bluuebunny/crossref_metadata_2025 huggingface dataset:
A Daily Rhythm in Glucose Tolerance
Authors: la Fleur Susanne E. , Kalsbeek Andries , Wortel Joke , Fekkes Madelon L. , Buijs Ruud M.
Published: March 2007

The suprachiasmatic nucleus (SCN), the biological clock, is responsible for a 24-h rhythm in plasma glucose concentrations, with the highest concentrations toward the beginning of the activity period. To investigate whether the SCN is also responsible for daily fluctuations in glucose uptake and to examine how these fluctuations relate to the rhythm in plasma glucose concentrations, SCN-intact rats and SCN-lesioned rats were injected intravenously with a glucose bolus at different time points. We found an increase in glucose uptake toward the beginning of the activity period, followed by a gradual reduction in glucose uptake toward the end of the activity period. The daily variation in glucose tolerance seemed not to be caused by fluctuations in insulin responses of the pancreas but by a daily variation in insulin sensitivity. Lesioning the SCN resulted in the disappearance of the daily fluctuation in glucose uptake and insulin sensitivity. Interestingly, SCN-lesioned rats showed an enhancement in glucose tolerance that could not be explained by higher insulin responses or enhanced insulin sensitivity. Therefore, these findings suggest a role for the SCN in insulin-independent glucose uptake. The present results further show that the daily rhythm in glucose tolerance follows the same pattern as the daily rhythm in plasma glucose concentrations. We hypothesized that the biological clock prepares the individual for the upcoming activity period by two separate mechanisms: increasing plasma glucose concentrations and making tissue more tolerant to glucose.
Protein Kinase C (PKC)-α Activation Inhibits PKC-ζ and Mediates the Action of PED/PEA-15 on Glucose Transport in the L6 Skeletal Muscle Cells
Authors: Condorelli Gerolama , Vigliotta Giovanni , Trencia Alessandra , Maitan Maria Alessandra , Caruso Matilde , Miele Claudia , Oriente Francesco , Santopietro Stefania , Formisano Pietro , Beguinot Francesco
Published: March 2007

Overexpression of the PED/PEA-15 protein in muscle and adipose cells increases glucose transport and impairs further insulin induction. Like glucose transport, protein kinase C (PKC)-α and -β are also constitutively activated and are not further stimulatable by insulin in L6 skeletal muscle cells overexpressing PED (L6PED). PKC-ζ features no basal change but completely loses insulin sensitivity in L6PED. In these cells, blockage of PKC-α and -β additively returns 2-deoxy-d-glucose (2-DG) uptake to the levels of cells expressing only endogenous PED (L6WT). Blockage of PKC-α and -β also restores insulin activation of PKC-ζ in L6PED cells, with that of PKC-α sixfold more effective than PKC-β. Similar effects on 2-DG uptake and PKC-ζ were also achieved by 50-fold overexpression of PKC-ζ in L6PED. In L6WT, fivefold overexpression of PKC-α or -β increases basal 2-DG uptake and impairs further insulin induction with no effect on insulin receptor or insulin receptor substrate phosphorylation. In these cells, overexpression of PKC-α blocks insulin induction of PKC-ζ activity. PKC-β is 10-fold less effective than PKC-α in inhibiting PKC-ζ stimulation. Expression of the dominant-negative K281→W PKC-ζ mutant simultaneously inhibits insulin activation of PKC-ζ and 2-DG uptake in the L6WT cells. We conclude that activation of classic PKCs, mainly PKC-α, inhibits PKC-ζ and may mediate the action of PED on glucose uptake in L6 skeletal muscle cells.
Evidence That Extrapancreatic GLUT2-Dependent Glucose Sensors Control Glucagon Secretion
Authors: Burcelin Rémy , Thorens Bernard
Published: March 2007

GLUT2−/− mice reexpressing GLUT1 or GLUT2 in their β-cells (RIPGLUT1 × GLUT2−/− or RIPGLUT2 × GLUT2−/− mice) have nearly normal glucose-stimulated insulin secretion but show high glucagonemia in the fed state. Because this suggested impaired control of glucagon secretion, we set out to directly evaluate the control of glucagonemia by variations in blood glucose concentrations. Using fasted RIPGLUT1 × GLUT2−/− mice, we showed that glucagonemia was no longer increased by hypoglycemic (2.5 mmol/l glucose) clamps or suppressed by hyperglycemic (10 and 20 mmol/l glucose) clamps. However, an increase in plasma glucagon levels was detected when glycemia was decreased to ≤1 mmol/l, indicating preserved glucagon secretory ability, but of reduced sensitivity to glucopenia. To evaluate whether the high-fed glucagonemia could be due to an abnormally increased tone of the autonomic nervous system, fed mutant mice were injected with the ganglionic blockers hexamethonium and chlorisondamine. Both drugs lead to a rapid return of glucagonemia to the levels found in control fed mice. We conclude that 1) in the absence of GLUT2, there is an impaired control of glucagon secretion by low or high glucose; 2) this impaired glucagon secretory activity cannot be due to absence of GLUT2 from α-cells because these cells do not normally express this transporter; 3) this dysregulation may be due to inactivation of GLUT2-dependent glucose sensors located outside the endocrine pancreas and controlling glucagon secretion; and 4) because fed hyperglucagonemia is rapidly reversed by ganglionic blockers, this suggests that in the absence of GLUT2, there is an increased activity of the autonomic nervous system stimulating glucagon secretion during the fed state.
A Neuronal Isoform of Nitric Oxide Synthase Expressed in Pancreatic β-Cells Controls Insulin Secretion
Authors: Lajoix Anne-Dominique , Reggio Hubbert , Chardès Thierry , Péraldi-Roux Sylvie , Tribillac Florence , Roye Michèle , Dietz Samuel , Broca Christophe , Manteghetti Michèle , Ribes Gérard , Wollheim Claes B. , Gross René
Published: March 2007

Evidence is presented showing that a neuronal isoform of nitric oxide synthase (NOS) is expressed in rat pancreatic islets and INS-1 cells. Sequencing of the coding region indicated a 99.8% homology with rat neuronal NOS (nNOS) with four mutations, three of them resulting in modifications of the amino acid sequence. Double-immunofluorescence studies demonstrated the presence of nNOS in insulin-secreting β-cells. Electron microscopy studies showed that nNOS was mainly localized in insulin secretory granules and to a lesser extent in the mitochondria and the nucleus. We also studied the mechanism involved in the dysfunction of the β-cell response to arginine and glucose after nNOS blockade with NG-nitro-l-arginine methyl ester. Our data show that miconazole, an inhibitor of nNOS cytochrome c reductase activity, either alone for the experiments with arginine or combined with sodium nitroprusside for glucose, is able to restore normal secretory patterns in response to the two secretagogues. Furthermore, these results were corroborated by the demonstration of a direct enzyme-substrate interaction between nNOS and cytochrome c, which is strongly reinforced in the presence of the NOS inhibitor. Thus, we provide immunochemical and pharmacological evidence that β-cell nNOS exerts, like brain nNOS, two catalytic activities: a nitric oxide production and an NOS nonoxidating reductase activity, both of which are essential for normal β-cell function. In conclusion, we suggest that an imbalance between these activities might be implicated in β-cell dysregulation involved in certain pathological hyperinsulinic states.
GLUT4 Is Reduced in Slow Muscle Fibers of Type 2 Diabetic Patients
Authors: Gaster Michael , Staehr Peter , Beck-Nielsen Henning , Schrøder Henrick D. , Handberg Aase
Published: March 2007

To gain further insight into the mechanisms underlying muscle insulin resistance, the influence of obesity and type 2 diabetes on GLUT4 immunoreactivity in slow and fast skeletal muscle fibers was studied. Through a newly developed, very sensitive method using immunohistochemistry combined with morphometry, GLUT4 density was found to be significantly higher in slow compared with fast fibers in biopsy specimens from lean and obese subjects. In contrast, in type 2 diabetic subjects, GLUT4 density was significantly lower in slow compared with fast fibers. GLUT4 density in slow fibers from diabetic patients was reduced by 9% compared with the weight-matched obese subjects and by 18% compared with the lean control group. The slow-fiber fraction was reduced to 86% in the obese subjects and to 75% in the diabetic subjects compared with the control group. Estimated GLUT4 contribution from slow fibers was reduced to 77% in the obese subjects and to 61% in type 2 diabetic patients compared with the control subjects. We propose that a reduction in the fraction of slow-twitch fibers, combined with a reduction in GLUT4 expression in slow fibers, may reduce the insulin-sensitive GLUT4 pool in type 2 diabetes and thus contribute to skeletal muscle insulin resistance.
Protein Metabolism in Clinically Stable Adult Cystic Fibrosis Patients With Abnormal Glucose Tolerance
Authors: Moran Antoinette , Milla Carlos , Ducret Rene , Nair K. Sreekumaran
Published: March 2007

Cystic fibrosis (CF) patients are reported to experience chronic protein catabolism. Since diabetes or impaired glucose tolerance (IGT) is common in CF, we hypothesized that their protein catabolic state is related to reduced insulin secretion or reduced insulin action. A total of 12 clinically stable adult CF patients with abnormal glucose tolerance and 12 age-, sex-, and lean body mass–matched healthy control subjects underwent protein turnover studies using l-[1-13C]leucine, l-[15N]phenylalanine, and l-[2H4]tyrosine, with and without exogenous insulin infusion. In the baseline fasting state, protein metabolism was entirely normal in CF patients, with no evidence of increased protein catabolism. In contrast, striking abnormalities were seen in CF patients when insulin was infused, since they did not experience normal suppression of the appearance rates of leucine, phenylalanine, or tyrosine (indexes of protein breakdown). At an insulin concentration of 45 ± 2 μU/ml, normal control subjects suppressed the leucine appearance rate by 19 ± 5% (P < 0.01), ketoisocaproate appearance rate by 10 ± 3% (P = 0.03), tyrosine appearance rate by 11 ± 2% (P = 0.03), and phenylalanine appearance rate by 6 ± 3% (P = 0.07). Phenylalanine conversion to tyrosine decreased by 22 ± 7% (P = 0.03). At a similar insulin concentration of 44 ± 3 μU/ml, normal suppression of amino acid appearance did not occur in CF. The leucine appearance rate decreased by 4 ± 2% (P = 0.65), ketoisocaproate appearance rate by 1 ± 2% (P = 0.94), tyrosine appearance rate by 0 ± 6% (P = 0.56), phenylalanine appearance rate by 5 ± 6% (P = 0.34), and phenylalanine conversion to tyrosine by 5 ± 6% (P = 0.95). Poor suppression of the amino acid appearance rate in CF was not related to previously documented glucose tolerance status (IGT or CF-related diabetes without fasting hyperglycemia), fasting insulin levels, the acute insulin response, insulin sensitivity, cytokine or counterregulatory hormone levels, resting energy expenditure, caloric intake, pulmonary function, or clinical status. Protein synthesis was not significantly affected by insulin infusion in either normal control subjects or CF patients. In conclusion, clinically stable adult CF patients have normal indexes of protein breakdown and synthesis in the fasting state. In contrast, elevation of plasma insulin to physiological postprandial levels fails to normally suppress indexes of protein breakdown. It is therefore likely that inability to spare protein during the postprandial state is the cause of protein catabolism in these patients.
Type 2 Diabetes Impairs Splanchnic Uptake of Glucose but Does Not Alter Intestinal Glucose Absorption During Enteral Glucose Feeding
Authors: Basu Ananda , Basu Rita , Shah Pankaj , Vella Adrian , Johnson C. Michael , Jensen Michael , Nair K. Sreekumaran , Schwenk W. Frederick , Rizza Robert A.
Published: March 2007

We have previously reported that splanchnic glucose uptake, hepatic glycogen synthesis, and hepatic glucokinase activity are decreased in people with type 2 diabetes during intravenous glucose infusion. To determine whether these defects are also present during more physiological enteral glucose administration, we studied 11 diabetic and 14 nondiabetic volunteers using a combined organ catheterization-tracer infusion technique. Glucose was infused into the duodenum at a rate of 22 μmol · kg−1 · min−1 while supplemental glucose was given intravenously to clamp glucose at ∼10 mmol/l in both groups. Endogenous hormone secretion was inhibited with somatostatin, and insulin was infused to maintain plasma concentrations at ∼300 pmol/l (i.e., twofold higher than our previous experiments). Total body glucose disappearance, splanchnic, and leg glucose extractions were markedly lower (P < 0.01) in the diabetic subjects than in the nondiabetic subjects. UDP-glucose flux, a measure of glycogen synthesis, was ∼35% lower (P < 0.02) in the diabetic subjects than in the nondiabetic subjects. This was entirely accounted for by a decrease (P < 0.01) in the contribution of extracellular glucose because the contribution of the indirect pathway to hepatic glycogen synthesis was similar between groups. Neither endogenous and splanchnic glucose productions nor rates of appearance of the intraduodenally infused glucose in the portal vein differed between groups. In summary, both muscle and splanchnic glucose uptake are impaired in type 2 diabetes during enteral glucose administration. The defect in splanchnic glucose uptake appears to be due to decreased uptake of extracellular glucose, implying decreased glucokinase activity. Thus, abnormal hepatic and muscle (but not gut) glucose metabolism are likely to contribute to postprandial hyperglycemia in people with type 2 diabetes.
Increased Fatty Acid Uptake and Altered Fatty Acid Metabolism in Insulin-Resistant Muscle of Obese Zucker Rats
Authors: Turcotte Lorraine Patricia , Swenberger Jason Richard , Zavitz Tucker Michelle , Yee Alice Jane
Published: March 2007

Altered muscle fatty acid (FA) metabolism may contribute to the presence of muscle insulin resistance in the genetically obese Zucker rat. To determine whether FA uptake and disposal are altered in insulin-resistant muscle, we measured palmitate uptake, oxidation, and incorporation into di- and triglycerides in isolated rat hindquarters, as well as muscle plasma membrane fatty acid–binding protein (FABPPM) content of lean (n = 16, fa/+) and obese (n = 15, fa/fa) Zucker rats (12 weeks of age). Hindquarters were perfused with 7 mmol/l glucose, 1,000 μmol/l albumin-bound palmitate, and albumin-bound [1-14C]palmitate at rest (no insulin). Glucose uptake was 42% lower in the obese than in the lean rats and indicated the presence of muscle insulin resistance. Fractional and total rates of palmitate uptake were 42 and 74% higher in the obese than in the lean rats and were associated with higher muscle FABPPM content (r2 = 0.69, P < 0.05). The percentage of palmitate oxidized was not significantly different between groups. FA disposal to storage was altered according to fiber type. When compared with lean rats, the rate of triglyceride synthesis in red muscle was 158% higher in obese rats, and the rate of palmitate incorporation into diglycerides in white muscle was 93% higher in obese rats. Pre- and postperfusion muscle triglyceride levels were higher in both red and white muscles of the obese rats. These results show that increased FA uptake and altered FA disposal to storage may contribute to the development of muscle insulin resistance in obese Zucker rats.
Insulin Sensitivity of Suppression of Endogenous Glucose Production Is the Single Most Important Determinant of Glucose Tolerance
Authors: Båvenholm Peter N. , Pigon Jan , Östenson Claes-Göran , Efendic Suad
Published: March 2007

Hyperglycemia results from an imbalance between endocrine pancreatic function and hepatic and extrahepatic insulin sensitivity. We studied 57 well-matched Swedish men with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), or mild diabetes. Oral glucose tolerance and insulin release were assessed during an oral glucose tolerance test (OGTT). Insulin sensitivity and glucose turnover were determined during a two-step euglycemic insulin clamp (infusion 0.25 and 1.0 mU · kg–1 · min–1). High-performance liquid chromatography–purified [6-3H]glucose was used as a tracer. During low-insulin infusion, the rate of endogenous glucose production (EGP) decreased more in subjects with NGT than in subjects with IGT or diabetes (δ rate of appearance [Ra] 1.25 ± 0.10 vs. 0.75 ± 0.14 vs. 0.58 ± 0.09 mg · kg–1 · min–1, P < 0.001). The corresponding rates of glucose infusion during the high-dose insulin infusion (M values) were 8.3 ± 0.6 vs. 5.4 ± 0.9 vs. 4.7 ± 0.4 mg · kg–1 · min–1 (P < 0.001). A total of 56% of the variation in glucose area under the curve (AUC) during OGTT (glucose AUC) was mainly explained by δ Ra (increase in multiple R2 0.42) but also by δ Rd (rate of disapperance) (increase in multiple R2 0.05), and the early insulin response during OGTT contributed significantly (increase in multiple R2 0.07). When M value was included in the model, reflecting extrahepatic insulin sensitivity, it contributed to 20% of the variation in glucose AUC, and together with the incremental insulin response (increase in multiple R2 0.21), it explained 45% of the variation. In conclusion, insulin sensitivity of suppression of EGP plays the most important role in the determination of blood glucose response during OGTT.
The Antihyperglycemic Drug α-Lipoic Acid Stimulates Glucose Uptake via Both GLUT4 Translocation and GLUT4 Activation
Authors: Konrad Daniel , Somwar Romel , Sweeney Gary , Yaworsky Karen , Hayashi Michiko , Ramlal Toolsie , Klip Amira
Published: March 2007

The cofactor of mitochondrial dehydrogenase complexes and potent antioxidant α-lipoic acid has been shown to lower blood glucose in diabetic animals. α-Lipoic acid enhances glucose uptake and GLUT1 and GLUT4 translocation in 3T3-L1 adipocytes and L6 myotubes, mimicking insulin action. In both cell types, insulin-stimulated glucose uptake is reduced by inhibitors of p38 mitogen-activated protein kinase (MAPK). Here we explore the effect of α-lipoic acid on p38 MAPK, phosphatidylinositol (PI) 3-kinase, and Akt1 in L6 myotubes. α-Lipoic acid (2.5 mmol/l) increased PI 3-kinase activity (31-fold) and Akt1 (4.9-fold). Both activities were inhibited by 100 nmol/l wortmannin. α-Lipoic acid also stimulated p38 MAPK phosphorylation by twofold within 10 min. The phosphorylation persisted for at least 30 min. Like insulin, α-lipoic acid increased the kinase activity of the α (2.8-fold) and β (2.1-fold) isoforms of p38 MAPK, measured by an in vitro kinase assay. Treating cells with 10 μmol/l of the p38 MAPK inhibitors SB202190 or SB203580 reduced the α-lipoic acid–induced stimulation of glucose uptake by 66 and 55%, respectively. In contrast, SB202474, a structural analog that does not inhibit p38 MAPK, was without effect on glucose uptake. In contrast to 2-deoxyglucose uptake, translocation of GLUT4myc to the cell surface by either α-lipoic acid or insulin was unaffected by 20 μmol/l of SB202190 or SB203580. The results suggest that inhibition of 2-deoxyglucose uptake in response to α-lipoic acid by inhibitors of p38 MAPK is independent of an effect on GLUT4 translocation. Instead, it is likely that regulation of transporter activity is sensitive to these inhibitors.
Hepatocyte Nuclear Factor-4α Involved in Type 1 Maturity-Onset Diabetes of the Young Is a Novel Target of AMP-Activated Protein Kinase
Authors: Leclerc Isabelle , Lenzner Claudia , Gourdon Laurence , Vaulont Sophie , Kahn Axel , Viollet Benoît
Published: March 2007

Mutations in the HNF4α gene are responsible for type 1 maturity-onset diabetes of the young (MODY1), which is characterized by a defect in insulin secretion. Hepatocyte nuclear factor (HNF)-4α is a transcription factor that plays a critical role in the transcriptional regulation of genes involved in glucose metabolism in both hepatocytes and pancreatic β-cells. Recent evidence has implicated AMP-activated protein kinase (AMPK) in the modulation of both insulin secretion by pancreatic β-cells and the control of glucose-dependent gene expression in both hepatocytes and β-cells. Therefore, the question could be raised as to whether AMPK plays a role in these processes by modulating HNF-4α function. In this study, we show that activation of AMPK by 5-amino-4-imidazolecarboxamide riboside (AICAR) in hepatocytes greatly diminished HNF-4α protein levels and consequently downregulates the expression of HNF-4α target genes. Quantitative evaluation of HNF-4α target gene expression revealed diminished mRNA levels for HNF-1α, GLUT2, l-type pyruvate kinase, aldolase B, apolipoprotein (apo)-B, and apoCIII. Our data clearly demonstrate that the MODY1/HNF-4α transcription factor is a novel target of AMPK in hepatocytes. Accordingly, it can be suggested that in pancreatic β-cells, AMPK also acts by decreasing HNF-4α protein level, and therefore insulin secretion. Hence, the possible role of AMPK in the physiopathology of type 2 diabetes should be considered.
Improved Glucose Tolerance and Acinar Dysmorphogenesis by Targeted Expression of Transcription Factor PDX-1 to the Exocrine Pancreas
Authors: Heller R. Scott , Stoffers Doris A. , Bock Troels , Svenstrup Kirsten , Jensen Jan , Horn Thomas , Miller Christopher P. , Habener Joel F. , Madsen Ole D. , Serup Palle
Published: March 2007

The homeodomain protein PDX-1 is critical for pancreas development and is a key regulator of insulin gene expression. PDX-1 nullizygosity and haploinsufficiency in mice and humans results in pancreatic agenesis and diabetes, respectively. At embryonic day (e) 10.5, PDX-1 is expressed in all pluripotential gut-derived epithelial cells destined to differentiate into the exocrine and endocrine pancreas. At e15, PDX-1 expression is downregulated in exocrine cells, but remains high in endocrine cells. The aim of this study was to determine whether targeted overexpression of PDX-1 to the exocrine compartment of the developing pancreas at e15 would allow for respecification of the exocrine cells. Transgenic (TG) mice were generated in which PDX-1 was expressed in the exocrine pancreas using the exocrine-specific elastase-1 promoter. These mice exhibited a marked dysmorphogenesis of the exocrine pancreas, manifested by increased rates of replication and apoptosis in acinar cells and a progressive fatty infiltration of the exocrine pancreas with age. Interestingly, the TG mice exhibited improved glucose tolerance, but absolute β-cell mass was not increased. These findings indicate that downregulation of PDX-1 is required for the proper maintenance of the exocrine cell phenotype and that upregulation of PDX-1 in acinar cells affects β-cell function. The mechanisms underlying these observations remain to be elucidated.
Activation of Acetyl-CoA Carboxylase by a Glutamate- and Magnesium-Sensitive Protein Phosphatase in the Islet β-Cell
Authors: Kowluru Anjaneyulu , Chen Hai-Qing , Modrick Lisa M. , Stefanelli Claudio
Published: March 2007

Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, a precursor in the biosynthesis of long-chain fatty acids, which have been implicated in physiological insulin secretion. The catalytic function of ACC is regulated by phosphorylation (inactive)−dephosphorylation (active). In this study we investigated whether similar regulatory mechanisms exist for ACC in the pancreatic islet β-cell. ACC was quantitated in normal rat islets, human islets, and clonal β-cells (HIT-15 or INS-1) using a [14C]bicarbonate fixation assay. In the β-cell lysates, ACC was stimulated by magnesium in a concentration-dependent manner. Of all the dicarboxylic acids tested, only glutamate, albeit ineffective by itself, significantly potentiated magnesium-activated ACC in a concentration-dependent manner. ACC stimulation by glutamate and magnesium was maximally demonstrable in the cytosolic fraction; it was markedly reduced by okadaic acid (OKA) in concentrations (<50 nmol/l) that inhibited protein phosphatase 2A (PP2A). Furthermore, pretreatment of the cytosolic fraction with anti-PP2A serum attenuated the glutamate- and magnesium-mediated activation of ACC, thereby suggesting that ACC may be regulated by an OKA-sensitive PP2A-like enzyme. Streptavidin-agarose chromatography studies have indicated that glutamate- and magnesium-mediated effects on ACC are attributable to activation of ACC’s dephosphorylation; this suggests that the stimulatory effects of glutamate and magnesium on ACC might involve activation of an OKA-sensitive PP2A-like enzyme that dephosphorylates and activates ACC. In our study, 5-amino-imidazolecarboxamide (AICA) riboside, a stimulator of AMP kinase, significantly inhibited glucose-mediated activation of ACC and insulin secretion from isolated β-cells. Together, our data provide evidence for a unique regulatory mechanism for the activation of ACC in the pancreatic β-cell, leading to the generation of physiological signals that may be relevant for physiological insulin secretion.
Effects of Acute Changes of Plasma Free Fatty Acids on Intramyocellular Fat Content and Insulin Resistance in Healthy Subjects
Authors: Boden Guenther , Lebed Brett , Schatz Melanie , Homko Carol , Lemieux Susan
Published: March 2007

The reason for the 3- to 4-h delay between a rise in plasma free fatty acid (FFA) levels and the development of insulin resistance remains unknown. In the current study, we have tested the hypothesis that the delay may be caused by the need for plasma FFAs to first enter muscle cells and to be re-esterified there before causing insulin resistance. To this end, we have determined intramyocellular triglyceride (IMCL-TG) content with proton nuclear magnetic resonance (1H-NMR) spectroscopy in healthy volunteers before and 4 h after lowering of plasma FFAs (with euglycemic-hyperinsulinemic clamping) or after increasing plasma FFAs (with lipid plus heparin infusions). Increasing plasma FFAs (from 516 to 1,207 μmol/l or from 464 to 1,857 μmol/l, respectively) was associated with acute increases in IMCL-TG from 100 to 109 ± 5% (P < 0.05) or to 133 ± 11% (P < 0.01), respectively, and with a significant increase in insulin resistance (P < 0.05 after 3.5 h). Lowering of plasma FFAs from 560 to 41 μmol/l was associated with a tendency for IMCL-TG to decrease (from 100 to 95 ± 3%). Changes in plasma FFAs correlated linearly with IMCL-TG (r = 0.74, P < 0.003). The demonstration that acute changes in plasma FFAs were accompanied by corresponding changes in IMCL-TG and with the development of insulin resistance, taken together with previous reports of a close correlation between IMCL-TG and insulin resistance, supported the notion that accumulation of IMCL-TG is a step in the development of FFA-induced insulin resistance.
Pharmacological Inhibition of Diabetic Retinopathy
Authors: Kern Timothy S. , Engerman Ronald L.
Published: March 2007

Effects of aminoguanidine and aspirin on the development of retinopathy have been examined in 5-year studies of diabetic dogs. Either agent was administered daily in doses of 20–25 mg · kg−1 · day−1. Because severity of hyperglycemia greatly influences development of the retinopathy, special effort was devoted to maintaining comparable glycemia in experimental and control groups. The retinal vasculature was isolated by the trypsin digest method, and retinopathy was assessed by light microscopy. Diabetes for 5 years resulted, as expected, in saccular capillary aneurysms, pericyte ghosts, acellular capillaries, retinal hemorrhages, and other lesions. Administration of aminoguanidine essentially prevented the retinopathy, significantly inhibiting the development of retinal microaneurysms, acellular capillaries, and pericyte ghosts compared with diabetic controls. Aspirin significantly inhibited the development of retinal hemorrhages and acellular capillaries over the 5 years of study, but had less effect on other lesions. Although diabetes resulted in significantly increased levels of advanced glycation end products (AGEs) (namely, pentosidine in tail collagen and aorta, and Hb-AGE), aminoguanidine had no significant influence on these parameters of glycation. Nitration of a retinal protein was significantly increased in diabetes and inhibited by aminoguanidine. The biochemical mechanism by which aminoguanidine has inhibited retinopathy thus is not clear. Aminoguanidine (but not aspirin) inhibited a diabetes-induced defect in ulnar nerve conduction velocity, but neither agent was found to influence kidney structure or albumen excretion.
Diabetes-Induced Accelerated Atherosclerosis in Swine
Authors: Gerrity Ross G. , Natarajan Rama , Nadler Jerry L. , Kimsey Troy
Published: March 2007

Patients with diabetes are at higher risk for atherosclerotic disease than nondiabetic individuals with other comparable risk factors. Studies examining mechanisms underlying diabetes-accelerated atherosclerosis have been limited by the lack of suitable humanoid animal models. In this study, diabetes was superimposed on a well-characterized swine model of atherosclerosis by injection of the β-cell cytotoxin streptozotocin (STZ), resulting in a >80% reduction in β-cells and an increase in plasma glucose to diabetic levels. Animals were maintained without exogenous insulin for up to 48 weeks. Plasma glucose and cholesterol levels and lesion extent and severity were quantified in swine with diabetes and hyperlipemia alone and in combination compared with controls. Diabetes had no effect on plasma cholesterol levels, but diabetic/hyperlipemic (D-HL) swine developed hypertriglyceridemia and showed a doubling in aortic sudanophilia over nondiabetic/hyperlipemic (N-HL) swine as early as 12 weeks (47.25 ± 4.5 vs. 24.0 ± 4.6%). At 20 weeks, coronary artery stenosis was significantly greater in D-HL than in N-HL animals (86 ± 10 vs. 46 ± 8%). Coronary lesions predominantly arose in the first 2–3 cm of the vessels and displayed humanoid morphology. Aortic lesions in D-HL swine had double the cholesterol content of those in N-HL swine, and incorporation of oleate into cholesteryl ester was significantly greater in grossly normal aortic areas of D-HL swine compared with N-HL and was attributed to similar elevated incorporation in monocytes. This large study demonstrates that a model of diabetes with humanoid characteristics, including hypertriglyceridemia and severe, accelerated atherosclerosis can be reproducibly induced and maintained in swine. This model should potentially be of great value in elucidating mechanisms underlying the accelerated atherosclerosis seen in human diabetic individuals.
Insulin Production by Human Embryonic Stem Cells
Authors: Assady Suheir , Maor Gila , Amit Michal , Itskovitz-Eldor Joseph , Skorecki Karl L. , Tzukerman Maty
Published: March 2007

Type 1 diabetes generally results from autoimmune destruction of pancreatic islet β-cells, with consequent absolute insulin deficiency and complete dependence on exogenous insulin treatment. The relative paucity of donations for pancreas or islet allograft transplantation has prompted the search for alternative sources for β-cell replacement therapy. In the current study, we used pluripotent undifferentiated human embryonic stem (hES) cells as a model system for lineage-specific differentiation. Using hES cells in both adherent and suspension culture conditions, we observed spontaneous in vitro differentiation that included the generation of cells with characteristics of insulin-producing β-cells. Immunohistochemical staining for insulin was observed in a surprisingly high percentage of cells. Secretion of insulin into the medium was observed in a differentiation-dependent manner and was associated with the appearance of other β-cell markers. These findings validate the hES cell model system as a potential basis for enrichment of human β-cells or their precursors, as a possible future source for cell replacement therapy in diabetes.
Complete Protection of Islets Against Allorejection and Autoimmunity by a Simple Barium-Alginate Membrane
Authors: Duvivier-Kali Valérie F. , Omer Abdulkadir , Parent Richard J. , O’Neil John J. , Weir Gordon C.
Published: March 2007

We describe a new technique for microencapsulation with high–mannuronic acid (high-M) alginate crosslinked with BaCl2 without a traditional permselective component, which allows the production of biocompatible capsules that allow prolonged survival of syngeneic and allogeneic transplanted islets in diabetic BALB/c and NOD mice for >350 days. The normalization of the glycemia in the transplanted mice was associated with normal glucose profiles in response to intravenous glucose tolerance tests. After explantation of the capsules, all mice became hyperglycemic, demonstrating the efficacy of the encapsulated islets. The retrieved capsules were free of cellular overgrowth and islets responded to glucose stimulation with a 5- to 10-fold increase of insulin secretion. Transfer of splenocytes isolated from transplanted NOD mice to NOD/SCID mice adoptively transferred diabetes, indicating that NOD recipients maintained islet-specific autoimmunity. In conclusion, we have developed a simple technique for microencapsulation that prolongs islet survival without immunosuppression, providing complete protection against allorejection and the recurrence of autoimmune diabetes.
Glucose Competence of the Hepatoportal Vein Sensor Requires the Presence of an Activated Glucagon-Like Peptide-1 Receptor
Authors: Burcelin Rémy , Da Costa Anabela , Drucker Daniel , Thorens Bernard
Published: March 2007

Activation of the hepatoportal glucose sensors by portal glucose infusion leads to increased glucose clearance and induction of hypoglycemia. Here, we investigated whether glucagon-like peptide-1 (GLP-1) could modulate the activity of these sensors. Mice were therefore infused with saline (S-mice) or glucose (P-mice) through the portal vein at a rate of 25 mg/kg · min. In P-mice, glucose clearance increased to 67.5 ± 3.7 mg/kg · min as compared with 24.1 ± 1.5 mg/kg · min in S-mice, and glycemia decreased from 5.0 ± 0.1 to 3.3 ± 0.1 mmol/l at the end of the 3-h infusion period. Coinfusion of GLP-1 with glucose into the portal vein at a rate of 5 pmol/kg · min (P–GLP-1 mice) did not increase the glucose clearance rate (57.4 ± 5.0 ml/kg · min) and hypoglycemia (3.8 ± 0.1 mmol/l) observed in P-mice. In contrast, coinfusion of glucose and the GLP-1 receptor antagonist exendin-(9-39) into the portal vein at a rate of 0.5 pmol/kg · min (P-Ex mice) reduced glucose clearance to 36.1 ± 2.6 ml/kg · min and transiently increased glycemia to 9.2 ± 0.3 mmol/l at 60 min of infusion before it returned to the fasting level (5.6 ± 0.3 mmol/l) at 3 h. When glucose and exendin-(9-39) were infused through the portal and femoral veins, respectively, glucose clearance increased to 70.0 ± 4.6 ml/kg · min and glycemia decreased to 3.1 ± 0.1 mmol/l, indicating that exendin-(9-39) has an effect only when infused into the portal vein. Finally, portal vein infusion of glucose in GLP-1 receptor−/− mice failed to increase the glucose clearance rate (26.7 ± 2.9 ml/kg · min). Glycemia increased to 8.5 ± 0.5 mmol/l at 60 min and remained elevated until the end of the glucose infusion (8.2 ± 0.4 mmol/l). Together, our data show that the GLP-1 receptor is part of the hepatoportal glucose sensor and that basal fasting levels of GLP-1 sufficiently activate the receptor to confer maximum glucose competence to the sensor. These data demonstrate an important extrapancreatic effect of GLP-1 in the control of glucose homeostasis.
Cytokine Induction of Fas Gene Expression in Insulin-Producing Cells Requires the Transcription Factors NF-κB and C/EBP
Authors: Darville Martine I. , Eizirik Décio L.
Published: March 2007

Fas-mediated cell death may play a role in the autoimmune destruction of pancreatic β-cells in type 1 diabetes. β-Cells do not express Fas under physiological conditions, but Fas mRNA and protein are induced in cytokine-exposed mouse and human islets, rendering the β-cells susceptible to Fas ligand–induced apoptosis. The aim of the present study was to investigate the molecular regulation of Fas by cytokines in rat β-cells and in insulin-producing RINm5F cells. Fas mRNA expression was increased 15-fold in fluorescence-activated cell sorting–purified rat β-cells exposed to interleukin (IL)-1β, whereas γ-interferon had no effect. Transfection experiments of rat Fas promoter-luciferase reporter constructs into purified rat β-cells and RINm5F insulinoma cells identified an IL-1β–responsive region between nucleotides −223 and −54. Inactivation of two adjacent NF-κB and C/EBP sites in this region abolished IL-1β–induced Fas promoter activity in RINm5F cells. Binding of NF-κB and C/EBP factors to their respective sites was confirmed by gel shift assays. In cotransfection experiments, NF-κB p65 transactivated the Fas promoter. NF-κB p50 and C/EBPβ overexpression had no effect by themselves on the Fas promoter activity, but when cotransfected with p65, each factor inhibited transactivation by p65. These results suggest a critical role for NF-κB and C/EBP factors in cytokine-regulation of Fas expression in insulin-producing cells.
Inverse Relationship Between Cytotoxicity of Free Fatty Acids in Pancreatic Islet Cells and Cellular Triglyceride Accumulation
Authors: Cnop Miriam , Hannaert Jean Claude , Hoorens Anne , Eizirik Décio L. , Pipeleers Daniel G.
Published: March 2007

Studies in Zucker diabetic fatty rats have led to the concept that chronically elevated free fatty acid (FFA) levels can cause apoptosis of triglyceride-laden pancreatic β-cells as a result of the formation of ceramides, which induce nitric oxide (NO)-dependent cell death. This “lipotoxicity” hypothesis could explain development of type 2 diabetes in obesity. The present study examines whether prolonged exposure to FFA affects survival of isolated normal rat β-cells and whether the outcome is related to the occurrence of triglyceride accumulation. A dose-dependent cytotoxicity was detected at 5–100 nmol/l of unbound oleate and palmitate, with necrosis occurring within 48 h and an additional apoptosis during the subsequent 6 days of culture. At equimolar concentrations, the cytotoxicity of palmitate was higher than that of oleate but lower than that of its nonmetabolized analog bromopalmitate. FFA cytotoxicity was not suppressed by etomoxir (an inhibitor of mitochondrial carnitine palmitoyltransferase I) or by antioxidants; it was not associated with inducible NO synthase expression or NO formation. An inverse correlation was observed between the percentage of dead β-cells on day 8 and their cellular triglyceride content on day 2. For equimolar concentrations of the tested FFA, oleate caused the lowest β-cell toxicity and the highest cytoplasmic triglyceride accumulation. On the other hand, oleate exerted the highest toxicity in islet non–β-cells, where no FFA-induced triglyceride accumulation was detected. In conditions without triglyceride accumulation, the lower FFA concentrations caused primarily apoptosis, both in islet β-cells and non–β-cells. It is concluded that FFAs can cause death of normal rat islet cells through an NO-independent mechanism. The ability of normal β-cells to form and accumulate cytoplasmic triglycerides might serve as a cytoprotective mechanism against FFA-induced apoptosis by preventing a cellular rise in toxic free fatty acyl moieties. It is conceivable that this potential is lost or insufficient in cells with a prolonged triglyceride accumulation as may occur in vivo.
IPF1/PDX1 Deficiency and β-Cell Dysfunction in Psammomys obesus, an Animal With Type 2 Diabetes
Authors: Leibowitz Gil , Ferber Sarah , Apelqvist Åsa , Edlund Helena , Gross David J. , Cerasi Erol , Melloul Danielle , Kaiser Nurit
Published: March 2007

The homeodomain transcription factor IPF1/PDX1 is required in β-cells for efficient expression of insulin, glucose transporter 2, and prohormone convertases 1/3 and 2. Psammomys obesus, a model of diet-responsive type 2 diabetes, shows markedly depleted insulin stores when given a high-energy (HE) diet. Despite hyperglycemia, insulin mRNA levels initially remained unchanged and then decreased gradually to 15% of the basal level by 3 weeks. Moreover, insulin gene expression was not increased when isolated P. obesus islets were exposed to elevated glucose concentrations. Consistent with these observations, no functional Ipf1/Pdx1 gene product was detected in islets of newborn or adult P. obesus using immunostaining, Western blot, DNA binding, and reverse transcriptase–polymerase chain reaction analyses. Other β-cell transcription factors (e.g., ISL-1, Nkx2.2, and Nkx6.1) were expressed in P. obesus islets, and the DNA binding activity of the insulin transcription factors RIPE3b1-Act and IEF1 was intact. Ipf1/Pdx1 gene transfer to isolated P. obesus islets normalized the defect in glucose-stimulated insulin gene expression and prevented the rapid depletion of insulin content after exposure to high glucose. Taken together, these results suggest that the inability of P. obesus islets to adapt to dietary overload, with depletion of insulin content as a consequence, results from IPF1/PDX1 deficiency. However, because not all animals become hyperglycemic on HE diet, additional factors may be important for the development of diabetes in this animal model.
Insulin Secretion and Glucose Kinetics During Exercise With and Without Pharmacological α1- and α2-Receptor Blockade
Authors: Aarnio Pauliina , Lauritsen Torsten , Dela Flemming
Published: March 2007

The mechanism behind exercise-induced decreases in plasma insulin concentrations was examined in eight healthy young men. In addition, the influence of specific α1- and α2-adrenoceptor blockade on glucose kinetics during exercise was studied. To test the hypothesis that exercise-induced decreases in insulin secretion are mediated via α2-adrenoceptors, all subjects exercised for 60 min on separate occasions under four conditions: with and without α1-receptor blockade (1 mg prazosin) and with and without or α2-receptor blockade (15 mg yohimbine). Glucose kinetics were measured using [3-3H]glucose. During exercise with α2-receptor blockade, the insulin concentration initially increased (first 20 min) then decreased, whereas it continually decreased in the corresponding control experiment. The C-peptide concentration did not change during exercise with α2-receptor blockade but decreased in the control experiment. During exercise with α1-receptor blockade and corresponding control experiments, insulin and C-peptide levels always decreased. With α1-receptor blockade, the glucose concentration increased (first 30 min) and then decreased, whereas it slightly decreased in all other experiments. In addition, with α1-receptor blockade, the glucose rate of appearance (Ra) increased rapidly (because of higher catecholamine concentrations in α1-receptor blockade versus control) and the glucose rate of disappearance (Rd) was higher compared with control. During exercise with α2-receptor blockade, the Ra and Rd were always lower compared with control. Therefore, we conclude that exercise-induced decreases in insulin secretion are mediated via α2-adrenoceptors and that blockade of α1- and α2-adrenoceptors during exercise elicits opposite responses in glucose Ra and Rd.
Growth Hormone Induces Cellular Insulin Resistance by Uncoupling Phosphatidylinositol 3-Kinase and Its Downstream Signals in 3T3-L1 Adipocytes
Authors: Takano Atsuko , Haruta Tetsuro , Iwata Minoru , Usui Isao , Uno Tatsuhito , Kawahara Junko , Ueno Eiichi , Sasaoka Toshiyasu , Kobayashi Masashi
Published: March 2007

Growth hormone (GH) is well known to induce in vivo insulin resistance. However, the molecular mechanism of GH-induced cellular insulin resistance is largely unknown. In this study, we demonstrated that chronic GH treatment of differentiated 3T3-L1 adipocytes reduces insulin-stimulated 2-deoxyglucose (DOG) uptake and activation of Akt (also known as protein kinase B), both of which are downstream effects of phosphatidylinositol (PI) 3-kinase, despite enhanced tyrosine phosphorylation of insulin receptor substrate (IRS)-1, association of IRS-1 with the p85 subunit of PI 3-kinase, and IRS-1–associated PI 3-kinase activity. In contrast, chronic GH treatment did not affect 2-DOG uptake and Akt activation induced by overexpression of a membrane-targeted form of the p110 subunit of PI 3-kinase (p110CAAX) or Akt activation stimulated by platelet-derived growth factor. Fractionation studies indicated that chronic GH treatment reduces insulin-stimulated translocation of Akt from the cytosol to the plasma membrane. Interestingly, chronic GH treatment increased insulin-stimulated association of IRS-1 with p85 and IRS-1–associated PI 3-kinase activity preferentially in the cytosol. These results indicate that cellular insulin resistance induced by chronic GH treatment in 3T3-L1 adipocytes is caused by uncoupling between activation of PI 3-kinase and its downstream signals, which is specific to the insulin-stimulated PI 3-kinase pathway. This effect of GH might result from the altered subcellular distribution of IRS-1–associated PI 3-kinase.
New Amino Acid Substitutions in the IRS-2 Gene in Finnish and Chinese Subjects With Late-Onset Type 2 Diabetes
Authors: Wang Hua , Rissanen Johanna , Miettinen Raija , Kärkkäinen Päivi , Kekäläinen Päivi , Kuusisto Johanna , Mykkänen Leena , Karhapää Pauli , Laakso Markku
Published: March 2007

We investigated the significance of the variants of the IRS-2 gene in patients with type 2 diabetes. The entire coding part of the IRS-2 gene was screened by single-strand conformation polymorphism analysis in 40 Chinese and 40 Finnish patients with late-onset type 2 diabetes. The association of the variants of the IRS-2 gene with type 2 diabetes was studied in 85 Finnish diabetic patients and 82 Finnish control subjects and in 100 Chinese diabetic patients and 85 Chinese control subjects. The four variants predicting structural changes in the insulin receptor substrate (IRS)-2 protein included an insertion of AAC (Asn) in the Asn repeat sequence centered around codons 29–36 (allele frequencies of 0 vs. 0.6% and 1.5 vs. 0%), the Ala157Thr substitution (0 vs. 0% and 0.5 vs. 0%), the Leu647Val substitution (0.6 vs. 0% and 0 vs. 0%), and the Gly1057Asp polymorphism (31 vs. 31% and 35 vs. 30%) (P = NS for all comparisons). Furthermore, six silent variants were observed (CGC147CGG, CCC155CCG, GCC156GCT, AGT723AGC, TGT816TGC, and CCC829CCT). The Gly1057Asp polymorphism was not associated with insulin resistance or impaired insulin secretion in Finnish subjects with normal glucose tolerance (n = 295) or impaired glucose tolerance (n = 38). These data indicate that structural variants of the IRS-2 gene were uncommon in Finnish and Chinese patients with type 2 diabetes. Thus, the variants in the coding part of the IRS-2 gene are unlikely to have a major role in the development of type 2 diabetes in Finnish or Chinese subjects.