bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Unusual Insulin of Psammomys obesus, a Rodent With Nutrition-Induced NIDDM-Like Syndrome
Authors: Kaiser Nurit , Bailyes Elaine M , Schneider Bruce S , Cerasi Erol , Steiner Donald F , Hutton John C , Gross David J
Published: September 2013

Psammomys obesus fed a high-calorie diet develops a NIDDM-like syndrome. The use of reverse-phase highperformance liquid chromatography (HPLC) to study Psammomys insulin biosynthesis and release revealed a very delayed elution time for the Psammomys insulin peak appearing near the position of human proinsulin. This unusual peak was initially thought to represent partially processed insulin on the basis of its molecular size and susceptibility to trimming by carboxypeptidase B (CpB). However, the findings of an active carboxypeptidase E (CpE) enzyme and the normal amidated forms of gastrin and cholecystokinin octapeptide (CCK-8) in Psammomys tissues were inconsistent with CpE-related aberrant processing of insulin. Moreover, amino acid sequencing of the delayed peak of Psammomys insulin revealed fully processed insulin with amino acid sequence as predicted by the cDNA. The unique presence of a B-30 phenylalanine residue, resulting in an increased hydrophobicity of the insulin molecule, probably underlies the marked delay in elution time on HPLC. The unusual structure of Psammomys insulin does not appear to contribute to the proinsulinemia observed in diabetic Psammomys since the HPLC-purified molecule did not inhibit PCI and PC2 convertase activities in an in vitro assay.
Proinsulin Conversion in GH3 Cells After Coexpression of Human Proinsulin With the Endoproteases PC2 and/or PC3
Authors: Kaufmann Jocelyne E , Irminger Jean-Claude , Mungall Juliette , Halban Philippe
Published: September 2013

Proinsulin conversion to insulin occurs in secretory granules of pancreatic β-cells. This processing has been suggested to require both the endoproteases PC2 and PC3 with each cleaving at only one of the two sites linking the insulin A- and B-chains with C-peptide. To evaluate this in an appropriate cellular setting, conversion of human proinsulin was followed in GH3 (rat pituitary) cells normally unable to convert this prohormone but equipped with the regulated secretory pathway. For this purpose, human proinsulin was expressed in GH3 cells, alone or in combination with PC2 and/or PC3, using recombinant adenoviruses. Cells were infected with the given adenoviruses and 24 h later were pulse-chased. Kinetics of proinsulin conversion were monitored by reverse-phase high-performance liquid chromatography. It was observed that while the two endoproteases do display a preference for a single site of cleavage (PC2 at the A-chain/C-peptide junction; PC3 at the B-chain/Cpeptide junction) and act in a synergistic manner to promote proinsulin conversion, either PC2 or PC3 alone can cleave at both sites to fully convert proinsulin to insulin. These results also show that a cell can be successfully infected by three different recombinant adenoviruses.
Circulating Leptin in Normal Children and During the Dynamic Phase of Juvenile Obesity: Relation to Body Fatness, Energy Metabolism, Caloric Intake, and Sexual Dimorphism
Authors: Lahlou Najiba , Landais Paul , De Boissieu Delphine , Bougnères Pierre-François
Published: September 2013

In 112 obese compared with 42 lean children, we found that serum leptin is elevated early in the evolution of childhood-onset obesity (28.4 ± 1.4 vs. 4.5 ± 0.4 ng/ml in lean children, P < 0.0001) and correlates with adiposity. Obese children also had higher serum leptin normalized to fat mass. Despite high serum leptin, obese children ingested 2–3 times more calories than did lean control subjects (P < 0.0001) and gained weight rapidly (10.2 ± 0.3 vs. 2.9 ± 0.1 kg/year in control subjects, P < 0.0001). Girls had higher leptin levels than did boys, in obese as well as in nonobese children, and showed a closer correlation between adiposity and serum leptin. Elevation of serum leptin was comparable before and after puberty in obese boys, but puberty further increased leptin levels in obese girls (36 ± 3 ng/ml), resulting in a clear sexual dimorphism with pubertal obese boys (22 ± 5 ng/ml, P < 0.005). In conclusion, increased serum leptin reflects but does not halt fat deposition in childhood obesity. After normalization to body adiposity, leptin was found to be increased independently by obesity status, female sex, and female sexual maturation.
Differences Between the Tolbutamide-Boosted and the Insulin-Modified Minimal Model Protocols
Authors: Saad Mohammed F , Steil Garry M , Kades Wagdy W , Ayad Maged F , Elsewafy Wagih A , Boyadjian Rima , Jinagouda Sujata D , Bergman Richard N
Published: September 2013

The insulin-modified frequently sampled Intravenous glucose tolerance test (FSIGTT) with minimal model analysis (MINMOD) was compared with the tolbutamide protocol and the glucose clamp in 35 nondiabetic subjects (age 38 ± 2 years [mean ± SE], BMI 27.2 ± 0.9 kg/m2). Each subject underwent two FSIGTTs, one with tolbutamide (300 mg) and the other with insulin (0.03 U/kg) and a euglycemic hyperinsulinemic clamp (40 mU · m−2 · min−1). Insulin sensitivity was determined from each FSIGTT with MINMOD and from the clamp. Insulin sensitivity indexes (SI) from the two FSIGTTs were significantly correlated (r = 0.77, P < 0.001), but SI(insuiin) was 29 ± 4% lower than SI(toibutamide). Both SI(insuiin) and Sictoibutamide) correlated significantly with SI(ciamp) (r = 0.70 and 0.71, P < 0.001 for each). Expressed in the same units (dl/min per µU/ml), SI(toibutamide) was on average 13 ± 6% lower than SI(ciamp) (4.51 ± 0.40 vs. 5.36 ± 0.36 × 10−2, P = 0.009), whereas SI(insulin) Was 44 ± 4% lower. SG(tolbutamide) and SG(insulin) were not different (1.88 ± 0.10 vs. 2.01 ± 0.09 × 10−2 min−1, P = 0.167) and were significantly correlated (r = 0.50, P = 0.002). Thus, insulin sensitivity estimates from both protocols correlate significantly with each other and with the clamp. They are quantitatively discrepant, however, possibly due to differences in the route of insulin delivery, saturation of insulin action, and/or tolbutamide-induced proinsulin release. Data obtained from these two MINMOD protocols are not directly comparable, and the same protocol must be used in any single cross-sectional or longitudinal study.
Increased Synthesis of Tumor Necrosis Factor-α in Uterine Explants From Pregnant Diabetic Rats and in Primary Cultures of Uterine Cells in High Glucose
Authors: Pampfer Serge , Vanderheyden Ivo , De Hertogh René
Published: September 2013

The production of tumor necrosis factor-α (TNF-α) was investigated in uterine explants from normal, diabetic, or insulin-treated diabetic pregnant rats. Explants from diabetic rats released more soluble TNF-α than did those in the other groups. The extent of this secretion was correlated with blood glucose concentration at the time of explantation. The concentration of cell membrane–associated TNF-α in the explants was not altered by diabetes. Daily insulin administration failed to normalize uterine TNF-α secretion despite correction of glycemia in the diabetic rats. Explants from normal pregnant rats cultured in vitro with increasing concentrations of D-glucose showed a dose-dependent increase in TNF-α secretion. The production of TNF-α in high glucose was also tested in primary cultures of uterine cells isolated from either immature or adult rats. TNF-α secretion was increased in high D-glucose but not in iso-osmolar concentrations of L-glucose, D-raffinose, D-galactose, or mannitol. Cell membraneassociated TNF-α was not influenced by high D-glucose. Semiquantitative reverse transcription–amplification of RNA extracted from primary cultures of uterine cells showed that the steady-state level of TNF-α transcripts was increased by high D-glucose but not by high L-glucose. The results are consistent with the hypothesis that hyperglycemia is instrumental in the overexpression of TNF-α in the diabetic uterus. Because TNF-α has a demonstrated negative impact on embryonic growth, enhanced TNF-α synthesis in the pregnant uterus may contribute to the embryopathy associated with maternal diabetes.
Value of Antibodies to Islet Protein Tyrosine Phosphatase–Like Molecule in Predicting Type 1 Diabetes
Authors: Hawa Mohammed , Rowe Rachael , Lan Michael S , Notkins Abner L , Pozzilli Paolo , Christie Michael R , Leslie R. David G
Published: September 2013

Islet antigens associated with type 1 diabetes include a recently identified protein tyrosine phosphatase–like molecule IA-2, which contains the intracellular fragment IA-2ic. To determine whether combinations of antibodies including those to IA-2 characterize and predict type 1 diabetes, we studied antibodies to IA-2, IA-2ic, glutamic acid decarboxylase (GAD65), and islet cell antibodies (ICAs) in 1) 60 newly diagnosed type 1 diabetic patients followed for 1 year, 2) 31 monozygotic twin pairs discordant for type 1 diabetes followed up to 12 years (11 twins developed diabetes), 3) 18 dizygotic twin pairs discordant for type 1 diabetes, and 4) normal healthy control subjects. Newly diagnosed type 1 diabetic patients frequently had antibodies to IA-2 (62%), IA-2ic (67%), GAD65 (77%), and ICAs (85%). The intracellular fragment of IA-2 probably contains the immunodominant epitope as 137 of 143 samples with IA-2 antibodies from type 1 diabetic patients also had IA-2ic antibodies. Monozygotic twins were usually discordant for antibody specificities. Concordance was higher in monozygotic than matched dizygotic twins for both antibody combinations (33 vs. 6%, P < 0.05) and the development of diabetes (33 vs. 0%, P < 0.01). In monozygotic twins, all the antibodies were highly predictive of type 1 diabetes (positive predictive values all >87%), although antibodies were also detected in twins at low risk of disease. In summary, IA-2 emerges as a major antigen associated with type 1 diabetes and distinct from GAD65. Type 1 diabetes–associated autoimmunity, which is probably induced by environmental factors, does not necessarily herald progression to the disease. However, genetic factors may influence the development of combinations of disease-associated antibodies and the progression to type 1 diabetes.
Atrial Natriuretic Peptide and Cyclic Nucleotides Affect Glucose-Induced Ca2+ Responses in Single Pancreatic Islet β-Cells Correlation With (Ca2+ + Mg2+)-ATPase Activity
Authors: Lee Bumsup , Laychock Suzanne G
Published: September 2013

Glucose stimulation of pancreatic islets is characterized by an initial decline in intracellular Ca2+ concentration ([Ca2+]i) (phase 0), followed by an increase in peak [Ca2+]i (phase 1). The effect of atrial natriuretic peptide (ANP) and cyclic nucleotides on the glucose-induced phase 0 [Ca2+]i was investigated by Fura-2 fluorescent imaging in single β-cells from isolated islets of rats maintained at 1.67 mmol/l glucose. ANP (1 nmol/1 to 1 μmol/l) inhibited the glucose (8.2 mmol/l)-induced phase 0 [Ca2+]i in a concentration-dependent manner. Forskolin, 8-bromo-cyclic AMP (8BrcAMP), and 8-bromo-cyclic guanosine monophosphate (8BrcGMP) also inhibited the glucose-induced phase 0 [Ca2+]i. The Ca2+ channel blocker, D 600, prevented the response to 8BrcAMP but not to ANP or 8BrcGMP on phase 0 [Ca2+]i. Thapsigargin (TG) also inhibited phase 0 [Ca2+]i by 90%. ANP, 8BrcGMP, and TG also reduced the time required for glucose to initiate the phase 1 increase in [Ca2+]i and each of these agents potentiated the effect of glucose on peak [Ca2+]i. Furthermore, sarco(endo)-plasmic reticulum (Ca2+ + Mg2+)-ATPase (SERCA) activity in RINm5F insulinoma cells was inhibited by 8BrcGMP and TG, but not 8BrcAMP. Thus, ANP and cGMP modulate [Ca2+]i regulation in pancreatic β-cells perhaps through mechanisms involving changes in SERCA activity and Ca2+ influx.
Differential Effects of Streptozotocin-Induced Diabetes on Cardiac Lipoprotein Lipase Activity
Authors: Rodrigues Brian , Cam Margaret C , Jian Kong , Lim Fiona , Sambandam Nandakumar , Shepherd Greg
Published: September 2013

Lipoprotein lipase (LPL) is an endothelial-bound enzyme that is rate determining for the clearance of triacylglycerol-rich lipoproteins. Previous studies using rats with streptozotocin (STZ)-induced diabetes have reported inconsistent effects on cardiac LPL activity or immunoreactive protein. To examine the contribution of the severity and duration of diabetes on cellular and heparin-releasable cardiac LPL activity, Wistar rats were administered a high (100 mg/kg; D100) or moderate (55 mg/kg; D55) dose of STZ, and LPL activity was examined at various times after diabetes induction. Heparin perfusion of the isolated Langendorff control heart induced the release of LPL activity as an initial fast phase followed by a slow phase of release. With increasing age, the second phase of LPL release became more pronounced. Severe STZ-induced diabetes reduced heparin-releasable LPL activity by 1 week in the D100 rats. However, in D55 rat hearts, peak heparin-releasable LPL activity was higher than that in control animals at 2 and 12 weeks after STZ injection, with a complete absence of the delayed phase at 12 weeks. The elevated heparin-releasable LPL peak could not be explained by an enhanced LPL synthesis because both cellular and surface-bound LPL activities in myocytes from D55 rats were low, relative to control. Chronic (12-day) insulin treatment of D55 rats prevented the rise in heparin-releasable LDL and the reduction in cell-associated LPL activity. Moreover, acute (90-min) treatment of D55 rats with rapid-acting insulin also reduced the heparin-releasable LPL activity to normal levels, although it had no effect on the low cellular LPL activity. When the heparin-releasable LPL pool was allowed to recover for 1 h after removal of the enzyme, D55 rat hearts continued to demonstrate a higher peak LPL activity after a second heparin perfusion. These studies demonstrate that in moderate but not severe diabetes, there is an augmented peak heparin-releasable LPL activity. Whether or not this enhanced heparin-releasable LPL activity has a pathological role in the diabetic heart has yet to be determined.
Pancreatic Islet Expression Studies and Polymorphic DNA Markers in the Genes Encoding Hepatocyte Nuclear Factor-3α, -3β, -3γ, -4γ, and -6
Authors: Vaisse Christian , Kim Jason , Espinosa Rafael , Le Beau Michelle M , Stoffel Markus
Published: September 2013

The genes encoding the functionally related hepatocyte nuclear factors HNF-1α and HNF-4α play a critical role in normal pancreatic β-cell function. Mutations in these liver-enriched transcription factors result in two forms of early-onset type 2 diabetes (maturity-onset diabetes of the young [MODY]), M0DY3 and M0DY1, which are characterized by impaired glucose-stimulated insulin secretion, early disease onset, and autosomal dominant inheritance. The transcriptional hierarchy of HNFs suggests that other proteins of the regulatory cascade might be responsible for other forms of MODY and/or late-onset type 2 diabetes. In this study, we show that HNF-3α, -3β, -3γ, -4γ, and -6 are expressed in pancreatic β-cells. We report the identification and characterization of simple tandem repeat DNA polymorphisms in the genes encoding HNF-3α, -3β, -3γ, -4γ, and -6 and the mapping of HNF-6 to chromosome bands 15q21.1–21.2 by fluorescence in situ hybridization. These markers will be useful to study the role of genetic variation in these genes in the pathogenesis of type 2 diabetes.
Prediction of Type 1 Diabetes Postpartum in Patients With Gestational Diabetes Mellitus by Combined Islet Cell Autoantibody Screening: A Prospective Multicenter Study
Authors: Füchtenbusch Martin , Ferber Karin , Standl Eberhard , Ziegler Anette-G
Published: September 2013

Women with gestational diabetes mellitus (GDM) have a considerable risk of developing diabetes later in life. To determine the predictive value of autoantibody markers in gestational diabetic pregnancy for the development of type 1 diabetes postpartum, we tested 437 patients with GDM (289 women treated with diet only [GDM-A] and 148 requiring insulin treatment during pregnancy [GDM-B]) for antibodies to islet cells (ICAs), GAD (GADAs), and tyrosine phosphatase ICA512/IA-2 (IA2As). We prospectively followed them with repeated oral glucose tolerance tests and antibody determinations for up to 7 years postpartum (mean, 1.6 years; range, 0–7.2 years). The cumulative risk of diabetes up to 5 years postpartum was 17% (95% CI 12–22%). The risk of type 1 diabetes was 3% (2–5%) by 9 months and 7% (4–9%) 2 years after delivery. At delivery, 8.5% of all patients were ICA+, 9.5% were GADA+, 6.2% were IA2A+, and 18.1% were positive for at least one antibody (12.6% for GDM-A vs. 30.4% for GDM-B, P < 0.0001). During follow-up, GADAs persisted in 75%, ICAs in 35%, and IA2As in 30% of the subjects positive for the respective marker at delivery. By 2 years postpartum, 29% (19–39%) of patients positive for at least one antibody developed type 1 diabetes, compared with 2% (1–4%) of antibody-negative patients (P < 0.0001). Thereby, the risk for type 1 diabetes 2 years postpartum increased with the number of antibodies present at delivery from 17% (6–28%) for one antibody, to 61% (30–91%) for two antibodies, and to 84% (55–100%) for 3 antibodies. Risk of progression to type 1 diabetes postpartum was also associated with the status of parity. Women with one or more pregnancies before the index pregnancy had a higher risk for type 1 diabetes 2 years after delivery (14.7% [4.9.–24.5%]) than women having their first (i.e., index) pregnancy (5% [2.9–7.1%]) (P < 0.006). A comparison of different prediction strategies showed that single antibody screening with GADA yielded the highest sensitivity of 63% (45–75%), compared with ICA (48% [31–65%]) and IA2A (34% [13–47%]). Combined screening with two autoantibodies increased sensitivity to 74% (58–90%) and 75% (60–92%) when using GADA plus ICA or GADA plus IA2A, respectively. Screening with all three markers improved sensitivity further to 82% (67–100%). β-cell autoantibodies determined at delivery in women with GDM are highly predictive for the development of type 1 diabetes postpartum. Autoantibody screening in pregnant women with GDM from populations at high risk for type 1 diabetes should therefore be considered to allow early diagnosis and appropriate therapy.
Targeted Disruption of the Tumor Necrosis Factor-α Gene: Metabolic Consequences in Obese and Nonobese Mice
Authors: Ventre John , Doebber Thomas , Wu Margaret , MacNaul Karen , Stevens Karla , Pasparakis Manolis , Kollias George , Moller David E
Published: September 2013

To address the hypothesis that tumor necrosis factor (TNF)-α has a role in obesity-associated insulin resistance or the regulation of in vivo lipid metabolism, mice with targeted disruption of the TNF-α gene were generated and studied. The absence of TNF-α protein in TNF-null (−/−) mice was confirmed. Lean or obese (gold-thioglucose [GTG]-injected) homozygous (−/−) mice were compared with lean or obese age- and sex-matched wild-type (+/+) mice derived from the same line at 13, 19, and 28 weeks of age. The following parameters were significantly affected in lean −/− versus +/+ mice: Body weight was not affected until week 28 (decreased by 14%); epididymal fat pad weight also decreased (25%) at this time, as did percentage body fat (16%), while percentage body protein was increased 13%. Fed plasma insulin levels decreased 47% (28 weeks), triglyceride levels decreased (all three ages; maximum 35% at 19 weeks), and fed plasma leptin decreased 33% (28 weeks). Fasting glucose was slightly (10%) reduced, but the glucose response to an oral glucose tolerance test (OGTT) was not affected. There was a trend (NS) toward increased total adipose tissue lipoprotein lipase in −/− versus +/+ mice. GTG-treat-ment resulted in obese −/− and +/+ mice with equal mean body weights (42 and 58% increased weight versus lean mice). The following parameters were significantly different in obese −/− mice: fasting plasma glucose decreased 13% (28 weeks), fed plasma insulin decreased 67% (28 weeks), and insulin response to OGTT was decreased by 50%. For both groups of obese mice, glucose levels during the OGTT were substantially increased compared with those in lean mice; however, mean stimulated glucose levels were 20% lower in obese −/− versus +/+ mice. We conclude 1) that TNF-α functions to regulate plasma triglycerides and body adiposity and 2) that although TNF-α contributes to reduced insulin sensitivity in older or obese mice, the absence of TNF-α is not sufficient to substantially protect against insulin resistance in the GTG hyperphagic model of rodent obesity.
Evidence Against a Direct Effect of Leptin on Glucose Transport in Skeletal Muscle and Adipocytes
Authors: Zierath Juleen R , Frevert Ernst U , Ryder Jeffrey W , Berggren Per-Olof , Kahn Barbara B
Published: September 2013

Recently, it has been proposed that leptin, the ob gene product, influences some steps in the insulin-signaling cascade. The purpose of the present study was to determine whether leptin exerts direct effects on glucose transport in insulin target tissues. Epitrochlearis muscles or isolated adipocytes from male SD rats were incubated in the absence or presence of recombinant leptin (3–1,000 ng/ml), and in the absence or presence of submaximal or maximal insulin concentrations. In skeletal muscle, insulin increased 3-O-methylglucose transport (1.88 ± 0.21, 4.06 ± 0.59, and 9.35 ± 1.90 umol ·μmol · ml−1 ·h−1, for 0, 0.6, and 12.0 nmol/1 insulin, respectively). Leptin exposure (300 ng/ml) for 2 h did not alter the basal, submaximal, or maximal response of glucose transport to insulin in skeletal muscle (1.50 ± 0.14,4.76 ± 0.58, and 9.04 ± 1.09 umol · ml−1 · h−1 for 0, 0.6, and 12.0 nmol/l insulin, respectively). Insulin increased glucose transport in rat adipocytes (0.194 ± 0.007,1.059 ± 0.029, and 3.367 ± 0.143 pmol [14C]glucose · 0.5 ml−1 cell suspension · min−1 for 0,0.8, and 80 nmol/l insulin, respectively); in vitro exposure to leptin (300 ng/ml) did not alter glucose transport (0.220 ± 0.006,1.269 ± 0.046, and 3.221 ± 0.285 pmol [14C]glucose · 0.5 ml−1 cell suspension · min−1 for 0, 0.8, and 80 nmol/1 insulin, respectively). Similar to our findings in the epitrochlearis muscle, leptin had no direct effect on basal or insulin-stimulated glucose uptake in soleus muscle from ob/ob or lean mice or adipocytes from normal mice. In summary, in vitro exposure of skeletal muscle or adipocytes to recombinant leptin did not alter glucose transport in the absence of insulin, nor did it affect the sensitivity or responsiveness of the glucose transport system to insulin.
Alterations in Cholesteryl Ester Transfer, Lipoprotein Lipase, and Lipoprotein Composition After Combined Pancreas-Kidney Transplantation
Authors: Bagdade John D , Teuscher Adrian U , Ritter Mary C , Eckel Robert H , Robertson R Paul
Published: September 2013

IDDM patients treated with conventional subcutaneous insulin have an abnormal increase in cholesteryl ester transfer (CET), the proatherogenic step in reverse-cholesterol transport that results in the enrichment of the apolipoprotein (apo) B-containing lipoproteins (VLDL, LDL) with cholesteryl ester (CE). This disturbance is closely linked to iatrogenic hyperinsulinemia and the nonphysiologic stimulation of lipoprotein lipase (LpL), a physiologic activator of CET, because lowering systemic insulin levels by administering insulin through the intraperitoneal insulin route normalizes LpL and CET. Hyperinsulinemia persists in IDDM patients who undergo successful pancreas-kidney transplantation (PKT) when their allografts are placed in the pelvis and drain into the iliac vein. Therefore, to determine whether hyperinsulinemia promotes CET in this setting, we studied CET, LpL, and insulin levels in 14 euglycemic normolipidemic IDDM PKT patients with nearnormal kidney function (creatinine 1.5 ± 0.4 mg/dl). Consistent with our prediction, the net mass of CE transferred from HDL to VLDL + LDL was significantly increased in the PKT group (P < 0.001) compared with nondiabetic renal transplant patients receiving the same immunosuppressive drugs and healthy control subjects. Both basal and arginine-stimulated insulin levels were increased above the kidney transplant group's levels and correlated with the mass of CE transferred at 2 h (r = 0.71, P < 0.05; r = 0.66, P < 0.05, respectively). Total basal LpL activities, LpL and hepatic triacylglycerol lipase activities, and LpL mass all tended to be higher than levels in healthy control subjects. Consistent with these changes in lipase activity, VLDL particle size was significantly reduced (P < 0.025) compared with that of control subjects. These findings indicate that PKT patients with systemically draining allografts have a persisting profile of potentially atherogenic disturbances in insulin levels, LpL, and CET that resemble IDDM patients treated with conventional subcutaneous insulin injections.
Selective Impairment in GLUT4 Translocation to Transverse Tubules in Skeletal Muscle of Streptozotocin-Induced Diabetic Rats
Authors: Dombrowski Luce , Roy Denis , Marette André
Published: September 2013

We previously reported that insulin induces the translocation of GLUT4 to both the plasma membrane and the transverse tubules (T-tubules) in rat skeletal muscle {Am J Physiol 270:E667–E676, 1996). The aim of the present study was to investigate whether the insulin-resistant glucose utilization of skeletal muscle from streptozotocin (STZ)-induced diabetic rats is linked to an impaired translocation of GLUT4 to the plasma membrane, the T-tubules, or both surface compartments. Whole-body insulin-mediated glucose disposal, assessed during a hyperinsulinemic-euglycemic clamp, was reduced by 48% (P < 0.01) in diabetic rats as compared with controls. Subcellular membrane fractions enriched with plasma membranes, T-tubules, or GLUT4-enriched intracellular membranes were isolated from hindlimb muscles of control and insulinstimulated rats, and GLUT4 content was measured by Western blot analysis. In the absence of insulin (unstimulated), GLUT4 content in muscle of diabetic rats was markedly lower (by ∼40%) in both the Ttubules and the intracellular membrane fraction as compared with controls. In contrast, the transporter protein levels were similar in the plasma membrane fraction. In skeletal muscle of control animals, the hyperinsulinemic clamp induced GLUT4 translocation from the intracellular membrane pool to both the plasma membrane and the T-tubule-enriched fractions (∼2.2-fold to ∼2.5-fold). Surprisingly, insulin increased plasma membrane GLUT4 content to comparable levels in control and diabetic rat skeletal muscle. However, insulin-mediated GLUT4 translocation to the T-tubules was significantly reduced in the same muscle. Wholebody insulin action was significantly correlated with GLUT4 protein levels in the T-tubules, but not with the transporter content in either plasma membranes or intracellular membranes. These results strongly suggest that peripheral resistance to insulin action on glucose disposal inSTZ-induced diabetic rats is caused by a selective impairment of GLUT4 translocation to skeletal muscle T-tubules.
Effect of Exercise Training and Food Restriction on Endothelium-Dependent Relaxation in the Otsuka Long-Evans Tokushima Fatty Rat, a Model of Spontaneous NIDDM
Authors: Sakamoto Sadaichi , Minami Kazushi , Niwa Yasuharu , Ohnaka Masaharu , Nakaya Yutaka , Mizuno Akira , Kuwajima Masamichi , Shima Kenji
Published: September 2013

We investigated whether endothelial function may be impaired in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model of spontaneous NIDDM. The effect of exercise training and food restriction on endothelial function was also studied. OLETF rats were divided into three groups at age 16 weeks: sedentary, exercise trained, and food restricted (70% of the food intake of sedentary rats). Otsuka Long-Evans Tokushima rats were used as the age-matched nondiabetic controls. Endothelium-dependent relaxation of the thoracic aorta induced by histamine was significantly attenuated in the sedentary or food-restricted rats, and exercise training improved endothelial function. Relaxation induced by sodium nitroprusside, a donor of nitric oxide, did not differ significantly among groups. Both exercise training and food restriction significantly suppressed plasma levels of glucose and insulin and serum levels of triacylglycerol and cholesterol and reduced the accumulation of abdominal fat. Insulin sensitivity, as measured by the hyperinsulinemic- euglycemic clamp technique, was significantly decreased in sedentary rats but was enhanced in exercise- trained and food-restricted rats. The urinary excretion of nitrite was significantly decreased in sedentary and food-restricted rats compared with nondiabetic rats and was significantly increased in exercise-trained rats. These results indicate that exercise training, but not food restriction, prevents endothelial dysfunction in NIDDM rats, presumably due to the exercise-induced increase in the production of nitric oxide.
Possibility of Distinct Insulin-Signaling Pathways Beyond Phosphatidylinositol 3-Kinase–Mediating Glucose Transport and Lipogenesis
Authors: Stevenson Ralph W , Kreutter David K , Andrews Kim M , Genereux Paul E , Gibbs E Michael
Published: September 2013

The aim of this study was to compare the effects of insulin and the insulinomimetic agent, englitazone, on functional end points and putative mediators of insulin action in 3T3-L1 adipocytes. Cells were incubated with englitazone for 48 h or with insulin for 10 or 30 min, or both, and 2-deoxy-D-[3H]glucose (2DG) uptake and lipogenesis (from [14C]glucose) were measured. Tyrosine phosphorylation of the insulin receptor (IR), insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), and pp60, and phosphatidylinositol (PI) 3-kinase activity (using PI as substrate) and mitogen-activated protein kinase (MAPK) activity were assayed in cell lysates. Englitazone increased 2DG uptake in a concentrationdependent (10–100 μmol/l) manner by up to sixfold, and preincubation with englitazone significantly enhanced insulin-stimulated 2DG uptake. However, englitazone had a biphasic effect on lipogenesis (163 ± 13% basal at 10 μmol/l vs. 96 ± 14% at 100 μmol/l), but when acetate was used as substrate, only concentrationdependent inhibition of lipogenesis occurred. In addition, englitazone decreased insulin-stimulated lipogenesis in a concentration-dependent manner. Englitazone did not increase IR, IRS-1/IRS-2, pp60, or MAPK phosphorylation, nor did it enhance insulin's stimulation of these parameters. Although englitazone alone did not activate PI 3-kinase, it did enhance the stimulation of the enzyme produced by a submaximally effective insulin concentration. Significant (63%) inhibition of insulin-stimulated lipogenesis occurred at a concentration of englitazone (30 μmol/l) that did not affect MAPK activation, which suggests that the drug's inhibitory effect on lipogenesis is not mediated by this pathway. Englitazone did not affect the expression of the peroxisome proliferator response element-containing fatty acyl CoA synthase gene, although it cannot be ruled out that expression of other lipogenic enzymes are altered by englitazone via peroxisome proliferator activated receptor-7 activation or by an alternate pathway. Thus englitazone stimulates 2DG uptake without affecting PI 3-kinase, but it can enhance both insulinstimulated 2DG uptake and PI 3-kinase activity. However, englitazone inhibits insulin-stimulated lipogenesis without inhibiting PI 3-kinase activity. Assuming activation of PI 3-kinase mediates insulin-stimulated 2-DG and lipogenesis, then the signaling pathways for each process diverge beyond PI 3-kinase.
RX871024 Induces Ca2+ Mobilization From Thapsigargin-Sensitive Stores in Mouse Pancreatic β-Cells
Authors: Efanova Ioulia B , Zaitsev Sergei V , Brown Graham , Berggren Per-Olof , Efendić Suad
Published: September 2013

The effects of RX871024, a compound with an imidazoline structure, on cytoplasmic-free Ca2+ concentration ([Ca2+]i) in mouse pancreatic β-cells were studied. RX871024 modulates [Ca2+]i; by at least two mechanisms. One mechanism involves closure ofATPregulated K+ channels, resulting in membrane depolarization, opening of voltage-gated L-type Ca2+ channels, and a subsequent increase in [Ca2+]i. Another mechanism, reported here for the first time, deals with RX871024-induced mobilization of Ca2+ from nonmitochondrial thapsigargin-sensitive intracellular stores. Reduced glutathione, inhibitors of cytochrome P-450, and monoaminooxidases A and B blocked this Ca2+ mobilization. It is concluded that the mechanism of RX871024-induced Ca2+ mobilization from intracellular stores involves changes in the oxidation/reduction state of the pancreatic β-cell and may be controlled by cytochrome P-450.
Severe Leptin Resistance in Brown Fat-Deficient Uncoupling Protein Promoter-Driven Diphtheria Toxin A Mice Despite Suppression of Hypothalamic Neuropeptide Y and Circulating Corticosterone Concentrations
Authors: Mantzoros Christos S , Frederich Robert C , Qu Daqing , Lowell Bradford B , Maratos-Flier Eleftheria , Flier Jeffrey S
Published: September 2013

Brown adipose tissue (BAT) has the capacity for uncoupled mitochondrial respiration and is proposed to be a key site for regulating energy expenditure in rodents. To better define the role of BAT in energy homeostasis, we previously created a line of transgenic mice with deficiency of BAT (UCP promoter-driven diphtheria toxin A transgenic mice [UCP-DTA]) mice. These mice develop obesity that initially is due to decreased energy expenditure and later accompanied by hyperphagia despite increased levels of circulating leptin. In addition, the obesity of these mice is accompanied by severe insulin-resistant diabetes and hyperlipidemia. To better define the basis for leptin resistance in this model, we treated UCP-DTA mice with leptin (300 ug i.p., b.i.d.) and compared their response with that of leptin-treated ob/ob and FVB control mice (30 μg i.p., b.i.d.). Leptin treatment of FVB and ob/ob mice decreased their body weight and food intake and improved their glucose homeostasis. In contrast, tenfold higher dosages of leptin had no effect on body weight, food intake, or circulating insulin or glucose concentrations of UCP-DTA mice. Hypothalamic neuropeptide Y (NPY) mRNA expression was lower in UCP-DTA mice than in littermate control FVB mice in the fed state, and increased progressively in response to food restriction as leptin levels fell. In parallel to the levels of hypothalamic NPY, corticosterone levels were initially suppressed and rose with food restriction. Thus food intake, body weight, and insulin and glucose homeostasis of UCP-DTA mice are all extraordinarily resistant to leptin, whereas hypothalamic NPY and the hypothalamopituitary adrenal (HPA) axis may remain under leptin control. Further elucidation of the mechanisms underlying leptin resistance in UCP-DTA mice may provide valuable insights into the basis for leptin resistance in human obesity.
Hyperinsulinemia Is Associated With the Incidence of Hypertension and Dyslipidemia in Middle-Aged Men
Authors: Salonen Jukka T , Lakka Timo A , Lakka Hanna-Maaria , Valkonen Veli-Pekka , Everson Susan A , Kaplan George A
Published: September 2013

Insulin resistance or compensatory hyperinsulinemia has been associated with hypertension and dyslipidemia in cross-sectional studies. In contrast, evidence from prospective population-based studies, which could establish the time order of the relationship, is sparse and inconsistent. Therefore, we investigated the associations of hyperinsulinemia with the incidence of hypertension and dyslipidemia in the Kuopio Ischemic Heart Disease Risk Factor Study, a population-based 4-year follow-up study of middle-aged men from eastern Finland. Out of 975 men who had no diabetes, 543 had resting systolic blood pressure (sBP) of <165 mmHg and resting diastolic blood pressure (dBP) of <95 mmHg at baseline and were not taking antihypertensive medication, and 764 had serum triglycerides of <2.3 mmol/l and HDL cholesterol of ≥1.0 mmol/l at baseline. In logistic regression models adjusted for age, baseline resting blood pressure, baseline lipids, obesity, weight change, and other risk factors, men with hyperinsulinemia (fasting insulin in the highest quintile, ≥12.0 mU/1) at baseline had a 2.0- fold (95% CI 1.1–3.5, P = 0.025) incidence of hypertension (sBP of≥165 or dBP of ≥95 mmHg), a 2.1-fold (95% CI 1.3–3.4, P = 0.002) incidence of dyslipidemia (serum HDL cholesterol of <1.0 mmol/l or serum triglycerides of ≥2.3 mmol/l), and a 2.6-fold (95% CI 1.1–6.3, P = 0.028) incidence of the combination of these disorders in 4 years, compared with normoinsulinemic men. These findings demonstrate the role of hyperinsulinemia in incident hypertension and dyslipidemia and suggest that both hypertension and dyslipidemia are associated with insulin metabolism disturbance, independently of obesity and body weight.
Troglitazone Upregulates LDL Receptor Activity in HepG2 Cells
Authors: Rayyes Osama Al , Florén Claes-Henrik
Published: September 2013

The aim of this in vitro study was to investigate the effect of troglitazone, a new oral antidiabetic agent, on LDL catabolism. HepG2 cells, which are cells from a well-differentiated cell line of hepatoma cells, were cultured and used to study LDL catabolism. Different concentrations of troglitazone, all within the therapeutic range for humans, were incubated in culture medium with 125I-labeled LDL to measure cell-associated and degraded 125I-LDL. Troglitazone increased cell-associated and degraded 125I-LDL by ∼30%. We also investigated if this effect occurred through a LDL receptor- mediated pathway or a non-LDL receptor pathway. By using dextran sulfate, a substance known to release bound LDL from its receptor, we found that troglitazone upregulated LDL receptor activity by ∼35%. In addition, we found that troglitazone increased the expression of the LDL receptor mRNA. The effect of troglitazone was comparable with that of a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, fluvastatin, with troglitazone having an upregulatory effect similar to that of fluvastatin. Insulin within human physiological concentrations also increased LDL receptor activity. We found that troglitazone and insulin had an additive effect on LDL catabolism. Also, the effect of troglitazone on LDL catabolism was studied in the presence of cyclosporine, an immunosuppressant drug that reduces LDL catabolism mainly by decreasing LDL receptor activity. The results showed that troglitazone can compensate for the reduced LDL receptor activity induced by cyclosporine, but that cyclosporine had a residual effect on the action of troglitazone. Thus troglitazone enhanced LDL binding, cell association, and degradation by increasing LDL receptor mRNA expression, with a subsequent increase in LDL receptor activity.
Prolonged Survival of Rat Islet and Skin Xenografts in Mice Treated with Donor Splenocytes and Anti-CD154 Monoclonal Antibody
Authors: Gordon Ethel J , Markees Thomas G , Phillips Nancy E , Noelle Randolph J , Shultz Leonard D , Mordes John P , Rossini Aldo A , Greiner Dale L
Published: September 2013

Treatment of C57BL/6 mice with one transfusion of BALB/c spleen cells and a brief course of anti-CD154 (anti-CD40 ligand) antibody permits BALB/c islet grafts to survive indefinitely and BALB/c skin grafts to survive for ∼50 days without further intervention. We now report adaptation of this protocol to the transplantation of islet and skin xenografts. We observed prolonged survival of rat islet xenografts in mice treated with donor-specific spleen cell transfusion and anti-CD154 monoclonal antibody (mAb). Challenge islet xenografts placed on these animals indicated that graft acceptance was species-specific but not strain specific. Spleen cells from recipients bearing intact grafts led to rejection of rat islet xenografts in scid mice, suggesting that graft acceptance was not due to complete clonal deletion of xenoreactive cells. We also observed prolonged survival of rat skin xenografts in mice treated with donor-specific transfusion and anti-CD154 mAb. Prolonged survival of skin xenografts was also species specific. We conclude that treatment with appropriately timed donor-specific transfusion and anti-CD154 mAb induces durable survival of both islet and skin xenografts in mice. Because this procedure is targeted directly at CD154, a co-activation molecule expressed predominantly by activated CD4+ T-cells, the results suggest that CD4+ cells have a major role in the cellular immune response to xenografts.
Splenic Macrophages From the NOD Mouse Are Defective in the Ability to Present Antigen
Authors: Piganelli Jon D , Martin Tracy , Haskins Kathryn
Published: September 2013

IDDM results from the destruction of pancreatic (β-cells by autoreactive T-cells that appear to avoid deletion early in development, possibly due to improper interaction with antigen-presenting cells (APCs) resident in the thymus or periphery. In the nonobese diabetic (NOD) mouse, there exists a defect in APC function characterized by its failure to fully mature upon stimulation. The NOD mouse thus provides an excellent model for the investigation of APC dysfunction and development and how these relate to the incidence of autoimmune diabetes. We initiated studies of APC function in the NOD mouse with respect to antigen processing and presentation, using a well-characterized antigen hen egg lysozyme (HEL) and comparing it with the closely related, major histocompatibility complex (MHC) (I-Ag7) identical, diabetes-resistant mouse strain NOR. Proliferation assays comparing NOD and NOR HEL-specific T-cells demonstrated that the T-cell proliferation response of the NOD mouse to both native and denatured forms of the antigen is lower than that of NOR. When crisscross proliferation experiments were conducted using purified Tcells and irradiated spleen cells as APCs from both strains, the results demonstrated that the defect in proliferation resided in the APC compartment of activation. The levels of intracellular glutathione (GSH) were compared in splenic macrophages from NOD and NOR mice; it was found that on antigenic stimulation, NOR macrophages produced significantly more intracellular GSH than did NOD macrophages, even under hyperglycemic (50 mmol/l glucose) conditions. The lower amount of GSH seen in the NOD may result in less efficient processing of antigen, and subsequently, lower levels of T-cell activation.
Fibroblast Growth Factor 2 Promotes Pancreatic Epithelial Cell Proliferation Via Functional Fibroblast Growth Factor Receptors During Embryonic Life
Authors: Bras Stéphanie Le , Miralles Francisco , Basmaciogullari Annie , Czernichow Paul , Scharfmann Raphael
Published: September 2013

Several investigators have postulated that soluble growth factors are involved in the early development of the pancreas. In many tissues in which soluble factors are implicated in development, these factors act on their target cells through tyrosine kinase receptors. Because we had some preliminary evidence that fibroblast growth factor receptors (FGFRs) were expressed in the early pancreas, we investigated the effect of fibroblast growth factors (FGFs) during embryonic pancreatic development. For that purpose, we first studied the distribution and the functionality of FGFRs during pancreatic organogenesis. FGFR1 and FGFR4 were shown to be expressed at a high level during early pancreatic development before embryonic day 16, their levels of expression decreasing thereafter. The functionality of FGFR was studied next. It was demonstrated in vitro that both FGF1 and FGF2 induce the expression of NGFI-A mRNA, a useful indicator of functional growth factor-signaling pathways. Finally, the effect of FGF2 on embryonic pancreatic epithelial cell proliferation was studied. It was shown that FGF2 induces the proliferation of pancreatic epithelial cells during embryonic life. Taken together, these data strongly suggest that FGFs are implicated in pancreatic development during embryonic life.
Temporal and Quantitative Correlations Between Insulin Secretion and Stably Elevated or Oscillatory Cytoplasmic Ca2+ in Mouse Pancreatic β-Cells
Authors: Jonas Jean-Christophe , Gilon Patrick , Henquin Jean-Claude
Published: September 2013

An increase in cytoplasmic Ca2+ in β-cells is a key step in glucose-induced insulin secretion. However, whether changes in cytoplasmic free Ca2+ ([Ca2+]i) directly regulate secretion remains disputed. This question was addressed by investigating the temporal and quantitative relationships between [Ca2+]i and insulin secretion. Both events were measured simultaneously in single mouse islets loaded with fura-PE3 and perifused with a medium containing diazoxide (to prevent any effect of glucose on the membrane potential) and either 4.8 or 30 mmol/l K+. Continuous depolarization with 30 mmol/l K+ in the presence of 15 mmol/l glucose induced a sustained rise in [Ca2+]i and insulin release. No oscillations of secretion were detected even after mathematical analysis of the data (pulse, spectral and sample distribution analysis). In contrast, alternating between 30 and 4.8 mmol/l K+ (1 min/2 min or 2.5 min/5 min) triggered synchronous [Ca2+]i and insulin oscillations of regular amplitude in each islet. A good correlation was found between [Ca2+]i and insulin secretion, and it was independent of the presence or absence of oscillations. This quantitative correlation between [Ca2+]i and insulin secretion was confirmed by experiments in which extracellular Ca2+ was increased or decreased (0.1–2.5 mmol/l) stepwise in the presence of 30 mmol/l K+. This resulted in parallel stepwise increases or decreases in [Ca2+]i and insulin secretion. However, while the successive [Ca2+]i levels were unaffected by glucose, each plateau of secretion was much higher in 20 than in 3 mmol/l glucose. In conclusion, in our preparation of normal mouse islets, insulin secretion oscillates only when [Ca2+]i oscillates in β-cells. This close temporal relationship between insulin secretion and [Ca2+]i changes attests of the regulatory role of Ca2+. There also exists a quantitative relationship that is markedly influenced by the concentration of glucose.
Insulin-Stimulated Akt Kinase Activity Is Reduced in Skeletal Muscle From NIDDM Subjects
Authors: Krook Anna , Roth Richard A , Jiang Xin Jian , Zierath Juleen R , Wallberg-Henriksson Harriet
Published: September 2013

The serine/threonine kinase Akt (PKB/Rac) has been implicated as playing a role in the insulin-signaling pathway to glucose transport. Little is known regarding the regulation of Akt kinase activity in insulin-sensitive tissues, such as skeletal muscle, or whether this regulation is altered in insulin-resistant states such as NIDDM. We examined the effect of insulin on Akt kinase activity in skeletal muscle from six NIDDM patients and six healthy subjects. Whole-body insulin sensitivity, assessed by the euglycemic-hyperinsulinemic clamp, was significantly lower in NIDDM subjects (P < 0.001), and this was accompanied by impaired in vitro insulin-stimulated glucose transport in skeletal muscle. In both groups, insulin induced a significant increase in Akt kinase activity, but the response to maximal insulin (60 nmol/1) was markedly reduced in skeletal muscle from NIDDM subjects (66% of control levels, P < 0.01). Impaired Akt kinase activity was not accompanied by decreased protein expression of Akt. Instead, a trend toward increased Akt expression was noted in skeletal muscle from NIDDM subjects (P < 0.1). These parallel defects in insulin-stimulated Akt kinase activity and glucose transport in diabetic skeletal muscle suggest that reduced Akt kinase activity may play a role in the development of insulin resistance in NIDDM.