bluuebunny/crossref_metadata_2025 huggingface dataset:
Rat Small Intestine Is an Insulin-Sensitive Gluconeogenic Organ
Authors: Croset Martine , Rajas Fabienne , Zitoun Carine , Hurot Jean-Marc , Montano Sandrine , Mithieux Gilles
Published: March 2007

At variance with the current view that only liver and kidney are gluconeogenic organs, because both are the only tissues to express glucose-6-phosphatase (Glc6Pase), we have recently demonstrated that the Glc6Pase gene is expressed in the small intestine in rats and humans and that it is induced in insulinopenic states such as fasting and diabetes. We used a combination of arteriovenous balance and isotopic techniques, reverse transcription–polymerase chain reaction, Northern blot analysis, and enzymatic activity assays. We report that rat small intestine can release neosynthesized glucose in mesenteric blood in insulinopenia, contributing 20–25% of total endogenous glucose production. Like liver glucose production, small intestine glucose production is acutely suppressed by insulin infusion. In the small intestine, glutamine and, to a much lesser extent, glycerol are the precursors of glucose, whereas alanine and lactate are the main precursors in liver. Accounting for these metabolic fluxes: 1) the phosphoenolpyruvate carboxykinase gene (required for the utilization of glutamine) is strongly induced at the mRNA and enzyme levels in insulinopenia; 2) the glycerokinase gene is expressed, but not induced; 3) the pyruvate carboxylase gene (required for the utilization of alanine and lactate) is repressed by 80% at the enzyme level in insulinopenia. These studies identify small intestine as a new insulin-sensitive tissue and a third gluconeogenic organ, possibly involved in the pathophysiology of diabetes.
Increased QTc Dispersion Is Related to Blunted Circadian Blood Pressure Variation in Normoalbuminuric Type 1 Diabetic Patients
Authors: Poulsen Per Løgstrup , Ebbehøj Eva , Arildsen Hanne , Knudsen Søren Tang , Hansen Klaus Würgler , Mølgaard Henning , Mogensen Carl Erik
Published: March 2007

A reduced nocturnal fall in blood pressure (BP) and increased QT dispersion both predict an increased risk of cardiovascular events in diabetic as well as nondiabetic subjects. The relationship between these two parameters remains unclear. The role of diabetic autonomic neuropathy in both QT dispersion and circadian BP variation has been proposed, but data have been conflicting. The aim of the present study was to describe associations between QT dispersion and circadian BP variation as well as autonomic function in type 1 diabetic patients. In 106 normoalbuminuric (urinary albumin excretion <20 μg/min) normotensive patients, we performed 24-h ambulatory BP (Spacelabs 90207) and short-term (three times in 5 min) power spectral analysis of RR interval oscillations, as well as cardiovascular reflex tests (deep breathing test, postural heart rate, and BP response). No patient had received (or had earlier received) antihypertensive or other medical treatment apart from insulin. In a resting 12-lead electrocardiogram, the QT interval was measured by the tangent method in all leads with well-defined T-waves. The measurement was made by one observer blinded to other data. The QT interval was corrected for heart rate using Bazett’s formula. The QTc dispersion was defined as the difference between the maximum and the minimum QTc interval in any of the 12 leads. When comparing patients with QTc dispersion below and above the median (43 ms), the latter had significantly higher night BP (114/67 vs. 109/62 mmHg, P < 0.003/P < 0.001), whereas day BP was comparable (129/81 vs. 127/79 mmHg). Diurnal BP variation was blunted in the group with QTc dispersion >43 ms with significantly higher night/day ratio, both for systolic (88.8 vs. 86.2%, P < 0.01) and diastolic (83.1 vs. 79.5%, P < 0.01) BP. The association between QTc dispersion and diastolic night BP persisted after controlling for potential confounders such as sex, age, duration of diabetes, urinary albumin excretion, and HbA1c. Power spectral analysis suggested an altered sympathovagal balance in patients with QTc dispersion above the median (ratio of low-frequency/high-frequency power: 1.0 vs. 0.85, P < 0.01). In normoalbuminuric type 1 diabetic patients, increased QTc dispersion is associated with reduced nocturnal fall in BP and an altered sympathovagal balance. This coexistence may be operative in the ability of these parameters to predict cardiovascular events.
The Peroxisome Poliferator–Activated Receptor-γ2 Pro12Ala Variant
Authors: Douglas Julie A. , Erdos Michael R. , Watanabe Richard M. , Braun Andi , Johnston Cristy L. , Oeth Paul , Mohlke Karen L. , Valle Timo T. , Ehnholm Christian , Buchanan Thomas A. , Bergman Richard N. , Collins Francis S. , Boehnke Michael , Tuomilehto Jaakko
Published: March 2007

Recent studies have identified a common proline-to-alanine substitution (Pro12Ala) in the peroxisome proliferator–activated receptor-γ2 (PPAR-γ2), a nuclear receptor that regulates adipocyte differentiation and possibly insulin sensitivity. The Pro12Ala variant has been associated in some studies with diabetes-related traits and/or protection against type 2 diabetes. We examined this variant in 935 Finnish subjects, including 522 subjects with type 2 diabetes, 193 nondiabetic spouses, and 220 elderly nondiabetic control subjects. The frequency of the Pro12Ala variant was significantly lower in diabetic subjects than in nondiabetic subjects (0.15 vs. 0.21; P = 0.001). We also compared diabetes-related traits between subjects with and without the Pro12Ala variant within subgroups. Among diabetic subjects, the variant was associated with greater weight gain after age 20 years (P = 0.023) and lower triglyceride levels (P = 0.033). Diastolic blood pressure was higher in grossly obese (BMI >40 kg/m2) diabetic subjects with the variant. In nondiabetic spouses, the variant was associated with higher fasting insulin (P = 0.033), systolic blood pressure (P = 0.021), and diastolic blood pressure (P = 0.045). These findings support a role for the PPAR-γ2 Pro12Ala variant in the etiology of type 2 diabetes and the insulin resistance syndrome.
Defective Glucose-Dependent Insulinotropic Polypeptide Receptor Expression in Diabetic Fatty Zucker Rats
Authors: Lynn Francis C. , Pamir Nathalie , Ng Eddy H.C. , McIntosh Christopher H.S. , Kieffer Timothy J. , Pederson Raymond A.
Published: March 2007

Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone that is released postprandially from the small intestine and acts in concert with glucagon-like peptide (GLP)-1 to potentiate glucose-induced insulin secretion from the pancreatic β-cell. In type 2 diabetes, there is a decreased responsiveness of the pancreas to GIP; however, the insulin response to GLP-1 remains intact. The literature suggests that the ineffectiveness of GIP in type 2 diabetes may be a result of chronic homologous desensitization of the GIP receptor. Yet, there has been no conclusive evidence suggesting that GIP levels are elevated in diabetes. The hypothesis of the present study is that one cause of decreased responsiveness to GIP in type 2 diabetes is an inappropriate expression of the GIP receptor in the pancreatic islet. This hypothesis was tested using a strain of diabetic fatty Zucker rats. The obese rats displayed basal GIP levels similar to the control animals; however, they were unresponsive to a GIP infusion (4 pmol · min–1 · kg–1), whereas the lean animals displayed a significant reduction in blood glucose (GIP levels, 50% control after 60 min, P < 0.05) as well as a significant increase in circulating insulin. GIP also potently stimulated first-phase insulin secretion from isolated perifused islets (10.3 ± 3.0 × basal), and GIP and GLP-1 potentiated insulin secretion from the perfused pancreas (6 × control area under the curve [AUC]) from lean animals. GIP yielded no significant effect in the Vancouver diabetic fatty Zucker (VDF) rat pancreases, whereas GLP-1 elicited an eightfold increase of insulin secretion from the perfused VDF pancreas. Islets from lean animals subjected to static incubations with GIP showed a 2.2-fold increase in cAMP, whereas GIP failed to increase islet cAMP in the VDF islets. Finally, the expression of both GIP receptor mRNA and protein was decreased in islets from VDF rats. These data suggest that the decreased effectiveness of GIP in the VDF rat and in type 2 diabetes may be a result of a decreased receptor expression in the islet.
Proteome Analysis of Interleukin-1β–Induced Changes in Protein Expression in Rat Islets of Langerhans
Authors: Larsen P. Mose , Fey S.J. , Larsen M.R. , Nawrocki A. , Andersen H.U. , Kähler H. , Heilmann C. , Voss M.C. , Roepstorff P. , Pociot F. , Karlsen A.E. , Nerup J.
Published: March 2007

The intracellular molecular events involved in the β-cell death process are complex but poorly understood. Cytokines, e.g., interleukin (IL)-1β, may play a crucial role in inducing this process. Protein synthesis is necessary for the deleterious effect of IL-1, and induction of both protective and deleterious proteins has been described. To characterize the rather complex pattern of islet protein expression in rat islets in response to IL-1, we have attempted to identify proteins of altered expression level after IL-1 exposure by 2D gel electrophoresis and mass spectrometry. Of 105 significantly changed (i.e., up- or downregulated or de novo–induced) protein spots, we obtained positive protein identification for 60 protein spots. The 60 identifications corresponded to 57 different proteins. Of these, 10 proteins were present in two to four spots, suggesting that posttranslatory modifications had occurred. In addition, 11 spots contained more than one protein. The proteins could be classified according to their function into the following groups: 1) energy transduction; 2) glycolytic pathway; 3) protein synthesis, chaperones, and protein folding; and 4) signal transduction, regulation, differentiation, and apoptosis. In conclusion, valuable information about the molecular mechanisms involved in cytokine-mediated β-cell destruction was obtained by this approach.
Altered Nephrogenesis Due to Maternal Diabetes Is Associated With Increased Expression of IGF-II/Mannose-6-Phosphate Receptor in the Fetal Kidney
Authors: Amri Kaouthar , Freund Nicole , Van Huyen J.P. Duong , Merlet-Bénichou Claudie , Lelièvre-Pégorier Martine
Published: March 2007

We have recently demonstrated that the exposure to hyperglycemia in utero impairs nephrogenesis in rat fetuses (Amri K et al., Diabetes 48:2240–2245, 1999). Diabetic pregnancy is commonly associated with alterations in the IGF system in fetal tissues. It has also been shown that both IGF-I and IGF-II are produced within developing metanephros and promote renal organogenesis. Therefore, we investigated the effect of maternal diabetes on IGFs and their receptors in developing fetal rat kidney. Diabetes was induced in pregnant rats by a single injection of streptozotocin on day 0 of gestation. We measured the amounts of IGF and their receptors, both proteins and mRNAs, in the metanephroi of fetuses issued from diabetic subjects and in age-matched fetuses from control subjects (14–20 days of gestation). IGF-II was produced throughout fetal nephrogenesis, whereas IGF-I protein was not detected, suggesting a critical role of IGF-II in kidney development. Fetal exposure to maternal diabetes caused no change in IGF production in the early stages of nephrogenesis. Similarly, the amounts of IGF-I receptor and insulin receptor were not altered. By contrast, there was an increase in production of IGF-II/mannose-6-phosphate receptor throughout nephrogenesis. Because this receptor plays an essential role in regulating the action of IGF-II, the altered nephrogenesis in fetuses exposed to maternal diabetes may be linked to a decrease in IGF-II bioavailability.
Regulation by Insulin of Gene Expression in Human Skeletal Muscle and Adipose Tissue
Authors: Ducluzeau Pierre-Henri , Perretti Noël , Laville Martine , Andreelli Fabrizio , Vega Nathalie , Riou Jean-Paul , Vidal Hubert
Published: March 2007

Defective regulation of gene expression may be involved in the pathogenesis of type 2 diabetes. We have characterized the concerted regulation by insulin (3-h hyperinsulinemic clamp) of the expression of 10 genes related to insulin action in skeletal muscle and in subcutaneous adipose tissue, and we have verified whether a defective regulation of some of them could be specifically encountered in tissues of type 2 diabetic patients. Basal mRNA levels (determined by reverse transcriptase–competitive polymerase chain reaction) of insulin receptor, insulin receptor substrate-1, p85α phosphatidylinositol 3-kinase (PI3K), p110αPI3K, p110βPI3K, GLUT4, glycogen synthase, and sterol regulatory-element-binding protein-1c (SREBP-1c) were similar in muscle of control (n = 17), type 2 diabetic (n = 9), type 1 diabetic (n = 9), and nondiabetic obese (n = 9) subjects. In muscle, the expression of hexokinase II was decreased in type 2 diabetic patients (P < 0.01). In adipose tissue, SREBP-1c (P < 0.01) mRNA expression was reduced in obese (nondiabetic and type 2 diabetic) subjects and was negatively correlated with the BMI of the subjects (r = −0.63, P = 0.02). Insulin (±1,000 pmol/l) induced a two- to threefold increase (P < 0.05) in hexokinase II, p85αPI3K, and SREBP-1c mRNA levels in muscle and in adipose tissue in control subjects, in insulin-resistant nondiabetic obese patients, and in hyperglycemic type 1 diabetic subjects. Upregulation of these genes was completely blunted in type 2 diabetic patients. This study thus provides evidence for a specific defect in the regulation of a group of important genes in response to insulin in peripheral tissues of type 2 diabetic patients.
Thiazolidinediones Increase Plasma-Adipose Tissue FFA Exchange Capacity and Enhance Insulin-Mediated Control of Systemic FFA Availability
Authors: Oakes Nicholas D. , Thalén Pia G. , Jacinto Severina M. , Ljung Bengt
Published: March 2007

We studied the effects of thiazolidinedione treatment (rosiglitazone 1 or 10 μmol · kg−1 · day−1 or darglitazone 1.3 μmol · kg−1 · day−1 for 3 weeks) on lipid metabolism in obese Zucker rats. In the basal 7-h fasted state, rosiglitazone (10 μmol · kg−1 · day−1) and darglitazone corrected the hypertriglyceridemia by increasing plasma triglyceride (TG) clearance and decreasing hepatic TG production, as assessed using Triton WR 1339. Free fatty acid (FFA) metabolism was assessed using 3H-palmitate tracer by estimating rates of plasma FFA appearance (Ra), whole-body FFA oxidation (Rox), and tissue-specific nonoxidative FFA disposal (Rfs). Basal Ra, plasma FFA levels, and clearance were increased by both thiazolidinediones. Detailed studies were conducted with darglitazone, which under basal conditions increased Ra (+114%), Rox (+51%), and Rfs in adipose tissues. During euglycemic clamps performed at insulin levels corresponding to those observed postprandially, darglitazone increased the glucose infusion rate from 4.7 to 13.3 mg · min−1 and, in contrast to the basal state, it decreased Ra (−67%), Rox (−84%), and Rfs in adipose tissue, muscle, and liver. We concluded that thiazolidinediones 1) ameliorate hypertriglyceridemia by lowered hepatic TG production and augmented TG clearance (two separate kinetic effects), 2) enhance insulin-mediated suppression of systemic FFA mobilization while increasing the capacity to mobilize FFA during fasting, 3) increase FFA trafficking into adipose tissue by increasing the ability of adipose tissue to take up and store FFA, and 4) enhance metabolic flexibility by improving glucoregulation under hyperinsulinemic conditions (possibly involving reduced skeletal muscle and liver exposure to fatty acids) and augmenting the capacity to utilize FFAs during fasting.
Diabetic Embryopathy in C57BL/6J Mice
Authors: Machado Antonio F. , Zimmerman Ernest F. , Hovland Jr. David N. , Weiss Robert , Collins Michael D.
Published: March 2007

Maternal diabetes (types 1 and 2) induces a broad array of congenital malformations, including neural tube defects (NTDs), in humans. One of the difficulties associated with studying diabetic embryopathy is the rarity of individual malformations. In an attempt to develop a sensitive animal model for maternal diabetes–induced NTDs, the present study uses chemically induced diabetes in an inbred mouse model with or without the splotch (Sp) mutation, a putatively nonfunctional allele of Pax3. Pax3 deficiency has been associated with an increase in NTDs. Female C57BL/6J mice, either with or without the Sp allele, were injected intravenously with alloxan (100 mg/kg), and plasma glucose was measured 3 days later. A wide range of hyperglycemia was induced, and these diabetic mice were bred to C57BL/6J males, some carrying the Sp allele. Gestational-day-18 fetuses were examined for developmental malformations. Fetuses from matings in which either parent carried the Sp allele were genotyped by polymerase chain reaction. Maternal diabetes significantly decreased fetal weight and increased the number of resorptions and malformations, including NTDs. A significant correlation was found between the level of maternal hyperglycemia and the malformation rate. The sex ratio for live fetuses in diabetic litters was significantly skewed toward male fetuses. Matings involving the Sp allele yielded litters with significantly higher percentages of maternal diabetes–induced spina bifida aperta but not exencephaly, and this increase was shown to be associated with the presence of a single copy of the Sp allele in affected fetuses. Thus, Pax3 haploinsufficiency in this murine model of diabetic embryopathy is associated with caudal but not cranial NTDs.
The HLA-DPB1–Associated Component of the IDDM1 and Its Relationship to the Major Loci HLA-DQB1, -DQA1, and -DRB1
Authors: Cucca Francesco , Dudbridge Frank , Loddo Miriam , Mulargia Anna P. , Lampis Rosannna , Angius Efisio , De Virgiliis Stefano , Koeleman Bobby P.C. , Bain Stephen C. , Barnett Anthony H. , Gilchrist Frances , Cordell Heather , Welsh Ken , Todd John A.
Published: March 2007

The major histocompatibility complex (MHC) HLA region on chromosome 6p21 contains the major locus of type 1 diabetes (IDDM1). Common allelic variants at the class II HLA-DRB1, -DQA1, and -DQB1 loci account for the major part of IDDM1. Previous studies suggested that other MHC loci are likely to contribute to IDDM1, but determination of their relative contributions and identities is difficult because of strong linkage disequilibrium between MHC loci. One prime candidate is the polymorphic HLA-DPB1 locus, which (with the DPA1 locus) encodes the third class II antigen–presenting molecule. However, the results obtained in previous studies appear to be contradictory. Therefore, we have analyzed 408 white European families (200 from Sardinia and 208 from the U.K.) using a combination of association tests designed to directly compare the effect of DPB1 variation on the relative predisposition of DR-DQ haplotypes, taking into account linkage disequilibrium between DPB1 and the DRB1, DQA1, and DQB1 loci. In these populations, the overall contribution of DPB1 to IDDM1 is small. The main component of the DPB1 contribution to IDDM1 in these populations appears to be the protection associated with DPB1*0402 on DR4-negative haplotypes. We suggest that the HLA-DP molecule itself contributes to IDDM1.
A Common Stromal Cell–Derived Factor-1 Chemokine Gene Variant is Associated With the Early Onset of Type 1 Diabetes
Authors: Dubois-Laforgue Danièle , Hendel Houria , Caillat-Zucman Sophie , Zagury Jean-François , Winkler Cheryl , Boitard Christian , Timsit José
Published: March 2007

Type 1 diabetes results from the autoimmune destruction of pancreatic β-cells. Although the disease shows a strong association with HLA class II alleles, other genes may influence the initiation or the rate of progression of the autoimmune process. The recruitment of mononuclear cells within the islets of Langerhans is a critical step in the pathogenesis of the disease. Because chemokines are cytokines that promote migration of mononuclear cells, we hypothesized that polymorphisms in chemokine receptor or chemokine genes, CCR5 and SDF1, may be involved in susceptibility to or clinical expression of type 1 diabetes. The frequencies of the CCR5-Δ32 and SDF1-3′A (801G→A in the 3′ untranslated region) variants were similar in 208 unrelated Caucasian patients with type 1 diabetes and in 120 Caucasian control subjects. They were not modified after stratification for the predisposing HLA-DR3 and -DR4 haplotypes. However, the SDF1-3′A variant was strongly associated with early onset (<15 years) of the disease (odds ratio 2.6, P = 0.0019). On average, the presence of the SDF1-3′A allele was associated with a 5-year reduction in the age at onset of diabetes (P = 0.0067). Our results suggest that stromal cell–derived factor-1 may be implicated in the aggressiveness of the autoimmune process leading to type 1 diabetes. These preliminary data require replication in other populations.
The Transplanted Fetal Endocrine Pancreas Undergoes an Inherent Sequential Differentiation Similar to That in the Native Pancreas
Authors: Lukinius Agneta , Korsgren Olle
Published: March 2007

This study examines, at the ultrastructural level, whether the fetal porcine endocrine pancreas (insulin, glucagon, somatostatin, and pancreatic polypeptide [PP]- and islet amyloid polypeptide [IAPP]-containing cells) develops normally after transplantation under the kidney capsule in athymic mice. We have thus used an in vivo pig-to-mouse model for the differentiation of the endocrine pancreas removed from its normal milieu. Islet-like cell clusters (ICCs) were prepared from the fetal porcine pancreas as previously described and transplanted under the renal capsule of athymic mice. At various times after transplantation, the endocrine pancreas was removed and the level of differentiation was compared with the native pancreas of the same biological age. At the ultrastructural level, several sequential steps could be identified based on the morphology and hormone content of the secretory granules of the endocrine cell examined. Applying this approach, we could demonstrate that the ontogeny of the transplanted fetal pig pancreas follows the same sequential differentiation as the native pancreas. The process seems to be under stringent control, apparently directly related to the biological age of the tissue, and independent not only of the new environment under the kidney capsule but also of the adult and xenogeneic milieu provided after transplantation to the athymic nude mouse. Therefore, all four major hormone-producing cells seem to develop normally after transplantation when compared with the development in the native pancreas. IAPP was produced by the pluripotent fetal endocrine cells as well as the adult α-, β-, and δ-cell granules in the native pancreas; however, in the transplanted pancreas, IAPP expression was demonstrated only in β-cells, δ-cells, and PP cells. No IAPP was found in granules of the α-cell lineage. The results suggest a sequential differentiation of all four major types of islet cells from a common pluripotent progenitor cell, which seems to be located in the pancreatic ducts. Therefore, the results presented strongly suggest that the ontogeny of the four major endocrine islet cells is determined by genetic information carried by the progenitor cells and not by the systemic or local environment.
Adenovirus Early Region 3 (E3) Immunomodulatory Genes Decrease the Incidence of Autoimmune Diabetes in NOD Mice
Authors: Efrat Shimon , Serreze David , Svetlanov Anton , Post Cristina M. , Johnson Ellis A. , Herold Kevan , Horwitz Marshall
Published: March 2007

The early three (E3) region of the adenovirus (Ad) encodes a number of immunomodulatory proteins that interfere with class I major histocompatibility–mediated antigen presentation and confer resistance to cytokine-induced apoptosis in cells infected by the virus. Transgenic expression of Ad E3 genes under the rat insulin II promoter (RIP-E3) in β-cells in nonobese diabetic (NOD) mice decreases the incidence and delays the onset of autoimmune diabetes. The immune effector cells of RIP-E3/NOD mice maintain the ability to infiltrate the islets and transfer diabetes into NOD-scid recipients, although at a significantly reduced rate compared with wild-type littermates. The islets of RIP-E3/NOD mice can be destroyed by adoptive transfer of splenocytes from wild-type NOD mice; however, the time to onset of hyperglycemia is delayed significantly, and 40% of these recipients were not diabetic at the end of the experiment. These findings suggest that expression of E3 genes in β-cells affects both the activation of immune effector cells and the intrinsic resistance of β-cells to autoimmune destruction.
Human Anti-CD38 Autoantibodies Raise Intracellular Calcium and Stimulate Insulin Release in Human Pancreatic Islets
Authors: Antonelli Alessandro , Baj Germano , Marchetti Piero , Fallahi Poupak , Surico Nicola , Pupilli Cinzia , Malavasi Fabio , Ferrannini Ele
Published: March 2007

CD38 is involved in transmembrane signaling in many cell types; anti-CD38 autoantibodies have been described in diabetic patients. We tested whether human anti-CD38 antibodies possess signaling properties by measuring their ability to raise intracellular calcium ([Ca2+]i) using the fluo-3-acetoxymethyl ester method in a human-derived T-cell line (Jurkat T-cells, expressing high levels of surface CD38) and in dispersed human islet cells from normal donors. In Jurkat T-cells, 11 of 19 anti–CD38-positive sera raised [Ca2+]i (by ≥20% of baseline), whereas no [Ca2+]i-mobilizing activity was found in 27 anti–CD38-negative sera (χ2 = 20.5, P < 0.0001). In dispersed human islet cells, 5 of 11 anti–CD38-positive sera (and none of three anti–CD38-negative sera) raised [Ca2+]i significantly. When preincubated with Staphylococcus aureus protein A to remove IgG, anti–CD38-positive sera showed a 70 ± 5% reduction in [Ca2+]i-mobilizing activity. Preincubation with CD38-transfected NIH-3T3 fibroblasts, but not with mock-transfected NIH-3T3 cells, abolished [Ca2+]i mobilization. In blocking experiments, preincubation with nonagonistic anti–CD38 monoclonal antibodies also prevented [Ca2+]i mobilization. In cultured human islets, anti–CD38-positive sera exhibiting [Ca2+]i-mobilizing activity in Jurkat T-cells (n = 6) significantly stimulated insulin release at 3.3 mmol/l glucose (median [interquartile range] 738 μU/ml [234], P = 0.0001 vs. 320 [52] μU/ml of control), whereas 6 anti-CD38-positive sera without [Ca2+]i-mobilizing activity and 10 anti–CD38-negative did not. In further incubations, the five anti–CD38-positive sera displaying [Ca2+]i-mobilizing activity in dispersed islet cells significantly stimulated insulin release at both 3.3 mmol/l glucose (2.2 ± 0.3% of insulin islet content, P < 0.002 vs. 1.2 ± 0.1% of control) and 16.7 mmol/l glucose (3.7 ± 0.3 vs. 2.3 ± 0.3%, P < 0.002). We conclude that human anti–CD38 autoantibodies with agonistic properties on the CD38 effector system occur in nature; in human islets, their [Ca2+]i-mobilizing activity is coupled with the ability to stimulate insulin release.
The Effect of Systemic Versus Portal Insulin Delivery in Pancreas Transplantation on Insulin Action and VLDL Metabolism
Authors: Carpentier André , Patterson Bruce W. , Uffelman Kristine D. , Giacca Adria , Vranic Mladen , Cattral Mark S. , Lewis Gary F.
Published: March 2007

Combined kidney-pancreas transplantation (KPT) with anastomosis of the pancreatic vein to the systemic circulation (KPT-S) or to the portal circulation (KPT-P) provides a human model in which the chronic effects of portal versus systemic insulin delivery on glucose and VLDL metabolism can be examined. Despite similar plasma glucose and C-peptide levels, KPT-S (n = 9) had an approximate twofold elevation of fasting and intravenous glucose–stimulated plasma insulin levels compared with both KPT-P (n = 7) and healthy control subjects (n = 15). The plasma free fatty acid (FFA) levels were elevated in both transplant groups versus control subjects, but the plasma insulin elevation necessary to lower plasma FFA by 50% was approximately two times higher in KPT-S versus KPT-P and control subjects. Endogenous glucose production was similar in KPT-S and KPT-P, despite ∼35% higher hepatic insulin levels in the latter, and was suppressed to a greater extent during a euglycemic-hyperinsulinemic clamp in KPT-S versus KPT-P. Total-body glucose utilization during the euglycemic-hyperinsulinemic clamp was ∼40% lower in KPT-S versus KPT-P, indicating peripheral tissue but not hepatic insulin resistance in KPT-S versus KPT-P. Both transplant groups had an approximate twofold elevation of triglyceride (TG)-rich lipoprotein apolipoprotein B (apoB) and lipids versus control subjects. Elevation of VLDL-apoB and VLDL-TG in both transplant groups was entirely explained by an ∼50% reduction in clearance of VLDL compared with healthy control subjects. In the presence of increased FFA load but in the absence of hepatic overinsulinization and marked hepatic insulin resistance, there was no elevation of VLDL secretion in KPT-S versus KPT-P and control subjects. These findings suggest that chronic systemic hyperinsulinemia and peripheral tissue insulin resistance with the consequent elevation of plasma FFA flux are insufficient per se to cause VLDL overproduction and that additional factors, such as hepatic hyperinsulinemia and/or gross insulin resistance, may be an essential prerequisite in the pathogenesis of VLDL overproduction in the common form of the insulin resistance syndrome.
Transgenic Overexpression of Leptin Rescues Insulin Resistance and Diabetes in a Mouse Model of Lipoatrophic Diabetes
Authors: Ebihara Ken , Ogawa Yoshihiro , Masuzaki Hiroaki , Shintani Mitsuyo , Miyanaga Fumiko , Aizawa-Abe Megumi , Hayashi Tatsuya , Hosoda Kiminori , Inoue Gen , Yoshimasa Yasunao , Gavrilova Oksana , Reitman Marc L. , Nakao Kazuwa
Published: March 2007

Lipoatrophic diabetes is caused by a deficiency of adipose tissue and is characterized by severe insulin resistance, hypoleptinemia, and hyperphagia. The A-ZIP/F-1 mouse (A-ZIPTg/+) is a model of severe lipoatrophic diabetes and is insulin resistant, hypoleptinemic, hyperphagic, and shows severe hepatic steatosis. We have also produced transgenic “skinny” mice that have hepatic overexpression of leptin (LepTg/+) and no adipocyte triglyceride stores, and are hypophagic and show increased insulin sensitivity. To explore the pathophysiological and therapeutic roles of leptin in lipoatrophic diabetes, we crossed LepTg/+ and A-ZIPTg/+ mice, producing doubly transgenic mice (LepTg/+:A-ZIPTg/+) virtually lacking adipose tissue but having greatly elevated leptin levels. The LepTg/+:A-ZIPTg/+ mice were hypophagic and showed improved hepatic steatosis. Glucose and insulin tolerance tests revealed increased insulin sensitivity, comparable to LepTg/+ mice. These effects were stable over at least 6 months of age. Pair-feeding the A-ZIPTg/+ mice to the amount of food consumed by LepTg/+:A-ZIPTg/+ mice did not improve their insulin resistance, diabetes, or hepatic steatosis, demonstrating that the beneficial effects of leptin were not due to the decreased food intake. Continuous leptin administration that elevates plasma leptin concentrations to those of LepTg/+:A-ZIPTg/+ mice also effectively improved hepatic steatosis and the disorder of glucose and lipid metabolism in A-ZIP/F-1 mice. These data demonstrate that leptin can improve the insulin resistance and diabetes of a mouse model of severe lipoatrophic diabetes, suggesting that leptin may be therapeutically useful in the long-term treatment of lipoatrophic diabetes.
Subcellular Localization of Insulin Receptor Substrate Family Proteins Associated With Phosphatidylinositol 3-Kinase Activity and Alterations in Lipolysis in Primary Mouse Adipocytes From IRS-1 Null Mice
Authors: Tsuji Youki , Kaburagi Yasushi , Terauchi Yasuo , Satoh Shinobu , Kubota Naoto , Tamemoto Hiroyuki , Kraemer Fredric B. , Sekihara Hisahiko , Aizawa Shinichi , Akanuma Yasuo , Tobe Kazuyuki , Kimura Satoshi , Kadowaki Takashi
Published: March 2007

To clarify the roles of insulin receptor substrate (IRS) family proteins in phosphatidylinositol (PI) 3-kinase activation and insulin actions in adipocytes, we investigated the intracellular localization of IRS family proteins and PI 3-kinase activation in response to insulin by fractionation of mouse adipocytes from wild-type and IRS-1 null mice. In adipocytes from wild-type mice, tyrosine-phosphorylated IRS-1 and IRS-2, which were found to associate with PI 3-kinase in response to insulin, were detected in the plasma membrane (PM) and low-density microsome (LDM) fractions. By contrast, tyrosine-phosphorylated IRS-3 (pp60), which was found to associate with PI 3-kinase, was predominantly localized in the PM fraction. In adipocytes from IRS-1–null mice, insulin-stimulated PI 3-kinase activity in anti-phosphotyrosine (αPY) immunoprecipitates in the LDM fraction was almost exclusively mediated via IRS-2 and was reduced to 25%; however, insulin-stimulated PI 3-kinase activity in the PM fraction was primarily mediated via IRS-3 and was reduced to 60%. To determine the potential functional impact of the distinct subcellular localization of IRSs and associating PI 3-kinase activity on adipocyte-specific metabolic actions, we examined lipolysis in IRS-1 null mice. The level of isoproterenol-induced lipolysis was increased 5.1-fold in adipocytes from IRS-1 null mice as compared with wild-type mice. Moreover, hormone-sensitive lipase (HSL) protein was increased 4.3-fold in adipocytes from IRS-1–null mice compared with wild-type mice, and HSL mRNA expression was also increased. The antilipolytic effect of insulin in IRS-1 null adipocytes, however, was comparable to that in wild-type mice. Thus, discordance between these two insulin actions as well as the transcriptional and translational effect (HSL mRNA and protein regulation) and the PM effect (antilipolysis) of insulin may be explained by distinct roles of both PI 3-kinase activity associated with IRS-1/IRS-2 and PI 3-kinase activity associated with IRS-3 in insulin actions related to their subcellular localization.
Requirement for p38 and p44/p42 Mitogen-Activated Protein Kinases in RAGE-Mediated Nuclear Factor-κB Transcriptional Activation and Cytokine Secretion
Authors: Yeh Chen-Hsiung , Sturgis Lydia , Haidacher Joe , Zhang Xue-Nong , Sherwood Sidney J. , Bjercke Robert J. , Juhasz Ondrej , Crow Michael T. , Tilton Ronald G. , Denner Larry
Published: March 2007

Advanced glycation end product (AGE) activation of the signal-transducing receptor for AGE (RAGE) has been linked to a proinflammatory phenotypic change within cells. However, the precise intracellular signaling pathways involved have not been elucidated. We demonstrate here that human serum albumin modified with Nε-(carboxymethyl)lysine (CML), a major AGE adduct that progressively accumulates with aging, diabetes, and renal failure, induced nuclear factor (NF)-κB–driven reporter gene expression in human monocytic THP-1 cells. The NF-κB response was blocked with a synthetic peptide corresponding to the putative ligand-binding domain of RAGE, with anti-RAGE antiserum, and by coexpression of truncated receptors lacking the intracellular domain. Signal transduction from RAGE to NF-κB involved the generation of reactive oxygen species, since reporter gene expression was blocked with the antioxidant N-acetyl-l-cysteine. CML-modified albumin produced rapid transient activation of tyrosine phosphorylation, extracellular signal-regulated kinase 1 and 2, and p38 mitogen-activated protein kinase (MAPK), but not c-Jun NH2-terminal kinase. RAGE-mediated NF-κB activation was suppressed by the selective p38 MAPK inhibitor SB203580 and by coexpression of a kinase-dead p38 dominant-negative mutant. Activation of NF-κB by CML-modified albumin increased secretion of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and monocyte chemoattractant protein-1) severalfold, and inhibition of p38 MAPK blocked these increases. These results indicate that p38 MAPK activation mediates RAGE-induced NF-κB–dependent secretion of proinflammatory cytokines and suggest that accelerated inflammation may be a consequence of cellular activation induced by this receptor.
Transcriptional Regulation of Plasminogen Activator Inhibitor Type 1 Gene by Insulin
Authors: Banfi Cristina , Eriksson Per , Giandomenico Giovanna , Mussoni Luciana , Sironi Luigi , Hamsten Anders , Tremoli Elena
Published: March 2007

Impairment of the fibrinolytic system, caused primarily by increases in the plasma levels of plasminogen activator inhibitor (PAI) type 1, are frequently found in diabetes and the insulin-resistance syndrome. Among the factors responsible for the increases of PAI-1, insulin has recently attracted attention. In this study, we analyzed the effects of insulin on PAI-1 biosynthesis in HepG2 cells, paying particular attention to the signaling network evoked by this hormone. Experiments performed in CHO cells overexpressing the insulin receptor indicate that insulin increases PAI-1 gene transcription through interaction with its receptor. By using inhibitors of the different signaling pathways evoked by insulin-receptor binding, it has been shown that the biosynthesis of PAI-1 is due to phosphatidylinositol (PI) 3-kinase activation, followed by protein kinase C and ultimately by mitogen-activated protein (MAP) kinase activation and extracellular signal–regulated kinase 2 phosphorylation. We also showed that this pathway is Ras-independent. Transfection of HepG2 cells with several truncations of the PAI-1 promoter coupled to a CAT gene allowed us to recognize two major response elements located in the regions between −804 and −708 and between −211 and −54. Electrophoretic mobility shift assay identified three binding sites for insulin-induced factors, all colocalized with putative Sp1 binding sites. Using supershifting antibodies, the binding of Sp1 could only be confirmed at the binding site located just upstream from the transcription start site of the PAI-1 promoter. A construct comprising four tandem repeat copies of the −93/−62 region of the PAI-1 promoter linked to CAT was transcriptionally activated in HepG2 cells by insulin. These results outline the central role of MAP kinase activation in the regulation of PAI-1 induced by insulin.
Evidence for a Major Role of Skeletal Muscle Lipolysis in the Regulation of Lipid Oxidation During Caloric Restriction In Vivo
Authors: Hagström-Toft Eva , Thörne Anders , Reynisdottir Signy , Moberg Erik , Rössner Stephan , Bolinder Jan , Arner Peter
Published: March 2007

A lipolytic process in skeletal muscle has recently been demonstrated. However, the physiological importance of this process is unknown. We investigated the role of skeletal muscle lipolysis for lipid utilization during caloric restriction in eight obese women before and after 11 days of very low–calorie diet (VLCD) (2.2 MJ per day). Subjects were studied with indirect calorimetry and microdialysis of skeletal muscle and adipose tissue in order to analyze substrate utilization and glycerol (lipolysis index) in connection with a two-step euglycemic-hyperinsulinemic (12 and 80 mU/m2 · min) clamp. Local blood flow rates in the two tissues were determined with 133Xe-clearance. Circulating free fatty acids and glycerol decreased to a similar extent during insulin infusion before and during VLCD, and there was a less marked insulin-induced reduction in lipid oxidation during VLCD. Adipose tissue glycerol release was hampered by insulin infusion to the same extent (∼40%) before and during VLCD. Skeletal muscle glycerol release was not influenced by insulin before VLCD. However, during VLCD insulin caused a marked (fivefold) (P < 0.01) increase in skeletal muscle glycerol release. The effect was accompanied by a fourfold stimulation of skeletal muscle blood flow (P < 0.01). We propose that, during short-term caloric restriction, the reduced ability of insulin to inhibit lipids, despite a preserved antilipolytic effect of the hormone in adipose tissue, is caused by an augmented mobilization of fat from skeletal muscle, and that a physiological role of muscle lipolysis provides a local source of fatty acids.
Alteration in Phosphorylation of P20 Is Associated With Insulin Resistance
Authors: Wang Yu , Xu Aimin , Ye Jiming , Kraegen Edward W. , Tse Cynthia A. , Cooper Garth J.S.
Published: March 2007

We have recently identified a small phosphoprotein, P20, as a common intracellular target for insulin and several of its antagonists, including amylin, epinephrine, and calcitonin gene-related peptide. These hormones elicit phosphorylation of P20 at its different sites, producing three phosphorylated isoforms: S1 with an isoelectric point (pI) value of 6.0, S2 with a pI value of 5.9, and S3 with a pI value of 5.6 (FEBS Letters 457:149–152 and 462:25–30, 1999). In the current study, we showed that P20 is one of the most abundant phosphoproteins in rat extensor digitorum longus (EDL) muscle. Insulin and amylin antagonize each other’s actions in the phosphorylation of this protein in rat EDL muscle. Insulin inhibits amylin-evoked phosphorylation of S2 and S3, whereas amylin decreases insulin-induced phosphorylation of S1. In rats made insulin resistant by dexamethasone treatment, levels of the phosphoisoforms S2 and S3, which were barely detectable in healthy rats in the absence of hormone stimulation, were significantly increased. Moreover, the ability of insulin to inhibit amylin-evoked phosphorylation of these two isoforms was greatly attenuated. These results suggested that alterations in the phosphorylation of P20 might be associated with insulin resistance and that P20 could serve as a useful marker to dissect the cellular mechanisms of this disease.
Effects of Pioglitazone on Adipose Tissue Remodeling Within the Setting of Obesity and Insulin Resistance
Authors: de Souza Christopher J. , Eckhardt Michele , Gagen Karen , Dong Mei , Chen Wei , Laurent Didier , Burkey Bryan F.
Published: March 2007

Obesity and dysfunctional energy partitioning can lead to the development of insulin resistance and type 2 diabetes. The antidiabetic thiazolidinediones shift the energy balance toward storage, leading to an increase in whole-body adiposity. These studies examine the effects of pioglitazone (Pio) on adipose tissue physiology, accumulation, and distribution in female Zucker (fa/fa) rats. Pio treatment (up to 28 days) decreased the insulin-resistant and hyperlipidemic states and increased food consumption and whole-body adiposity. Magnetic resonance imaging (MRI) analysis and weights of fat pads demonstrated that the increase in adiposity was not only limited to the major fat depots but also to fat deposition throughout the body. Adipocyte sizing profiles, fat pad histology, and DNA content show that Pio treatment increased the number of small adipocytes because of both the appearance of new adipocytes and the shrinkage and/or disappearance of existing mature adipocytes. The remodeling was time dependent, with new small adipocytes appearing in clusters throughout the fat pad, and accompanied by a three- to fourfold increase in citrate synthase and fatty acid synthase activity. The appearance of new fat cells and the increase in fat mass were depot specific, with a rank order of responsiveness of ovarian > retroperitoneal > subcutaneous. This differential depot effect resulted in a redistribution of the fat mass in the abdominal region such that there was an increase in the visceral:subcutaneous ratio, as confirmed by MRI analysis. Although the increased adiposity is paradoxical to an improvement in insulin sensitivity, the quantitative increase of adipose mass should be viewed in context of the qualitative changes in adipose tissue, including the remodeling of adipocytes to a smaller size with higher lipid storage potential. This shift in energy balance is likely to result in lower circulating free fatty acid levels, ultimately improving insulin sensitivity and the metabolic state.
Bezafibrate Reduces mRNA Levels of Adipocyte Markers and Increases Fatty Acid Oxidation in Primary Culture of Adipocytes
Authors: Cabrero Àgatha , Alegret Marta , Sánchez Rosa M. , Adzet Tomás , Laguna Juan C. , Vázquez Manuel
Published: March 2007

The molecular mechanisms by which peroxisome proliferator–activated receptor (PPAR) activation by fibrates reduces fat deposition and improves insulin sensitivity are not completely understood. We report that exposure of a rat primary culture of adipocytes for 24 h to the PPAR activator bezafibrate increased the mRNA levels of crucial genes involved in peroxisomal and mitochondrial β-oxidation. The mRNA levels of the peroxisomal β-oxidation rate-limiting enzyme acyl-CoA oxidase and of the muscle-type carnitine palmitoyl transferase I (M-CPT-I), which determines the flux of mitochondrial β-oxidation, increased by 1.6-fold (P < 0.02) and 4.5-fold (P = 0.001), respectively. These changes were accompanied by an increase in the transcript levels of the uncoupling protein-2 (UCP-2; 1.5-fold induction; P < 0.05) and UCP-3 (3.7-fold induction; P < 0.001), mitochondrial proteins that reduce ATP yield and may facilitate the oxidation of fatty acids. Furthermore, bezafibrate increased the mRNA levels of the fatty acid translocase (2-fold induction; P < 0.01), suggesting a higher fatty acid uptake into adipocytes. In agreement with these changes, bezafibrate caused a 1.9-fold induction (P < 0.02) in 9,10-[3H]palmitate oxidation. Moreover, bezafibrate reduced the mRNA expression of several adipocyte markers, including PPARγ (30% reduction; P = 0.05), tumor necrosis factor-α (33% reduction; P < 0.05), and the ob gene (26% reduction). In contrast, adipocyte fatty acid binding protein mRNA levels increased (1.5-fold induction; P < 0.01), pointing to a mobilization of fatty acids to mitochondria and peroxisomes. The reduction of the adipocyte markers caused by bezafibrate was accompanied by an increase in the mRNA levels of the preadipocyte marker Pref-1 (1.6-fold induction; P < 0.01). Some of the changes observed in the primary culture of rat adipocytes also were studied in the epididymal white adipose tissue of bezafibrate-treated rats for 7 days. In vivo, M-CPT-I mRNA levels increased (4.5-fold induction; P = 0.001) in epididymal white adipose tissue of bezafibrate-treated rats. Similarly, fatty acid translocase (2.6-fold induction; P = 0.002) and Pref-1 (5.6-fold induction) mRNA levels increased, although differences in the latter were not significant because of huge individual variations. These results indicate that exposure of adipocytes to bezafibrate, independent of its hepatic effects, increases the degradation of fatty acids, reducing their availability to synthesize triglycerides. As a result, some degree of dedifferentiation of adipocytes to preadipocyte-like cells is achieved. These changes may be involved in the reduction in fat depots and in the improvement of insulin sensitivity observed after bezafibrate treatment.