bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the LIM/Homeodomain Gene Islet-1 and Single Nucleotide Screening in NIDDM
Authors: Riggs Andrew C , Tanizawa Yukio , Aoki Minoru , Wasson Jon , Ferrer Jorge , Rabin Daniel U , Vaxillaire Martine , Froguel Philippe , Permutt M Alan
Published: September 2013

Islet-1 (Isl-1) is a unique transcription factor that binds to the enhancer region of the insulin gene. To evaluate this gene in non-insulin-dependent diabetes mellitus (NIDDM), a full-length human Isl-1 cDNA was isolated and the genomic structure was characterized. The cDNA [2,395 bp plus additional poly(A) residues] contained an open reading frame from an initiator methionine at nucleotide 240 to an opal stop codon at nucleotide 1,286 (GenBank accession number UO7559), encoding a predicted protein of 349 amino acids (39 kDa). From their ends, 23 additional clones were sequenced, revealing 15 incomplete cDNAs and 8 intron-containing partially processed precursors. As determined by Northern blotting and reverse transcriptase–polymerase chain reaction analysis, Isl-1 was most abundantly expressed as a 2.4-kb mRNA in human islets, with a restricted pattern of expression in other adult human tissues. Analysis of genomic clones revealed that Isl-1 is encoded by six exons, varying in size from 168 bp (exon 5) to 1,230 bp (exon 6). Exons 2 and 3 each encode a LIM domain, while the homeodomain is completely contained within exon 4. The sequence of the proximal promoter region, including 426 bp upstream of the 5′ -end of the cDNA, revealed two potential regulatory elements, GCCAGCCGG (–414 to –406) and GCCACAGG (–357 to – 350), each differing by one base form a homologous sequence in the insulin gene (GCCACCGG) that has been shown to be a major positive regulatory element (Boam DSW, Clark AR, Docherty L: Positive and negative regulation of the human insulin gene by multiple trans-acting factors. J Biol Chem 265:8285–8296, 1990). A search by single-strand conformational polymorphism analysis for variants in the Isl-1 gene was undertaken in NIDDM patients. Three variants were identified in the cDNA, none of which alter the predicted amino acid sequence. No variants were found in the promoter. The results of these studies thus indicate that the Isl-1 gene is not a major contributor to NIDDM susceptibility in this Northern European Caucasian population.
Amelioration of High-Fat Feeding–Induced Insulin Resistance in Skeletal Muscle With the Antiglucocorticoid RU486
Authors: Kusunoki Masataka , Cooney Gregory J , Hara Tsutomu , Storlien Leonard H
Published: September 2013

Fat feeding produces whole-body insulin resistance and decreased glucose uptake in muscle tissue of rats. To examine the effect of glucocorticoid blockade on the insulin resistance caused by high-fat feeding, four groups of rats were fed diets high in starch (70% of calories) or fat (59% of calories) for 4 weeks with or without the antiglucocorticoid RU486 (69.8 μmol · kg−1 · day−1) in the food. Whole-body insulin action was assessed by the euglycemic clamp technique at an upper physiological insulin level with bolus 2-[3H]deoxyglucose to determine individual tissue insulin-stimulated glucose uptake. Whole-body glucose utilization (clamp glucose infusion rate [GIR]) was decreased by high-fat feeding (GIR 68.3 ± 12.2 vs. 182.6 ± 12.8 μmol · kg−1 · min−1 for the starch-fed group; P < 0.001). Addition of RU486 to the diet significantly improved (GIR 133.9 ± 12.8 μmol · kg−1 · min−1; P < 0.01), but did not fully reverse, the insulin resistance caused by fat feeding. RU486 was without effect in the starch-fed rats. In skeletal muscles, RU486 ameliorated 62 and 68% of the insulin resistance produced by fat feeding in red quadriceps and extensor digitorum longus hindlimb muscles, respectively, but had no effect in heart or white adipose tissue. These results suggest that glucocorticoids play, in a tissue-specific manner, a role in the maintenance and/or production of insulin resistance produced by high-fat feeding.
Apoptotic Cell Death Triggered by Nitric Oxide in Pancreatic β-Cells
Authors: Kaneto Hideaki , Fujii Junichi , Geuk Seo Han , Suzuki Keiichiro , Matsuko Taka-aki , Masahiro Nakamura , Tatsumi Haruyuki , Yamasaki Yoshimitsu , Kamada Takenobu , Taniguchi Naoyuki
Published: September 2013

Nitric oxide (NO) is believed to be an effector molecule that mediates interleuMn (IL)-1β-induced destruction and dysfunction of pancreatic β-cells. We have demonstrated that both exogenous NO and NO generated endogenously by IL-1β brought about apoptosis of isolated rat pancreatic islet cells as well as pancreatic β-cell tumorderived cell line HIT. This apoptosis was characterized by cleavage of DNA into nucleosomal fragments of 180–200 bp and morphologically by nuclear shrinkage, chromatic condensation, and apoptotic body formation. The EL-1β-induced internucleosomal DNA cleavage occurred in a time- and dose-dependent manner. Actinomycin D, cycloheximide, and nitric oxide synthase inhibitors inhibited the DNA cleavage, which was correlated with the amount of NO produced, indicating that NO produced by HIT cells themselves could mediate the apoptosis. Furthermore, in the presence of tumor necrosis factor (TNF)-α, large amounts of NO were produced by IL-1β and DNA cleavage occurred more noticeably, although TNF-β alone did not generate NO. Streptozotocin (STZ), a diabetogenic reagent containing a nitroso moiety, also released NO and induced internucleosomal DNA cleavage in HIT cells. These results suggest that NO-induced internucleosomal DNA cleavage is an important initial step in the destruction and dysfunction of pancreatic (β-cells induced by inflammatory stimulation or treatment with STZ.
Cloning and Expression of Cytokine-Inducible Nitric Oxide Synthase cDNA From Rat Islets of Langerhans
Authors: Karlsen Allan E , Andersen Henrik U , Vissing Henrik , Mose Larsen Peter , Fey Stephen J , Cuartero Beatriz , Madsen Ole D , Petersen Jacob S , Mortensen Steen B , Mandrup-Poulsen Thomas , Boel Esper , Nerup Jørn
Published: September 2013

An inducible nitric oxide (NO) synthase isoform (iNOS) is specifically induced in the β-cells of interleuldn (IL)-1β–exposed rat islets, suggesting a role for NO in the pathogenesis of type I diabetes. The aim of this study was to clone and characterize iNOS cDNA from cytokineexposed islets. Neither NO production nor iNOS transcription could be detected in rat islets or in rat insulinoma RIN-5AH β-cells cultured in the absence of cytokines. Addition of IL-1α alone or in combination with tumor necrosis factor-α induced a concentration- and time-dependent expression of the iNOS gene and associated NO production (measured as nitrite) from both islets and RIN cells. iNOS transcripts were cloned by reverse transcriptase-polymerase chain reaction from the cytokine-exposed rat islets and RIN cells, and DNA sequence analysis revealed a near 100% identity to the recently published iNOS cDNA cloned from cytokineexposed rat hepatocytes and smooth muscle cells. Recombinant rat islet iNOS was transiently and stably expressed in human kidney 293 fibroblasts, and the high enzymatic activity was inhibited by addition of the Larginine analogs, Nω-nitro-L-arginine methyl ester and aminoguanidine. Two-dimensional gel electrophoresis revealed the recombinant iNOS as a series of spots with the expected molecular mass of 131 kDa and pi values in the range of 6.8 to 7.0. In conclusion, the IL-1β-induced iNOS cloned and expressed from rat islets and RIN cells is encoded by the same transcript as the iNOS induced in other cell types.
Glucagon-Like Peptide I Increases Cytoplasmic Calcium in Insulin-Secreting βTC3-Cells by Enhancement of Intracellular Calcium Mobilization
Authors: Gromada Jesper , Dissing Steen , Bokvist Krister , Renström Erik , Frøkjær-Jensen Jørgen , Wulff Birgitte S , Rorsman Patrik
Published: September 2013

In the insulin-secreting β-cell line (STC3, stimulation with 11.2 mmol/1 glucose caused a rise in the intracellular free Ca2+ concentration ([Ca2+]i) in only 18% of the tested cells. The number of glucose-responsive cells increased after pretreatment of the cells with glucagon-like peptide I (GLP-I)(7–36)amide and at 10−11 mol/1; 84% of the cells responded to glucose with a rise in [Ca2+]i. GLP-I(7–36)amide induces a rapid increase in [Ca2+]i only in cells exposed to elevated glucose concentrations (≥5.6 mmol/1). The action of GLP-I(7–36)amide and forskolin involved a 10-fold increase in cytoplasmic cAMP concentration and was mediated by activation of protein kinase A. It was not associated with an effect on the membrane potential but required some (small) initial entry of Ca2+ through voltage-dependent L-type Ca2+ channels, which then produced a further increase in [Ca2+]i by mobilization from intracellular stores. The latter effect reflected Ca2+-induced Ca2+ release and was blocked by ryanodine. Similar increases in [Ca2+]i were also observed in voltageclamped cells, although there was neither activation of a background (Ca2+-permeable) inward current nor enhancement of the voltage-dependent L-type Ca2+ current. These observations are consistent with GLP-I(7–36) amide inducing glucose sensitivity by promoting mobilization of Ca2+ from intracellular stores. We propose that this novel action of GLP-I(7–36)amide represents an important factor contributing to its insulinotropic action.
Diabetic Ketoacidosis in Obese African-Americans
Authors: Umpierrez Guillermo , Casals Mary M C , Gebhart Suzanne S P , Mixon Peggy S , Clark W Scott , Phillips Lawrence
Published: September 2013

Our preliminary data indicate that 15% of African-American patients presenting with diabetic ketoacidosis (DKA) are obese. To determine underlying mechanisms, we analyzed the clinical characteristics and indexes of insulin secretion and insulin sensitivity in 35 obese patients with DKA, 22 obese patients with hyperglycemia, 10 lean patients with DKA, and 10 obese nondiabetic subjects. Studies were performed 1 day after resolution of DKA and after 12 weeks of follow-up. At presentation, both obese DKA and obese hyperglycemic patients had no detectable insulin response to intravenous glucose, but they did respond to glucagon administration. The acute insulin response (AIR) to glucagon in obese DKA patients (0.9 ± 0.1 ng/ml) was lower than in obese hyperglycemic subjects (1.5 ± 0.1 ng/ml, P < 0.01), but significantly greater than in lean patients with DKA (0.1 ± 0.1 ng/ml, P < 0.01). After 12 weeks of follow-up, the AIR to glucose improved in both groups of obese diabetic patients but remained significantly lower than in nondiabetic control subjects (both/* < 0.01). In contrast, the AIR to glucagon was not significantly different from that in obese control subjects. Insulin sensitivity was decreased in both groups of obese diabetic patients at presentation and improved after follow-up to levels similar to those in obese nondiabetic control subjects. Reactivity with islet cell antibodies was not detected in any of the patients. During follow-up, 25 of 35 obese DKA and 16 of 22 hyperglycemic patients were able to discontinue insulin therapy, with continued good metabolic control. Our results indicate that in African-Americans, obese patients with DKA represent a subset of type II diabetes. Although impaired insulin secretion and insulin action were found at presentation, decreased pancreatic insulin reserve appears to be the primary defect in the development of DKA in obese patients.
Glucose-Induced Insulin Receptor Tyrosine Phosphorylation in Insulin-Secreting β-Cells
Authors: Rothenberg Paul L , Willison L David , Wolf Bryan A
Published: September 2013

In the βTC3 insulin-secreting β-cell line, glucose rapidly induces the tyrosine phosphorylation of the 97-kDa insulin receptor β-subunit. Phosphorylation is transient, with fourfold stimulation by 2 min and subsequent dephosphorylation to basal levels by 10–15 min. Elevating the extracellular KC1 concentration equipotently initiates receptor phosphorylation. Preventing insulin secretion with 1 μmol/l epinephrine or by removing extracellular Ca2+ blocks the effect. In the absence of glucose-induced secretion, exogenous insulin also stimulated insulin receptor autophosphorylation transiently and with an ED50 of 4 × 10−9 mol/1. In addition, functional insulin-like growth factor I (IGF-I) receptors are also expressed by these β-cells, as indicated by IGF-I-induced receptor tyrosine phosphorylation (ED50 = 5 × 10−9 mol/1) and also by detection of hybrid insulin/IGF-I receptor autophosphorylation at 10−7 mol/1 IGF-I. Both glucose and insulin stimulate the tyrosine phosphorylation of the insulin receptor substrate (IRS) IRS-1 and increase by two- to fivefold the rapid association of IRS-1 with the 85-kDa α-subunit of the phosphatidylinositol-3-kinase, as determined by co-immunoprecipitation assays. These results demonstrate that in these β-cells, glucose-induced insulin secretion activates the β-cell surface insulin receptor tyrosine kinase and its intracellular signal transduction pathway, suggesting a new autocrine mechanism for the regulation of β-cell function.
Exposure of Adhesion Molecules on Activated Platelets in Patients with Newly Diagnosed IDDM Is Not Normalized by Near-Normoglycemia
Authors: Tschoepe Diethelm , Driesch Elke , Schwippert Barbara , Nieuwenhuis H-Karel , Gries Friedrich Arnold
Published: September 2013

It has been suggested that platelet hyperactivity contributes to the early evolution of diabetic vascular disease per se. This study directly evaluates the level of intravascular platelet activation in newly diagnosed IDDM patients before and after tight metabolic control. Platelet activation was determined by the Duesseldorf-III flow cytometry assay in 21 recent-onset hyperglycemic IDDM patients before insulin, after 3 days of treatment with intravenous insulin, and after 14 and 60 days of intensified conventional insulin therapy. The intravasal platelet activation status was quantified by the percentage of platelets exposing the activation-dependent molecules CD62 (P-selectdn), thrombospondin (TSP), and CD63 (GP53) as well as the activated fibrinogen receptor (GPIIB/IIIA). Fifty matched normal subjects served as control subjects. Fourteen patients completed the 60-day study design. After initial recompensation, near-normoglycemic control was achieved after 14 days (fasting blood glucose, 117.0 ± 19.0 mg/dl), and the HbA1 concentration was 7.6 ± 1.2% after 60 days. CD62+ (4.0 ± 4.5%), TSP+ (2.0 ± 1.8%), CD63+ (11.0 ± 7.0%), and activated-GPIIB/IIIA+ (7.6 ± 7.7%) platelet levels were initially 5, 3.3, 5.7, and 2.8 times higher than the mean level of normal. There was no correlation with any of the nearly normalized metabolic parameters. Thus, more activated platelets circulate in newly diagnosed IDDM patients, which supports the assumption of a prethrombotic condition even in disease stages without apparent vascular damage. Metabolic control does not appear to be successful in attenuating activated cellular hemostasis.
Insulin-Stimulated Muscle Glucose Clearance in Patients With NIDDM: Effects of One-Legged Physical Training
Authors: Dela Flemming , Larsen Jens Jørn , Mikines Kári J , Ploug Thorkil , Petersen Lone Nørgaard , Galbo Henrik
Published: September 2013

Physical training increases insulin action in skeletal muscle in healthy men. In non-insulin-dependent diabetes mellitus (NIDDM), only minor improvements in whole-body insulin action are seen. We studied the effect of training on insulin-mediated glucose clearance rates (GCRs) in the whole body and in leg muscle in seven patients with NIDDM and in eight healthy control subjects. One-legged training was performed for 10 weeks. GCR in whole body and in both legs were measured before, the day after, and 6 days after training by hyperinsulinemic (28, 88, and 480 mU · min−1 · m−2), isoglycemic clamps combined with the leg balance technique. On the 5th day of detraining, one bout of exercise was performed with the nontraining leg. Muscle biopsies were obtained before and after training. Whole-body GCRs were always lower (P < 0.05) in NIDDM patients compared with control subjects and increased (P < 0.05) in response to training. In untrained muscle, GCR was lower (P < 0.05) in NIDDM patients (13 ± 4, 91 ± 9, and 148 ± 12 ml/min) compared with control subjects (56 ± 12, 126 ± 14, and 180 ± 14 ml/min). It Increased (P < 0.05) in both groups in response to training (43 ± 10, 144 ± 17, and 205 ± 24 [NIDDM patients] and 84 ± 10, 212 ± 20, and 249 ± 16 ml/min [control subjects]). Acute exercise did not increase leg GCR. In NIDDM patients, the effect of training was lost after 6 days, while the effect lasted longer in control subjects. Training increased (P < 0.05) muscle lactate production and glucose storage as well as glycogen synthase (GS) mRNA in both groups. We conclude that training increases insulin action in skeletal muscle in control subjects and NIDDM patients, and in NIDDM patients normal values may be obtained. The increase in trained muscle cannot fully account for the increase in whole-body GCR. Improvements in GCR involve enhancement of insulin-mediated increase in muscle blood flow and the ability to extract glucose. They are accompanied by enhanced nonoxidative glucose disposal and increases in GS mRNA. The improvements in insulin action are short-lived.
Frequency of Mutations of Insulin Receptor Gene in Japanese Patients With NIDDM
Authors: Kan Masaharu , Kanai Fumihiko , Iida Mitsuru , Jinnouchi Hideaki , Todaka Mikio , Imanaka Takanobu , Ito Kimio , Nishioka Yasuhiko , Ohnishi Tetsuo , Kamohara Seika , Hayashi Hideki , Murakami Takashi , Kagawa Susumu , Sano Hiroyuki , Hashimoto Naotake , Yoshida Sho , Makino Hideichi , Ebina Yousuke
Published: September 2013

To examine the prevalence of abnormalities in the insulin receptor structure gene in Japanese Patients with non-insulin-dependent diabetes mellitus (NIDDM), a population of 51 patients with NIDDM was screened for mutations in this gene. Patient genomic DNAs of both alleles corresponding to 22 exons of the gene were amplified by polymerase chain reaction (PCR). The PCR products on pUC19 were sequenced. Three patients with heterozygous missense mutation Thr831→Ala831 in exon 13 and one patient with heterozygous missense mutation Tyr1334→Cys1334 in exon 22 of the ²-subunits were identified. Linkage analysis of one of the families plus statistical studies showed that the mutation Thr831→Ala831 is possibly responsible for the onset of NIDDM. In COS cells transiently expressing both mutant receptor cDNAs and a cDNA of a Mr 85,000 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), the mutation Tyr1334→Cys1334 impaired binding of the receptor with the Mr 85,000 subunit of PI 3-kinase, but linkage analysis of the family showed that the mutation did not cosegregate with NIDDM in the pedigree. Therefore, one missense mutation (Thr831→Ala831) in the insulin receptor, as found in three patients, is possibly involved in the etiology of a subset of the 51 NIDDM patients.
Skeletal Muscle Kallikrein: Potential Role in Metabolic Regulation
Authors: Mayfield Ronald K , Shimojo Nobuo , Jaffa Ayad A
Published: September 2013

Skeletal muscle glucose metabolism appears to be regulated by locally derived factors as well as by systemically circulating hormones. Local factors may be particularly important during exercise, when substrate demand can increase rapidly. Numerous studies in perfused limbs suggest that the kallikrein-kinin system may participate in the regulation of substrate delivery and utilization by skeletal muscle. Evidence also suggests that kinins mediate the increase in insulin sensitivity after administration of converting enzyme inhibitors. Tissue kallikrein has been isolated and purified from rat skeletal muscles, and its level is highest in muscle with high oxidative activity. In other tissues, kallikrein synthesis is under the influence of insulin. It has not been possible to demonstrate effects of kallikrein or kinins on glucose metabolism in isolated skeletal muscle or cardiocytes. Therefore modulation of glucose metabolism by kallikrein or kinins may only be observed in intact perfused tissues or organs.
Kininase II-Type Enzymes: Their Putative Role in Muscle Energy Metabolism
Authors: Dragović Tomislav , Minshall Richard , Jackman Herbert L , Wang Li-Xiu , Erdös Ervin G
Published: September 2013

Because of the importance of bradykinin in improving heart function in some conditions or in enhancing glucose uptake by skeletal muscle, we investigated kininases in these tissues. In P3 fraction of the heart and skeletal muscles, angiotensin I-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) are the major kininases, as determined first with specific substrates and second with bradykinin. ACE activity was highest in guinea pig heart (2.7 ± 0.07 μmol·h−1 · mg protein−1) but decreased in other species in this order: dog atrium, rat heart, dog ventricle, and human atrium. The specific activity of NEP was lower: 0.45 μmol · h−1 · mg protein−1 in cultured neonatal cardiac myocytes and varying between 0.12 and 0.05 μmol · h−1 · mg protein−1 in human, dog, rat, and guinea pig heart. In the skeletal muscle P3, ACE was most active in guinea pig and rat (1.2 and 1.1 μmol · h−1 · mg protein−1, respectively) but less so in dog (0.09 μmol · h−1 · mg protein−1). NEP activity was higher in dog P3 (0.28 μmol · h−1 · mg protein−1) but lower in rat and guinea pig (0.19 and 0.1 μmol · h−1 · mg protein−1, respectively). Continuous density gradient centrifugation enriched NEP activity in dog and rat (from 0.3 to 1.0 and 0.49 μmol · h−1 · mg protein−1, respectively). Immunoprecipitation with antiserum to purified NEP proved the specificity of the rat enzyme. Bradykinin (0.1 mmol/1) was inactivated in the presence and absence of inhibitors by rat skeletal muscle NEP, as measured by high-performance liquid chromatography. Here, 36% of the activity was caused by NEP and 19% by ACE. In radioimmunoassay (bradykinin 10 nmol/1), 46 and 55% of kininase in rat and dog skeletal muscle P3, respectively, was due to ACE; 36 and 28%, respectively, was due to NEP. Aside from these enzymes, an aminopeptidase in rat P3 also inactivates bradykinin. Thus, in conclusion, heart and skeletal muscle membranes contain kininase II-type enzymes, but their activity depends on the species.
Substrate Metabolism, Hormone Interaction, and Angiotensin-Converting Enzyme Inhibitors in Left Ventricular Hypertrophy
Authors: Zhu Yi-chun , Zhu Yi-zhun , Spitznagel Heidi , Gohlke Peter , Unger Thomas
Published: September 2013

Left ventricular hypertrophy is considered to be an independent risk factor giving rise to ischemia, arrhythmias, and left ventricular dysfunction. Slow movement of intracellular calcium contributes to the impaired contraction and relaxation function of hypertrophied myocardium. Myofibril content may also be shifted to fetal-type isoforms with decreased contraction and relaxation properties in left ventricular hypertrophy. Myocyte hypertrophy and interstitial fibrosis are regulated independently by mechanical and neurohumoral mechanisms. In severely hypertrophied myocardium, capillary density is reduced, the diffusion distance for oxygen, nutrients, and metabolites is increased, and the ratio of energy-production sites to energy-consumption sites is decreased. The metabolic state of severely hypertrophied myocardium is anaerobic, as indicated by the shift of lactate dehydrogenase marker enzymes. Therefore, the hypertrophied myocardium is more vulnerable to ischemie events. As a compensatory response to severe cardiac hypertrophy and congestive heart failure, the ADP/ATP carrier is activated and atrial natriuretic peptide is released to increase high-energy phosphate production and reduce cardiac energy consumption by vasodilation and sodium and fluid elimination. However, in severely hypertrophied and failing myocardium, vasoconstrictor and sodium- and fluid-retaining factors, such as the renin-angiotensin system, aldosterone, and sympathetic nerve activity, play an overwhelming role. Angiotensin-converting enzyme inhibitors (ACEIs) are able to prevent cardiac hypertrophy and improve cardiac function and metabolism. Under experimental conditions, these beneficial effects can be ascribed mainly to bradykinin potentiation, although a contribution of the ACEI-induced angiotensin II reduction cannot be excluded.
Diabetogenic T-Cell Clones
Authors: Haskins Kathryn , Wegmann Dale
Published: September 2013

The role of T-cells in the pathogenesis of IDDM has been an area of much interest, and investigators have recently acquired new tools for studies on T-cells with the advent of T-cell clones that are reactive with islet antigens. Derived from NOD mice, diabetogenic T-cell lines and clones have for the most part been CD4+ and T-helper 1 (Th1)-like in their cytokine production. Some CD8+ cytotoxic clones have also been reported, although these have generally not transferred diabetes in the absence of CD4+ T-cells. The T-cell clones that have been described can also be separated on the basis of their antigen reactivity. While many of the T-cell lines and clones described react with islets, isolated islet cells, or islet membrane preparations, others have known antigen specificities, reacting with defined islet cell proteins such as insulin, GAD, and heat shock proteins. Particularly in the case of insulin-reactive clones, diabetogenicity has also been demonstrated. In light of the many possible T-cell reactivities that may arise from the islet lesion, the question of whether there is a dominant initiating antigen is a particularly intriguing one.
The β3-Adrenergic Receptor Gene Trp64Arg Mutation is Overrepresented in Obese Women: Effects on Weight, BMI, Abdominal Fat, Blood Pressure, and Reproductive History in an Elderly Australian Population
Authors: Kurabayashi Takumi , Carey David G P , Morrison Nigel A
Published: September 2013

A tryptophan to arginine (Trp64Arg) mutation in the β3-adrenergic receptor (β3-AR) gene has been implicated in diabetes and obesity. We investigated the relationship of the β3-AR gene mutation with total body weight, BMI, central abdominal fat, blood pressure (BP), and reproductive history in 686 elderly subjects (429 women, 257 men; mean age 69.8 ± 6.9 [±SD] years) from a cross section of a normal population in Australia. About 14% of the test population were heterozygote carriers of the Trp64Arg mutation; however, significant effects on clinical parameters were only observed in women. The frequency of the mutation was significantly increased in obese women compared with lean women (BMI ≥ 27: 20% compared with BMI < 27: 11%, P = 0.02). Significantly higher total body weight (67.5 ± 12.9 vs. 64.1 ± 12.2 kg, P = 0.03) and BMI (26.3 ± 4.7 vs. 25.1 ± 4.5 kg/m2, P = 0.03) was observed in heterozygote women compared with normal subjects (homozygous for tryptophan). Central abdominal fat was not significantly different, except in women under 70 years, where heterozygotes had 16% higher abdominal fat compared with normal subjects. Female heterozygotes had significantly higher diastolic BP, even after adjustment for age and BMI (88.9 ± 11.1 vs. 84.2 ± 10.8 mmHg, P = 0.003) and a longer reproductive life, with an earlier menarche (12.8 ± 1.3 vs. 13.4 ± 1.5 years, P = 0.006), a higher gravidity (4.4 ± 2.4 vs. 3.5 ± 2.1, P = 0.01), and higher parity (3.8 ± 2.0 vs. 3.0 ± 1.9, P = 0.005). Clearly, the β3-AR mutation has pleiotrophic effects on a number of physiological systems, including BMI, BP, and reproductive history, perhaps suggesting evolutionary reasons for its maintenance in the population.
Diadenosine Polyphosphates: A Novel Class of Glucose-Induced Intracellular Messengers in the Pancreatic β-Cell
Authors: Ripoll Cristina , Martin Franz , Rovira Juan Manuel , Pintor Jesús , Miras-Portugal María Teresa , Soria Bernat
Published: September 2013

Diadenosine polyphosphates are a group of low-weight compounds that increase after exposure to a wide variety of oxidants and have been suggested to act as “alarmones,” alerting the cell to the onset of metabolic stress. We demonstrate here that glucose at concentrations that induce insulin release produce a 30- to 70-fold increase in the concentration of diadenosine triphosphate (Ap3A) and tetraphosphate (Ap4A) in β-cells. Furthermore, Ap3A and Ap4A, at the concentrations found in glucose-stimulated cells, are effective inhibitors of the ATP-regulated K+ channels when applied to the intracellular side of excised membrane patches from cultured β-cells. We suggest that Ap3A and Ap4A act as second messengers mediating a glucose-induced blockade of the pancreatic β-cell ATP-regulated potassium channel.
Evidence for Dissociation of Insulin Stimulation of Blood Flow and Glucose Uptake in Human Skeletal Muscle: Studies Using [15O]H2O, [18F]fluoro-2-deoxy-D-glucose, and Positron Emission Tomography
Authors: Raitakari Maria , Nuutila Pirjo , Ruotsalainen Ulla , Laine Hanna , Teräs Mika , Iida Hidehiro , Mäkimattila Sari , Utriainen Tapio , Oikonen Vesa , Sipilä Hannu , Haaparanta Merja , Solin Olof , Wegelius Uno , Knuuti Juhani , Yki-Järvinen Hannele
Published: September 2013

We determined the effect of insulin on muscle blood flow and glucose uptake in humans using [15O]H2O, [18F]fluoro-2-deoxy-D-glucose ([18F]FDG), and positron emission tomography (PET). Femoral muscle blood flow was measured in 14 healthy volunteers (age 34 ± 8 years, BMI 24.6 ± 3.4 kg/m2 [means ± SD]) before and at 75 min during a 140-min high-dose insulin infusion (serum insulin 2,820 ± 540 pmol/l) under normoglycemic conditions. A dynamic scan of the femoral region was performed using PET for 6 min after injection of [15O]H2O to determine the 15O concentration in tissue. Regional femoral muscle blood flow was calculated using an autoradiographic method from the dynamic data obtained with PET and [15O]H2O. Femoral muscle glucose uptake was measured during hyperinsulinemia immediately after the flow measurement using PET-derived [18F]FDG kinetics and a three-compartment model. Whole-body glucose uptake was quantitated using the euglycemic insulin clamp technique. In the basal state, 84 ± 8% of blood flow was confined to skeletal muscle. Insulin increased leg blood flow from 29 ± 14 to 54 ± 29 ml · kg−1 leg · min−1 (P < 0.001) and muscle flow from 31 ± 18 to 58 ± 35 ml · kg−1 muscle · min−1 (P < 0.005). Under insulin-stimulated conditions, 81 ± 8% of blood flow was in muscle tissue (NS versus basal). Skeletal muscle explained 70 ± 25% of the increase in leg blood flow. No correlation was observed between blood flow and glucose uptake when analyzed individually in identical regions of interest within femoral muscles. These data demonstrate that skeletal muscle accounts for most of the insulin-induced increase in blood flow. Insulin-stimulated rates of blood flow and glucose uptake do not colocalize in the same regions of muscle tissue, suggesting that insulin's hemodynamic and metabolic effects are differentially regulated.
β-Cell Growth and Mass Are Preserved in Long-Term Syngeneic Islet Transplantation in Streptozocin-Induced Diabetic Lewis Rats
Authors: Nácher Victor , Raurell Mercè , Merino J Francisco , Aranda Olga , Soler Joan , Montana Eduard
Published: September 2013

We determined β-cell replication and mass in basal and stimulated conditions in long-term transplanted islets. Three groups of streptozocin-induced diabetic Lewis rats were transplanted with 1,000 islets (500 islets under left and right kidney capsules). At 2 (Tx-2), 5 (Tx-5), or 9 (Tx-9) months after transplantation, one of the two grafts (basal) was harvested; 14 days later, the contralateral graft (stimulated) was also harvested. Normoglycemia was achieved and maintained in all transplanted rats, although the capacity to respond to a glucose challenge deteriorated slightly 9 months after transplantation. β-cell replication remained stable in Tx-2, Tx-5, and Tx-9 basal grafts and was similar to replication in a control group of nontransplanted rats (0.28 ± 0.06%); replication increased in Tx-2 (0.90 ± 0.23%, P < 0.05) and Tx-9 (0.72 ± 0.09%, P < 0.05) stimulated grafts. β-cell mass in basal grafts was similar to the initially transplanted mass (1.24 ± 0.06 mg) and increased in stimulated grafts in Tx-2 (1.91 ± 0.38 mg, P < 0.05) and Tx-5 (1.73 ± 0.27 mg, P = 0.01) groups, compared with basal grafts, and in Tx-2 and Tx-9 groups (1.92 ± 0.30 mg, P < 0.05), compared with initially transplanted mass. Therefore, β-cell replication and mass were preserved up to 9 months after syngeneic transplantation, and β-cells maintained the capacity to respond to increased metabolic demand, suggesting that replication is not a limiting factor in the survival of transplanted islets.
Insulin Sensitivity and Abdominal Obesity in African-American, Hispanic, and Non-Hispanic White Men and Women: The Insulin Resistance and Atherosclerosis Study
Authors: Karter Andrew J , Mayer-Davis Elizabeth J , Selby Joe V , D'Agostino Ralph B , Haffner Steven M , Sholinsky Phyliss , Bergman Richard , Saad Mohammed F , Hamman Richard F
Published: September 2013

Increased abdominal obesity has been related to lower insulin sensitivity (SI), independent of overall obesity, but it has been suggested that this relationship may be weaker in non-whites. In the Insulin Resistance and Atherosclerosis Study (IRAS), SI was estimated using a minimal model analysis of the frequently sampled intravenous glucose tolerance test in 1,625 men and women aged 40–69 years. Subjects included African-Americans, Hispanics, and non-Hispanic whites from Oakland and Los Angeles, CA, San Antonio, TX, and the San Luis Valley, CO. Minimum waist circumference was significantly (P = 0.0001) associated with SI after adjusting for age, sex, height, BMI, glucose tolerance status, ethnicity, and clinic. This relationship was significantly (P = 0.0001) stronger in subjects with normal glucose tolerance (NGT) (β = -0.030, P = 0.0001) than in those with impaired glucose tolerance (IGT) (β = -0.010, P = 0.02; NIDDM: β = -0.013, P = 0.0001). There were no significant ethnic differences in effect size across the spectrum of glucose tolerance. Waist circumference was also positively related to fasting insulin, an indirect measure of insulin sensitivity, in NGT (P = 0.0001), IGT (P = 0.0003), and NIDDM (P = 0.0002). The waist-fasting insulin relationship was significantly weaker in African-Americans, relative to non-Hispanic whites, in NGT and IGT (tests of statistical interaction: P = 0.04 and P = 0.02, respectively). In general, these patterns were similar in models specifying waist-to-hip ratio (WHR), rather than waist circumference, as the independent variable. While some ethnic variability exists, a negative relationship between abdominal obesity and insulin sensitivity was confirmed for all three ethnic groups across the spectrum of glucose tolerance.
The βHC-9 Pancreatic β-Cell Line Preserves the Characteristics of Progenitor Mouse Islets
Authors: Noda Mitsuhiko , Komatsu Mitsuhisa , Sharp Geoffrey W G
Published: September 2013

βHC-9 is a pancreatic β-cell line that is derived from the hyperplastic islets of transgenic mice that express the simian virus 40 tumor antigen gene in the islets. This cell secretes insulin in response to glucose in a concentration-dependent manner. Maximal and half-maximal concentrations were ∼ 20 and ∼ 10 mmol/l, respectively, with a maximal fractional release that averaged 3.7% of the total cellular insulin content per 60 min. The cellular insulin content was 3–9% of the content of mouse islet cells. Under perifusion conditions, high glucose concentrations induced a sharp first phase that lasted ∼ 10 min and a succeeding second phase of sustained release, as exhibited by mouse islets. The cells did not show a rising second phase as seen with rat islets. This biphasic response was obtained without the need for activators of protein kinase A such as forskolin or 3-isobutyl-1-methylxanthine. The dose-dependency and the phasic response to glucose were essentially invariable up to passage 38 but thereafter declined. The cells respond to various well-known stimulators of insulin secretion, including leucine and arginine; to modulators such as carbachol, glucagon-like peptide I, and pituitary adenylyl cyclase activating polypeptide; and to the inhibitors norepinephrine, somatostatin, and galanin. The pharmacological agents glibenclamide, 12-O-tetradecanoylphorbol-13-acetate, and KCl stimulate and forskolin potentiates insulin release. Mannoheptulose, 2-deoxyglucose, and nitrendipine inhibit glucose-stimulated insulin release from the cells. The intracellular Ca2+ concentration was raised by high glucose and by glibenclamide. In conclusion, this cell line preserves the fundamental characteristics of the progenitor normal mouse islets very well. Although several cell lines have been reported to have glucose-responsive insulin secretion, few demonstrate clear biphasic secretion as this cell line displays. In this context, this cell line should serve as a potent tool for studying the mechanisms of insulin secretion, especially the important phasic secretion.
PDX-1 Induces Insulin and Glucokinase Gene Expressions in αTC1 Clone 6 Cells in the Presence of Betacellulin
Authors: Watada Hirotaka , Kajimoto Yoshitaka , Miyagawa Jun-ichiro , Hanafusa Toshiaki , Hamaguchi Kazuyuki , Matsuoka Taka-aki , Yamamoto Koji , Matsuzawa Yuji , Kawamori Ryuzo , Yamasaki Yoshimitsu
Published: September 2013

The pancreatic β- and α-cells are developmentally related to each other but reveal diverse gene expression patterns. Among the two important transcription factors for insulin gene expression, IEF1 is present both in α- and β-cells, but PDX-1/IPF1/STF-1/IDX-1, a homeodomain-containing transcription factor, is present in β-cells but not in α-cells. To elucidate the function of PDX-1 in the expression of β-cell-specific genes, we established stable αTC1 clone 6 (αTC1.6)-derived transfectants expressing PDX-1 and examined the changes in the gene expression patterns in them. The exogenous expression of PDX-1 in αTC1.6 cells alone could induce islet amyloid polypeptide (IAPP) mRNA expression in the cells but not the expression of insulin, glucokinase, or GLUT2 gene. However, when βcellulin was added to the medium, the PDX-1-expressing αTC1.6 cells, but not the control αTC1.6 cells, came to express insulin and glucokinase mRNAs. This did not occur with other growth factors such as epidermal growth factor, transforming growth factor α, and insulin-like growth factor I. GLUT2 mRNA remained undetectable in the PDX-1–expressing αTC1.6 cells. These observations demonstrate the potency of PDX-1 for the expression of the insulin, glucokinase, and IAPP genes and suggest that certain regulatory factors, which can partially be modified by βcellulin, also contribute to the β-cell specificity of gene expression.
A Subtractive Cloning Approach to the Identification of mRNAs Specifically Expressed in Pancreatic β-cells
Authors: Neophytou Pavlos I , Muir Elizabeth M , Hutton Johnxs C
Published: September 2013

A polymerase chain reaction-based subtractive hybridization procedure was applied to cDNAs prepared from mouse insulinoma (βTC3) and glucagonoma (αTC2) cell lines to construct a library of cDNAs that are highly expressed in pancreatic β-cells. An analysis of 555 randomly chosen clones in the library showed that 80 were derived from abundant mRNAs and were accounted for by 29 distinct sequences. Of these, 17 were identical or homologous to known mammalian cDNAs or expressed sequence tags. Genes known to be highly expressed in β-cells were represented at a high frequency, namely insulin (15 of 80 clones), islet amyloid polypeptide (8 of 80 clones), proinsulin convertase 1 (6 of 80 clones), and neuropeptide Y (2 of 80 clones). Many of the novel cDNA sequences that were highly represented in the library showed a relative specificity to β-cells compared with other tissues, including glucagonoma, liver, kidney, brain, 3T3 fibroblasts, and AtT20 corticotrophs, and warrant further investigation. When combined with functional or immunological screening procedures, the approach will be useful for the isolation of β-cell–specific molecules for immunological and genetic investigations of β-cell function and pathology.
Selective Damage to Sensorimotor Perivascular Nerves in the Mesenteric Vessels of Diabetic Rats
Authors: Belai Abebech , Milner Pam , Aberdeen Judy , Burnstock Geoffrey
Published: September 2013

The perivascular innervation of the superior mesenteric artery and vein was examined using immunohistochemical and immunoassay techniques in rats 8 weeks after induction of diabetes with streptozotocin (STZ). Increased density of innervation and fluorescence intensity was noted for substance P– and calcitonin gene-related peptide–immunoreactive nerves in the diabetic vessels. A slight increase in the density of vasoactive intestinal polypeptide-immunoreactive nerve fibers innervating the mesenteric artery was also noted. However, there was no change in the density of neuropeptide Y– and dopamine β-hydroxylase–immunoreactive nerve fibers, although the fluorescence intensity of neuropeptide Y–immunoreactive nerve fibers was reduced in diabetic rat vessels. Immunoassays showed that the levels of substance P– and calcitonin gene–related peptide were increased > 10-fold in the diabetic mesenteric vein, while levels of neuropeptide Y and vasoactive intestinal polypeptide were unchanged. In summary, there is a marked increase in nerve fibers containing sensory neuropeptides in mesenteric vessels of STZ-induced diabetic rats, which, in view of the reported impaired sensorimotor function in these vessels, is likely to reflect a neuropathic change.
Induction of Allogeneic Islet Survival by Intrahepatic Islet Preimmunization and Transient Immunosuppression
Authors: Goss John A , Flye M Wayne , Lacy Paul E
Published: September 2013

Induction of tolerance to fully major histocompatibility complex (MHC)-mismatched rat islet allografts implanted at two different islet transplant sites (liver and kidney capsule [KC]) was examined. Streptozotocin-induced diabetic Lewis (RT11) rats remained hyperglycemic (> 200 mg/dl) after intrahepatic preimmunization by injection of 200 low-temperature cultured (24°C for 7 days) Wistar-Furth (WF, RT1u) rat islets into the portal vein with one injection (1 ml) of rat antilymphocyte serum intraperitoneally. Three weeks later, 1,200 WF islets that had been cultured to remove passenger lymphoid cells were transplanted into the liver via the portal vein or under the KC. The intrahepatic transplants survived 60.2 ± 11.9 days, and all six of the KC transplants maintained normoglycemia for > 100 days after the preimmunization regimen. In contrast, survival of fresh islet transplants was not significantly improved by this preimmunization protocol at either transplantation site. This study demonstrates that indefinite islet allograft survival can be achieved across a full MHC mismatch by intrahepatic preimmunization with a small number of cultured donor islets and a brief period of immunosuppression followed by transplantation of low-temperature cultured donor islets.
Evidence for an Anaplerotic/Malonyl-CoA Pathway in Pancreatic β-Cell Nutrient Signaling
Authors: Brun Thierry , Roche Enrique , Assimacopoulos-Jeannet Françoise , Corkey Barbara E , Kim Ki-Han , Prentki Marc
Published: September 2013

A metabolic model of fuel sensing has been proposed in which malonyl-CoA and long-chain acyl-CoA esters may act as coupling factors in nutrient-induced insulin release (Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney J, Corkey BE: Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem 267:5802–5810, 1992). To gain further insight into the control of malonyl-CoA content in islet tissue, we have studied the short- and long-term regulation of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in the β-cell. These enzymes catalyze the formation of malonyl-CoA and its usage for de novo fatty acid biogenesis. ACC mRNA, protein, and enzymatic activity are present at appreciable levels in rat pancreatic islets and clonal β-cells (HIT cells). Glucose addition to HIT cells results in a marked increase in ACC activity that precedes the initiation of insulin release. Fasting does not modify the ACC content of islets, whereas it markedly downregulates that of lipogenic tissues. This indicates differential regulation of the ACC gene in lipogenic tissues and the islets of Langerhans. FAS is very poorly expressed in islet tissue, yet ACC is abundant. This demonstrates that the primary function of malonyl-CoA in the β-cells is to regulate fatty acid oxidation, not to serve as a substrate for fatty acid biosynthesis. The anaplerotic enzyme pyruvate carboxylase, which allows the replenishment of citric acid cycle intermediates needed for malonyl-CoA production via citrate, is abundant in islet tissue. Glucose causes an elevation in β (HIT)-cell citrate that precedes secretion, and only those nutrients that can elevate citrate induce effective insulin release. The results provide new evidence in support of the model and explain why malonyl-CoA rises markedly and rapidly in islets upon glucose stimulation: 1) glucose elevates citrate, the precursor of malonyl-CoA; 2) glucose enhances ACC enzymatic activity; and 3) malonyl-CoA is not diverted to lipids. The data suggest that ACC is a key enzyme in metabolic signal transduction of the β-cell and provide evidence for the concept that an anaplerotic/malonyl-CoA pathway is implicated in insulin secretion.
Immunological and Metabolic Effects of Prophylactic Insulin Therapy in the NOD-scid/scid Adoptive Transfer Model of IDDM
Authors: Bowman Mark A , Campbell Lalita , Darrow Bethany L , Ellis Tamir M , Suresh Alaparthy , Atkinson Mark A
Published: September 2013

Prophylactic insulin therapy prevents IDDM in spontaneous animal models of the disease and has shown promise in preventing the disease in humans. Although large clinical trials have been formed to use this therapy, a comparative analysis of the efficiency of different pharmaceutical forms and doses of insulin in preventing IDDM has not been performed, and the mechanism underlying the observed prevention of disease is unknown. In the NOD-scid/scid adoptive transfer model of IDDM (10(7) new-onset NOD splenocytes injected intravenously into 6- to 8-week NOD/scid-scid recipients; insulitis develops at 6–9 days post-transfer and 100% IDDM by 32 days post-transfer), life-table (log-rank) analyses revealed that IDDM can be delayed (compared with insulin-free diluent, once daily, n = 8) with equivalent efficiency by prophylactic administration (–9–50 days post-transfer) of high (metabolism-altering) doses of short-acting (0.5 U, once daily, regular, n = 13) or long-acting (0.5 U, once daily, ultralente, n = 9) insulin as well as non-metabolism-altering low-dose insulin (0.02 U, once daily, regular, n = 8). Furthermore, IDDM was delayed with somatostatin (0.2 μg, twice daily, n = 11), an agent that suppresses endogenous insulin production. No significant difference was seen between the preventative effects of these agents. In an assessment of when therapies can be initiated and still maintain clinical efficiency, only prophylactic somatostatin therapy delayed IDDM (n = 10, P = 0.02) when initiated at 14 days post-transfer, whereas the short-acting insulin regimen did not retard the onset of IDDM (n = 8, P = 0.25) compared with diluent-treated controls. The 24-h urinary C-peptide levels were significantly reduced with short-acting (−56%, P = 0.01) and long-acting (–67%, P = 0.02) insulin products and somatostatin (–59%, P = 0.02) compared with diluent-treated controls. These results indicate that both immunological and metabolic (i.e., β-cell rest) factors may contribute to the beneficial effects of prophylactic insulin therapy.