Michael , Claussen Claus D. , Schick Fritz , Häring Hans U. , Jacob Stephan
Published: March 2007

An increased intramyocellular lipid (IMCL) content, as quantified by 1H-magnetic resonance spectroscopy (1H-MRS), is associated with reduced insulin sensitivity. At present, it is unclear which factors determine IMCL formation and how rapidly IMCL accumulation can be induced. We therefore studied the impact of hyperinsulinemia and elevated circulating nonesterified fatty acid (NEFA) levels on IMCL formation and insulin sensitivity. We further evaluated the influence of a high-fat diet on IMCL storage. In the infusion protocol, 12 healthy male subjects underwent a 6-h hyperinsulinemic-euglycemic glucose clamp with concomitant infusion of Intralipid plus heparin. IMCL was quantified by 1H-MRS in soleus (SOL) and tibialis anterior (TA) muscle at baseline and then every hour. IMCL levels started to increase significantly after 2 h, reaching a maximum of 120.8 ± 3.4% (SOL) and 164.2 ± 13.8% (TA) of baseline after 6 h (both P < 0.05). In parallel, the glucose infusion rate (GIR) decreased progressively, reaching a minimum of 60.4 ± 5.4% of baseline after 6 h. Over time, the GIR was strongly correlated with IMCL in TA (r = −0.98, P ≤ 0.003) and SOL muscle (r = −0.97, P ≤ 0.005). In the diet protocol, 12 male subjects ingested both a high-fat and low-fat diet for 3 days each. Before and after completion of each diet, IMCL levels and insulin sensitivity were assessed. After the high-fat diet, IMCL levels increased significantly in TA muscle (to 148.0 ± 16.9% of baseline; P = 0.005), but not in SOL muscle (to 114.4 ± 8.2% of baseline; NS). Insulin sensitivity decreased to 83.3 ± 5.6% of baseline (P = 0.033). There were no significant changes in insulin sensitivity or IMCL levels after the low-fat diet. The effects of the high-fat diet showed greater interindividual variation than those of the infusion protocol. The data from the lipid infusion protocol suggest a functional relationship between IMCL levels and insulin sensitivity. Similar effects could be induced by a high-fat diet, thereby underlining the physiological relevance of these observations.
Exercise Training Improves Insulin-Mediated Capillary Recruitment in Association With Glucose Uptake in Rat Hindlimb
Authors: Rattigan Stephen , Wallis Michelle G. , Youd Joanne M. , Clark Michael G.
Published: March 2007

Exercise training is considered to be beneficial in the treatment and prevention of insulin insensitivity, and much of the effect occurs in muscle. We have recently shown that capillary recruitment by insulin in vivo is associated with and may facilitate insulin action to increase muscle glucose uptake. In the present study, we examined the effect of 14 days of voluntary exercise training on euglycemic-hyperinsulinemic clamped (10 mU · min−1 · kg−1 for 2 h), anesthetized rats. Whole-body glucose infusion rate (GIR), hindleg glucose uptake, femoral blood flow (FBF), vascular resistance, and capillary recruitment, as measured by metabolism of infused 1-methylxanthine (1-MX), were assessed. In sedentary animals, insulin caused a significant (P < 0.05) increase in FBF (1.6-fold) and capillary recruitment (1.7-fold) but a significant decrease in vascular resistance. In addition, hindleg glucose uptake was increased (4.3-fold). Exercise training increased insulin-mediated GIR (24%), hindleg glucose uptake (93%), and capillary recruitment (62%) relative to sedentary animals. Neither capillary density nor total xanthine-oxidase activity in skeletal muscle were increased as a result of the training regimen used. We concluded that exercise training improves insulin-mediated increases in capillary recruitment in combination with augmented muscle glucose uptake. Increased insulin-mediated glucose uptake may in part result from the improved hemodynamic control attributable to exercise training.
Interleukin-1β Stimulation of c-Jun NH2-Terminal Kinase Activity in Insulin-Secreting Cells
Authors: Major Christopher D. , Wolf Bryan A.
Published: March 2007

Cytokines have been shown to have dramatic effects on pancreatic islets and insulin-secreting β-cell lines. It is well established that cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and γ-interferon (IFN-γ) inhibit β-cell function and are cytotoxic to human and rodent pancreatic islets in vitro. Despite the pleiotropic effects of cytokines on β-cells, the specific signal transduction pathways and molecular events involved in β-cell dysfunction remain largely unresolved. In this report, we have examined IL-1β stimulation of c-Jun NH2-terminal kinase (JNK) activity in insulin-secreting clonal cell lines. We demonstrate that IL-1β transiently activates 46- and 54-kDa isoforms of JNK in cultured RINm5F β-cells. Furthermore, IL-1β stimulation of JNK activity is specific, because TNF-α and IFN-γ were without effect. Stable overexpression of JNK1 in RINm5F cells increased levels of activated JNK without affecting kinase activity. JNK-interacting protein (JIP) associates with endogenous as well as overexpressed JNK, suggesting that JIP may serve to regulate JNK activity. Finally, we demonstrate that activated JNK is fully retained in cytoplasmic and membrane compartments without any nuclear translocation. Together, these data indicate that IL-1β–stimulated JNK activity may be distinctly targeted to cytoplasmic and/or membrane compartments in clonal insulin-producing cells, and that JIP may serve to localize JNK activity to specific substrates.
Phenotyping of Individual Pancreatic Islets Locates Genetic Defects in Stimulus Secretion Coupling to Niddm1i Within the Major Diabetes Locus in GK Rats
Authors: Lin Jian-Man , Ortsäter Henrik , Fakhrai-Rad Hossein , Galli Joakim , Luthman Holger , Bergsten Peter
Published: March 2007

The major diabetes quantitative trait locus (Niddm1), which segregates in crosses between GK rats affected with spontaneous type 2–like diabetes and normoglycemic F344 rats, encodes at least two different diabetes susceptibility genes. Congenic strains for the two subloci (Niddm1f and Niddm1i) have been generated by transfer of GK alleles onto the genome of F344 rats. Whereas the Niddm1f phenotype implicated insulin resistance, the Niddm1i phenotype displayed diabetes related to insulin deficiency. Individual islets from 16-week-old congenic rats were characterized for insulin release and oxygen tension (pO2). In the presence of 3 mmol/l glucose, insulin release from Niddm1f and Niddm1i islets was ∼5 pmol · g−1 · s−1 and pO2 was 120 mmHg. Similar recordings were obtained from GK and F344 islets. When glucose was raised to 11 mmol/l, insulin release increased significantly in Niddm1f and F344 islets but was essentially unchanged in islets from GK and Niddm1i. The high glucose concentration lowered pO2 to the same extent in islets from all strains. Addition of 1 mmol/l tolbutamide to the perifusion medium further increased pulsatile insulin release threefold in all islets. The pulse frequency was ∼0.4 min−1. α-Ketoisocaproate (11 mmol/l) alone increased pulsatile insulin release eightfold in islets from Niddm1f, Niddm1i, and control F344 rats but had no effect on insulin release from GK islets. These secretory patterns in response to α-ketoisocaproate were paralleled by reduction of pO2 in Niddm1f, Niddm1i, and control F344 islets and no change of pO2 in GK islets. The results demonstrate that Niddm1i carries alleles of gene(s) that reduce glucose-induced insulin release and that are amenable to molecular identification by genetic fine mapping.
Protection From Obesity and Insulin Resistance in Mice Overexpressing Human Apolipoprotein C1
Authors: Jong Miek C. , Voshol Peter J. , Muurling Martin , Dahlmans Vivian E.H. , Romijn Johannes A. , Pijl Hanno , Havekes Louis M.
Published: March 2007

Apolipoprotein (APO) C1 is a 6.6-kDa protein present in plasma and associated with lipoproteins. Using hyperinsulinemic-euglycemic clamp tests, we previously found that in APOC1 transgenic mice, the whole-body insulin-mediated glucose uptake is increased concomitant with a decreased fatty acid uptake. These latter results are confirmed in the present study, showing that APOC1 transgenic mice exhibit a 50% reduction in the uptake of the fatty acid analog 15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid in white adipose tissue stores. We next investigated whether APOC1 overexpression can modulate the initiation and/or development of obesity and insulin resistance. When crossbred on the genetically obese ob/ob background, APOC1 transgenic mice were fully protected from the development of obesity compared with ob/ob only mice, as reflected by a strong reduction in body weight (21 ± 4 vs. 44 ± 7 g), total adipose tissue stores (15 ± 3 vs. 25 ± 3% body wt), and average adipocyte size (7,689 ± 624 vs. 15,295 ± 1,289 μm2). Although less pronounced, APOC1 overexpression also reduced body weight on a wild-type background, solely due to a reduction in adipose tissue. Furthermore, despite elevated plasma free fatty acid and triglyceride levels, APOC1 overexpression significantly improved insulin sensitivity in ob/ob mice, as demonstrated by a strong reduction in plasma glucose and insulin levels, as well as a better performance in the glucose tolerance test. In conclusion, a marked reduction in the uptake of fatty acids into adipocytes may underlie the protection from obesity and insulin resistance in transgenic mice overexpressing human APOC1.
Diabetes-Associated Sustained Activation of the Transcription Factor Nuclear Factor-κB
Authors: Bierhaus Angelika , Schiekofer Stephan , Schwaninger Markus , Andrassy Martin , Humpert Per M. , Chen Jiang , Hong Mei , Luther Thomas , Henle Thomas , Klöting Ingrid , Morcos Michael , Hofmann Marion , Tritschler Hans , Weigle Bernd , Kasper Michael , Smith Mark , Perry George , Schmidt Ann-Marie , Stern David M. , Häring Hans-Ulrich , Schleicher Erwin , Nawroth Peter P.
Published: March 2007

Activation of the transcription factor nuclear factor-κB (NF-κB) has been suggested to participate in chronic disorders, such as diabetes and its complications. In contrast to the short and transient activation of NF-κB in vitro, we observed a long-lasting sustained activation of NF-κB in the absence of decreased IκBα in mononuclear cells from patients with type 1 diabetes. This was associated with increased transcription of NF-κBp65. A comparable increase in NF-κBp65 antigen and mRNA was also observed in vascular endothelial cells of diabetic rats. As a mechanism, we propose that binding of ligands such as advanced glycosylation end products (AGEs), members of the S100 family, or amyloid-β peptide (Aβ) to the transmembrane receptor for AGE (RAGE) results in protein synthesis–dependent sustained activation of NF-κB both in vitro and in vivo. Infusion of AGE-albumin into mice bearing a β-globin reporter transgene under control of NF-κB also resulted in prolonged expression of the reporter transgene. In vitro studies showed that RAGE-expressing cells induced sustained translocation of NF-κB (p50/p65) from the cytoplasm into the nucleus for >1 week. Sustained NF-κB activation by ligands of RAGE was mediated by initial degradation of IκB proteins followed by new synthesis of NF-κBp65 mRNA and protein in the presence of newly synthesized IκBα and IκBβ. These data demonstrate that ligands of RAGE can induce sustained activation of NF-κB as a result of increased levels of de novo synthesized NF-κBp65 overriding endogenous negative feedback mechanisms and thus might contribute to the persistent NF-κB activation observed in hyperglycemia and possibly other chronic diseases.
Diabetes-Associated HLA-DQ Genes and Birth Weight
Authors: Stene Lars C. , Magnus Per , Rønningen Kjersti S. , Joner Geir
Published: March 2007

An association has recently been described between increased birth weight and increased risk of childhood-onset type 1 diabetes. Whether this relationship is explained by genes associated with both increased birth weight and increased risk of type 1 diabetes is unknown. In the present study, we tested the association between birth weight and HLA-DQ genotypes known to confer risk for type 1 diabetes among 969 nondiabetic children randomly selected from the Norwegian population. We found that HLA genotypes previously shown to confer risk for type 1 diabetes were associated with reduced birth weight (the mean difference in birth weight between the DQB1*0602/DQB1*0602 and DQ8/DQ2 genotypes was 354 g [95% CI 105–604]), which was opposite of that expected if HLA genes explained the birth weight–type 1 diabetes association.
Overexpression of 1-Acyl-Glycerol-3-Phosphate Acyltransferase-αEnhances Lipid Storage in Cellular Models of Adipose Tissue and Skeletal Muscle
Authors: Ruan Hong , Pownall Henry J.
Published: March 2007

Plasma nonesterified fatty acids (NEFA) at elevated concentrations antagonize insulin action and thus may play a critical role in the development of insulin resistance in type 2 diabetes. Plasma NEFA and glucose concentrations are regulated, in part, by their uptake into peripheral tissues. Cellular energy uptake can be increased by enhancing either energy transport or metabolism. The effects of overexpression of 1-acylglycerol-3-phosphate acyltransferase (AGAT)-α, which catalyzes the second step in triglyceride formation from glycerol-3-phosphate, was studied in 3T3-L1 adipocytes and C2C12 myotubes. In myotubes, overexpression of AGAT-α did not affect total [14C]glucose uptake in the presence or absence of insulin, whereas insulin-stimulated[14C]glucose conversion to cellular lipids increased significantly(33%, P = 0.004) with a concomitant decrease (-30%, P =0.005) in glycogen formation. [3H]oleic acid (OA) uptake in AGAT-overexpressing myotubes increased 34% (P = 0.027) upon insulin stimulation. AGAT-α overexpression in adipocytes increased basal (130%, P = 0.04) and insulin-stimulated (27%, P = 0.01)[3H]OA uptake, increased insulin-stimulated glucose uptake (56%, P = 0.04) and conversion to cellular lipids (85%, P =0.007), and suppressed basal (-44%, P = 0.01) and isoproterenol-stimulated OA release (-45%, P = 0.03) but not glycerol release. Our data indicate that an increase in metabolic flow to triglyceride synthesis can inhibit NEFA release, increase NEFA uptake, and promote insulin-mediated glucose utilization in 3T3-L1 adipocytes. In myotubes,however, AGAT-α overexpression does not increase basal cellular energy uptake, but can enhance NEFA uptake and divert glucose from glycogen synthesis to lipogenesis upon insulin stimulation.
Effects of Chronic Central Nervous System Administration of Agouti-Related Protein in Pair-Fed Animals
Authors: Small Caroline J. , Kim Min S. , Stanley Sarah A. , Mitchell John R.D. , Murphy Kevin , Morgan David G.A. , Ghatei Mohammad A. , Bloom Stephen R.
Published: March 2007

The melanocortin receptor (MC3-R and MC4-R) antagonist, agouti-related protein (AGRP), is a potent stimulant of food intake. We examined the effect of chronic intracerebroventricular (ICV) AGRP treatment on energy metabolism and pituitary function in ad libitum fed rats and rats administered AGRP and then pair-fed to a saline control group. Chronic ICV AGRP (83-132)administration (1 nmol/day for 7 days) significantly increased food intake and body weight in ad libitum fed animals compared with saline-treated controls(body weight on day 7: 272 ± 6 [saline] vs. 319 ± 8 g [AGRP ad libitum fed]; P < 0.001). A significant increase in the epididymal fat pad weight, interscapular brown adipose tissue (BAT) weight, and plasma leptin was also observed in the ad libitum fed group. In the AGRP pair-fed group, a significant increase in the epididymal fat pad weight, BAT weight,and plasma leptin was again observed, suggesting that AGRP caused metabolic changes independent of increased food intake. BAT uncoupling protein 1 (UCP-1)content was significantly decreased compared with saline controls in both the AGRP ad libitum fed (21 ± 8% of saline control; P < 0.01)and AGRP pair-fed groups (24 ± 7% of saline control; P <0.01). Plasma thyroid-stimulating hormone (TSH) was significantly suppressed compared with saline controls in both the AGRP ad libitum fed and AGRP pair-fed groups (3.5 ± 0.3 [saline] vs. 2.7 ± 0.4 [AGRP ad libitum fed] vs. 2.1 ± 0.2 ng/ml [AGRP pairfed]; P < 0.01). This study demonstrates that independent of its orexigenic effects, chronic AGRP treatment decreased BAT UCP-1, suppressed plasma TSH, and increased fat mass and plasma leptin, suggesting that it may play a role in energy expenditure.
Regulation of Glycogen Synthase Kinase-3 in Human Skeletal Muscle
Authors: Wojtaszewski Jørgen F.P. , Nielsen Pernille , Kiens Bente , Richter Erik A.
Published: March 2007

Studies of skeletal muscle from rodents performed both in vivo and in vitro suggest a regulatory role of glycogen synthase kinase (GSK) 3 in glycogen synthase (GS) activation in response to insulin. Recently, hyper-insulinemic clamp studies in humans support such a role under nearly physiological conditions. In addition, in rats the activation of GS in skeletal muscle during treadmill running is time-related to the deactivation of GSK3. We investigated whether GSK3 was deactivated in human muscle during low-(∼50% Vo2max for 1.5 h) and high-intensity (∼75%Vo2max for 1 h) bicycle exercise as well as food intake. We observed a small but significant increase in GSK3α (10-20%) activity in biopsies obtained from vastus lateralis after both low- and high-intensity exercise, whereas GSK3β activity was unaffected. Subsequent food intake increased Aktphosphorylation (∼2-fold) and deactivated GSK3α(∼40%), whereas GSK3β activity was unchanged. GS activity increased in response to both exercise and food intake. We conclude that GSK3α but not GSK3β may have a role in the regulation of GS activity in response to meal-associated hyperinsulinemia in humans. However, in contrast to findings in muscle from rats, exercise does not deactivate GSK3 in humans, suggesting a GSK3-independent mechanism in the regulation of GS activity in muscle during physical activity.
Double-Stranded RNA—Dependent Protein Kinase Is Not Required for Double-Stranded RNA—Induced Nitric Oxide Synthase Expression or Nuclear Factor-κB Activation by Islets
Authors: Blair Libby A. , Heitmeier Monique R. , Scarim Anna L. , Maggi Leonard B. , Corbett John A.
Published: March 2007

Environmental factors, such as viral infection, have been implicated in the destruction of β-cells during the development of autoimmune diabetes. Double-stranded RNA (dsRNA), produced during viral replication, is an active component of a viral infection that stimulates antiviral responses in infected cells. Previous studies have shown that treatment of rat islets with dsRNA in combination with γ-interferon (IFN-γ) results in a nitric oxide-dependent inhibition of glucose-stimulated insulin secretion. This study examines the role of nuclear factor-κB (NF-κB) and the dsRNA-dependent protein kinase (PKR) in dsRNA + IFN-γ-induced nitric oxide synthase (iNOS) expression and nitric oxide production by rat, mouse,and human islets. Treatment of rat and human islets with dsRNA in the form of polyinosinic-polycytidylic acid (poly IC) and IFN-γ resulted in iNOS expression and nitric oxide production. Inhibitors of NF-κB activation—the proteasome inhibitor MG-132 and the antioxidant pyrrolidinedithiocarbamate (PDTC)—prevented poly IC +IFN-γ-induced iNOS expression and nitric oxide production. Incubation of rat islets for 3 h or human islets for 2 h with poly IC alone or poly IC +IFN-γ resulted in NF-κB nuclear translocation and degradation of the NF-κB inhibitor protein, IκB, events that are prevented by MG-132. PKR has been shown to participate in dsRNA-induced NF-κB activation in a number of cell types, including mouse embryonic fibroblasts. However, poly IC stimulated NF-κB nuclear translocation and IκB degradation to similar levels in islets isolated from mice devoid of PKR(PKR-/-) and wild-type mice (PKR+/+). Furthermore, the genetic absence of PKR did not affect dsRNA + IFN-γ-induced iNOS expression, nitric oxide production, or the inhibitory actions of these agents on glucose-stimulated insulin secretion. These results suggest that 1) NF-κB activation is required for dsRNA + IFN-γ-induced iNOS expression, 2) PKR is not required for either dsRNA-induced NF-κB activation or dsRNA + IFN-γ-induced iNOS expression by islets, and 3) PKR is not required for dsRNA + IFN-γ-induced inhibition of glucose-stimulated insulin secretion by islets.