bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial DNA in β-Cells Is a Sensitive Target for Damage by Nitric Oxide
Authors: Wilson Glenn L , Patton Nancy J , LeDoux Susan P
Published: September 2013

Increasing evidence indicates that nitric oxide (NO) may play a role in immune-mediated injury to β-cells. One site for the action of this agent is the mitochondrion. Although the exact targets for damage within this organelle have yet to be fully elucidated, a potential location for injury is mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by NO. Both exogenous NO generation (spermine/NO adduct [sper/NO]) and endogenous production of NO (IL-1β) were studied. To study the effects of exogenously produced NO, neonatal rat islet cells in monolayers were exposed to varying doses of sper/NO for 30 min. Total cellular DNA was isolated and treated with alkali to produce strand breaks at abasic sites resulting from exposure to NO. Damage to mtDNA was evaluated using a quantitative Southern blot technique. The results showed that sper/NO caused dose-dependent damage to mtDNA. Additionally, mtDNA was found to be more sensitive to injury generated by either source than a similarly sized fragment of nuclear DNA. To evaluate the effects of endogenously produced NO, β-cell cultures were treated with IL-1β for 18 h. Other cultures were treated with IL-1β and an inhibitor of the inducible form of nitric oxide synthase, aminoguanidine. DNA was evaluated as described for the sper/NO studies. IL-1β caused appreciable damage to mtDNA, and this damage was reduced in mtDNA from cultures treated with IL-1β and aminoguanidine. These studies show that mtDNA is a sensitive target for NO generated both endogenously and exogenously and that this DNA is more vulnerable to NO-induced damage than nuclear DNA.
Energetic Requirement of Insulin Secretion Distal to Calcium Influx
Authors: Rustenbeck Ingo , Herrmann Cornelia , Grimmsmann Thomas
Published: September 2013

A number of agents that inhibit oxidative phosphorylation by different mechanisms (carbonyl cyanide m-chlorophenylhydrazone [CCCP], sodium azide, oligomycin) induced an increase of cytoplasmic Ca2+ concentration ([Ca2+]i) in pancreatic β-cells, as measured by microfluorimetry with digital imaging. All three agents are known inhibitors of insulin secretion, and the secretory response to 20 mmol/l glucose was found to be abolished in spite of elevated [Ca2+]i. Two reasons could account for this dissociation between increase of [Ca2+]i and insulin secretion: 1) the increase did not take place at a site critical for exocytosis, 2) a threshold concentration of a metabolism-derived factor like ATP exists for the induction of exocytosis. The increase of [Ca2+]i by CCCP and sodium azide involved release of Ca2+ from internal stores, whereas oligomycin induced a slow D 600–inhibitable Ca2+ influx. Because CCCP and sodium azide, but not oligomycin, decreased the mitochondrial membrane potential concomitantly with the increase of [Ca2+]i, release of Ca2+ from the mitochondria most probably plays a decisive role for the internal mobilization. A Ca2+ influx induced by 40 mmol/l K+ or 250 μmol/l tolbutamide was unimpaired in the presence of oligomycin, but oligomycin completely abolished insulin secretion in response to these agents. While CCCP and sodium azide opened ATP-sensitive K+ channels, oligomycin was virtually ineffective, although it could be shown to significantly reduce β-cell ATP production. By comparison of the effects of different inhibitors of oxidative phosphorylation, we conclude that the initiation of exocytosis in β-cells is particularly sensitive to a decrease of energy metabolism, more than ATP-sensitive K+ channels or voltage-dependent Ca2+ channels. Thus, any increase of [Ca2+]i in β-cells that occurs in a situation of a decreased ATP supply is unlikely to elicit a secretory response.
Dyslipidemia and Hyperglycemia Predict Coronary Heart Disease Events in Middle-Aged Patients With NIDDM
Authors: Lehto Seppo , Rönnemaa Tapani , Haffher Steven M , Pyörälä Kalevi , Kallio Veikko , Laakso Markku
Published: September 2013

Patients with NIDDM are at increased risk for coronary heart disease (CHD). However, information on the predictive value of cardiovascular risk factors and the degree of hyperglycemia with respect to the risk for CHD in diabetic patients is still limited. Therefore, we carried out a prospective study on risk factors for CHD, including a large number of NIDDM patients. At baseline, risk factor levels of CHD were determined in 1,059 NIDDM patients (581 men and 478 women), aged from 45 to 64 years. These patients were followed up to 7 years with respect to CHD events. Altogether, 158 NIDDM patients (97 men [16.7%] and 61 women [12.8%]) died of CHD and 256 NIDDM patients (156 men [26.8%] and 100 women [20.9%]) had a serious CHD event (death from CHD or nonfatal myocardial infarction). A previous history of myocardial infarction, low HDL cholesterol level (<1.0 mmol/l), high non-HDL cholesterol (>5.2 mmol/l), high total triglyceride level (>2.3 mmol/l), and high fasting plasma glucose (>13.4 mmol/l) were associated with a twofold increase in the risk of CHD mortality or morbidity, independently of other cardiovascular risk factors. High calculated LDL cholesterol level (≥4.1 mmol/l) was significantly associated with all CHD events. The simultaneous presence of high fasting glucose (>13.4 mmol/l) with low HDL cholesterol, low HDL–to–total cholesterol ratio, or high total triglycerides further increased the risk for CHD events up to threefold. Our 7-year follow-up study provides evidence that dyslipidemia and poor glycemic control predict CHD mortality and morbidity in patients with NIDDM.
Leptin Directly Alters Lipid Partitioning in Skeletal Muscle
Authors: Muoio Deborah M , Dohn G Lynis , Fiedorek Frederick T , Tapscott Edward B , Coleman Rosalind A
Published: September 2013

Leptin, an adipocyte-derived hormone that directly regulates both adiposity and energy homeostasis, decreases food intake and appears to partition metabolic fuels toward utilization and away from storage. Because skeletal muscle expresses the leptin receptor and plays a major role in determining energy metabolism, we studied leptin's effects on glucose and fatty acid (FA) metabolism in isolated mouse soleus and extensor digitorum longus (EDL) muscles. One muscle from each animal served as a basal control. The contralateral muscle was treated with insulin (10 mU/ml), leptin (0.01–10 μg/ml), or insulin plus leptin, and incorporation of [14C]glucose or [14C]oleate into CO2 and into either glycogen or triacylglycerol (TAG) was determined. Leptin increased soleus muscle FA oxidation by 42% (P < 0.001) and decreased incorporation of FA into TAG by 35% (P < 0.01) in a dose-dependent manner. In contrast, insulin decreased soleus muscle FA oxidation by 40% (P < 0.001) and increased incorporation into TAG by 70% (P < 0.001). When both hormones were present, leptin attenuated both the antioxidative and the lipogenic effects of insulin by 50%. Less pronounced hormone effects were observed in EDL muscle. Leptin did not alter insulin-stimulated muscle glucose metabolism. These data demonstrate that leptin has direct and acute effects on skeletal muscle.
Hemodynamic Actions of Insulin in Rat Skeletal Muscle: Evidence for Capillary Recruitment
Authors: Rattigan Stephen , Clark Michael G , Barrett Eugene J
Published: September 2013

Insulin-induced increases in blood flow are hypothesized to enhance overall glucose uptake by skeletal muscle. Whether the insulin-mediated changes in blood flow are associated with altered blood flow distribution and increased capillary recruitment in skeletal muscle is not known. In the present study, the effects of insulin on hemodynamic parameters in rat skeletal muscle in vivo were investigated. Mean arterial blood pressure, heart rate, femoral blood flow, hind leg vascular resistance, and glucose uptake were measured in control and euglycemic insulin-clamped (10 mU · min−1 · kg−1) anesthetized rats. Blood flow distribution within the hind leg muscles was assessed by measuring the metabolism of 1-methylxanthine (1-MX), an exogenously added substrate for capillary xanthine oxidase. Insulin treatment had no effect on heart rate but significantly increased arterial blood pressure (12 mmHg) and femoral blood flow (80%) and decreased hind leg vascular resistance (31%). Changes were similar in magnitude and in time of onset to those reported in humans. Insulin treatment increased hind leg glucose uptake approximately fourfold and also increased hind leg 1-MX metabolism by 50%, suggesting increased exposure to endothelial xanthine oxidase. To ascertain whether the increased 1-MX metabolism was simply due to increased bulk femoral blood flow, epinephrine was infused at a dose (0.125 pg · min−1 · kg−1) chosen to match the insulin-induced increase in femoral blood flow. This dose of epinephrine had no significant effects on arterial blood pressure or heart rate but increased femoral blood flow and lowered hind leg vascular resistance to a similar extent as insulin. Epinephrine did not significantly alter 1-MX metabolism as compared with control animals. These results demonstrate that insulin increases total hind leg blood flow and metabolism of 1-MX, suggesting a recruitment of capillary blood flow in rat hind leg not mimicked by epinephrine.
Pioglitazone Induces In Vivo Adipocyte Differentiation in the Obese Zucker fa/fa Rat
Authors: Haliakon Sophie , Doaré Liliane , Foufelle Fabienne , Kergoat Micheline , Guerre-Millo Michèle , Berthault Marie-France , Dugail Isabelle , Morin Joëlle , Auwerx Johan , Ferré Pascal
Published: September 2013

Thiazolidinediones are potent antidiabetic compounds, in both animal and human models, which act by enhancing peripheral sensitivity to insulin. Thiazolidinediones are high-affinity ligands for peroxisome proliferator-activated receptor-γ, a key factor for adipocyte differentiation, and they are efficient promoters of adipocyte differentiation in vitro. Thus, it could be questioned whether a thiazolidinedione therapy aimed at improving insulin sensitivity would promote the recruitment of new adipocytes in vivo. To address this problem, we have studied the in vivo effect of pioglitazone on glucose metabolism and gene expression in the adipose tissue of an animal model of obesity with insulin resistance, the obese Zucker (fa/fa) rat. Pioglitazone markedly improves insulin action in the obese Zucker (fa/fa) rat, but doubles its weight gain after 4 weeks of treatment. The drug induces a large increase of glucose utilization in adipose tissue, where it stimulates the expression of genes involved in lipid metabolism such as the insulin-responsive GLUT, fatty acid synthase, and phospho-enolpyruvate carboxykinase genes, but decreases the expression of the ob gene. These changes are related to both an enhanced adipocyte differentiation, as shown by the large increase in the number of small adipocytes in the retroperitoneal fat pad, and a direct effect of pioglitazone on specific gene expression (phosphoenolpyruvate carboxykinase and ob genes) in mature adipocytes.
Role of the Glucagon Receptor COOH-Terminal Domain in Glucagon-Mediated Signaling and Receptor Internalization
Authors: Buggy Joseph J , Heurich Rainer O , MacDougall Margit , Kelley Keith A , Livingston James N , Yoo-Warren Heeja , Rossomando Anthony J
Published: September 2013

The binding of glucagon to its hepatic receptor is known to result in a number of effects, including the intracellular accumulation of cAMP, the mobilization of intracellular Ca2+, and the endocytosis of glucagon and its receptor into intracellular vesicles. In this study, we begin to define the functional role of the COOH-terminal tail of the human glucagon receptor in glucagon-stimulated signal transduction and receptor internalization. We have created and expressed in Chinese hamster ovary (CHO) cells five truncation mutants in which the COOH-terminal 24, 56, 62, 67, and 73 amino acids have been removed. Cells expressing relevant truncated receptors were assayed for cell surface expression by immunofluorescence, for ligand-binding properties, for cAMP and Ca2+-mediated signal transduction properties, and for receptor endocytosis. In addition, a mutant receptor containing seven serine-to-alanine mutations in the COOH-terminal tail was studied. Our results reveal the following: 1) a region of the COOH-terminal tail that is required for proper cell surface expression, 2) the COOH-terminal 62 amino acids, which comprise the majority of the tail, are not required for ligand binding, cAMP accumulation, or Ca2+ mobilization, and 3) phosphorylation of the COOH-terminal tail is crucial for glucagon-stimulated receptor endocytosis.
Effects of Metformin on Lactate Uptake and Gluconeogenesis in the Perfused Rat Liver
Authors: Radziuk Jerry , Zhang Zi , Wiernsperger Nicolas , Pye Susan
Published: September 2013

To directly assess the effects of the biguanide, metformin, on hepatic gluconeogenesis, it was added at high therapeutic levels (90 pg/ml) to the medium perfusing an isolated rat liver. Lactate (1 mg/min) was infused simultaneously along with [14C]lactate with or without [3H]lactate. [6-3H]glucose was added at the beginning of the perfusion in studies where [3H]lactate was not infused. Glucose levels decreased relative to control studies (metformin dose = 0) and lactate concentrations increased in this closed system. Quantitative analysis of the relationship between labeled glucose and lactate indicated that the flux of carbon from lactate to glucose and CO2 was halved, whereas reflux from glucose to lactate increased by ∼80%. This was corroborated by measurement of labeled lactate extraction as well as glucose, CO2, and lactate production across the liver. Glycogen content of the liver fell by 60% relative to control and was greater for the gluconeogenic pathway. These data are consistent with an inhibitory action of metformin on gluconeogenesis, which is due to a primary inhibition of hepatic lactate uptake.
Localization of the ATP-Sensitive K+ Channel Subunit Kir6.2 in Mouse Pancreas
Authors: Suzuki Masakazu , Fujikura Keiko , Inagaki Nobuya , Seino Susumu , Takata Kuniaki
Published: September 2013

Kir6.2, a member of the inward rectifier K+ channel family, is a component of the ATP-sensitive K+ (KATP) channel considered to play a key role in glucose-induced insulin secretion. We studied the distribution of Kir6.2 in mouse pancreas at the cellular level. The sites of Kir6.2 mRNA expression were determined by in situ hybridization histochemistry with a digoxigenin (DIG)-labeled antisense cRNA probe. The hybridization signal was unevenly present throughout the islets of Langerhans, while no distinct signal was detected in exocrine acinar cells. This distribution was confirmed by another cRNA probe complementary to a different region of Kir6.2 mRNA. In situ hybridization and immunofluorescence staining of serial sections with the anti-insulin, the anti-glucagon, and the anti-somato-statin antibodies showed Kir6.2 mRNA to be present in α-, β-, and δ-cells. Furthermore, immunofluorescence staining with antibody raised against Kir6.2 revealed that Kir6.2 protein is localized within the pancreatic islets and is not found in exocrine pancreas. Kir6.2 was further shown to be located together with insulin, glucagon, or somatostatin. The positive staining of Kir6.2 appeared concentrated along the contour of each islet cell, suggesting that Kir6.2 is at the plasma membrane of islet cells. These results suggest that Kir6.2, as a component of KATP channels, is an important molecule in the regulation of all the release of insulin, glucagon, and somatostatin.
The TNF-α Gene Neo I Polymorphism Influences the Relationship Among Insulin Resistance, Percent Body Fat, and Increased Serum Leptin Levels
Authors: Fernández-Real J M , Gutierrez C , Ricart W , Casamitjana R , Fernández-Castañer M , Vendrell J , Richart C , Soler J
Published: September 2013

Tumor necrosis factor-α (TNF-α), acting as a modulator of gene expression in adipocytes, is implicated in the development of insulin resistance and obesity. The aim of this study was to investigate whether the Nco I polymorphism of the TNF-a gene influences the relationship among insulin resistance, percent body fat, and serum leptin levels. A sample of 38 subjects (19 men, mean age 36.2 ± 1.9 years, BMI 28.8 ± 1.2 kg/m2, range 22.2–35.7; and 19 women, age 34.9 ± 1.4 years, BMI 28.1 ± 0.8 kg/m2, range 19–37.9) was divided into two groups on the basis of the Nco I genotype. Twenty-three subjects were (+/+) homozygotes for the presence of the Nco I restriction site that is associated with a guanine at position −308 of the TNF-a promoter. Of the other subjects, 12 were (+/−) heterozygotes and 3 (−/−) homozygotes for the absence of the restriction site, resulting from a guanine-to-adenine substitution at position −308 of the TNF-a promoter. This substitution (termed TNF-2) leads to higher rate of transcription of TNF-a than the wild-type allele TNF-1 in vitro. TNF-1 (+/+) and TNF-2 (+/− and −/−) groups of subjects were comparable in sex, age, BMI, waist-to-hip ratio, and several skinfold measurements. Basal serum insulin was greater (14.2 ± 2 vs. 9.2 ± 0.9 mlM, P = 0.041) in the TNF-2 group in the presence of comparable serum glucose concentration. The integrated area under the curve of serum insulin concentrations, measured in response to a 75-g oral glucose challenge, and the percent body fat, measured by bioelectric impedance, were significantly increased in TNF-2 subjects (226.8 ± 33 vs. 139.4 ± 17.8 mU/l, P = 0.032; 33.6 ± 2.8 vs. 24.9 ± 2%, P = 0.01). TNF-2 subjects also showed a decreased insulin sensitivity index, as determined by the frequently sampled intravenous glucose tolerance test with minimal model analysis (1.9 ± 0.4 vs. 3.05 ± 0.3 min−1 · mU−1 · 1−1 P = 0.03). These differences were more marked among women. Paralleling the known relationship between insulin and leptin levels, serum leptin concentration was clearly increased in the TNF-2 group (19.6 ± 3.4 vs. 11.1 ± 1.5 ng/ml, P = 0.03). Therefore, (+/−) heterozygotes and (−/−) homozygotes may be more susceptible to developing insulin resistance and increased percent body fat. Results of the present study suggest that TNF-αNco I polymorphism may exacerbate the alterations in leptin levels normally found among insulin-resistant subjects.
Advanced Glycation End Product-Induced Activation of NF-κB is Suppressed by α-Lipoic Acid in Cultured Endothelial Cells
Authors: Bierhaus Angelika , Chevion Shlomit , Chevion Mordechai , Hofmann Marion , Quehenberger Peter , Illmer Thomas , Luther Thomas , Berentshtein Eduard , Tritschler Hans , Müller Martin , Wahl Peter , Ziegler Reinhard , Nawroth Peter P
Published: September 2013

Depletion of cellular antioxidant defense mechanisms and the generation of oxygen free radicals by advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic vascular complications. Here we demonstrate that incubation of cultured bovine aortic endothelial cells (BAECs) with AGE albumin (500 nmol/l) resulted in the impairment of reduced glutathione (GSH) and ascorbic acid levels. As a consequence, increased cellular oxida-tive stress led to the activation of the transcription factor NF-KB and thus promoted the upregulation of various NF-KB-controlled genes, including endothelial tissue factor. Supplementation of the cellular antiox-idative defense with the natural occurring antioxidant α-lipoic acid before AGE albumin induction completely prevented the AGE albumin–dependent depletion of reduced glutathione and ascorbic acid. Electrophoretic mobility shift assays (EMSAs) revealed that AGE albumin-mediated NF-KB activation was also reduced in a time- and dose-dependent manner as long as α-lipoic acid was added at least 30 min before AGE albumin stimulation. Inhibition was not due to physical interactions with protein DNA binding, since α-lipoic acid, directly included into the binding reaction, did not prevent binding activity of recombinant NF-KB. Western blots further demonstrated that α-lipoic acid inhibited the release and translocation of NF-KB from the cytoplasm into the nucleus. As a consequence, α-lipoic acid reduced AGE albumin-induced NF-KB mediated transcription and expression of endothelial genes relevant in diabetes, such as tissue factor and endothelin-1. Thus, supplementation of cellular antioxidative defense mechanisms by extracellularly administered α-lipoic acid reduces AGE albumin-induced endothelial dysfunction in vitro.
Mutations in the Hepatocyte Nuclear Factor-1α/MODY3 Gene in Japanese Subjects With Early- and Late-Onset NIDDM
Authors: Iwasaki Naoko , Oda Naohisa , Ogata Makiko , Hara Manami , Hinokio Yoshinori , Oda Yukie , Yamagata Kazuya , Kanematsu Sachiko , Ohgawara Hisako , Omori Yasue , Bell Graeme I
Published: September 2013

Recent studies have shown that mutations in the hepatocyte nuclear factor (HNF)-1α gene are the cause of maturity-onset diabetes of the young type 3 (MODY3). We have screened 193 unrelated Japanese subjects with NIDDM for mutations in this gene: 83 with early-onset NIDDM (diagnosis at <30 years of age) and 110 with late-onset NIDDM (diagnosis >30 years of age). All of the members of the latter group also had at least one sibling with NIDDM. The 10 exons, flanking introns, and promoter region were amplified using polymerase chain reaction and were sequenced directly. Mutations were found in 7 of the 83 (8%) unrelated subjects with early-onset NIDDM. The mutations were each different and included four missense mutations (L12H, R131Q, K205Q, and R263C) and three frameshift mutations (P379fsdelCT, T392fsdelA, and L584S585fsinsTC). One of the 110 subjects with late-onset NIDDM was heterozygous for the missense mutation G191D. This subject, who was diagnosed with NIDDM at 64 years of age, also had a brother with NIDDM (age at diagnosis, 54 years) who carried the same mutation, suggesting that this mutation contributed to the development of NIDDM in these two siblings. None of these mutations were present in 50 unrelated subjects with normal glucose tolerance (100 normal chromosomes). Mutations in the HNF-1α gene occur in Japanese subjects with NIDDM and appear to be an important cause of early-onset NIDDM in this population. In addition, they are present in about 1% of subjects with late-onset NIDDM.
Novel Glucosensor for Hypoglycemic Detection Localized to the Portal Vein
Authors: Hevener Andrea L , Bergman Richard N , Donovan Casey M
Published: September 2013

In this investigation, we sought to constrain the locus of essential portohepatic glucosensors and test the hypothesis that they reside strictly in the portal vein and not the liver. Male Wistar rats were chronically cannulated in the carotid artery (sampling), jugular vein (infusion), and portal vein, either adjacent to (PORADJ, 0.6 ± 0.1 cm, n = 6) or upstream from (PORUPS, 2.7 ± 0.1 cm, n = 8) the liver. Animals were exposed to one of three protocols distinguished by the site of glucose infusion: PORUPS, PORADJ, or peripheral (PER). Systemic hypoglycemia (2.4 ± 0.1 mmol/1) was induced via jugular vein insulin infusion (50 mU · kg−1 · min−1). Arterial plasma catecholamines were assessed at basal (−30 and 0 min) and during sustained hypoglycemia (60, 75, 90, 105 min). By design, hepatic glucose was significantly elevated during PORUPS and PORADJ versus PER (4.3 ± 0.1 vs. 2.4 ± 0.1 mmol/l, respectively; P < 0.05). There were no significant differences between protocols in arterial glucose or insulin concentrations (9,372 ± 1,798 pmol/l). When liver and systemic glucose concentrations were allowed to fall concomitantly (PER), epinephrine was elevated 16-fold above basal levels (3.0 ± 0.6 vs. 46.4 ± 4.3 nmol/l, P < 0.001). When portohepatic normoglycemia was maintained during PORUPS, a 67% suppression in the epinephrine response versus that during PER was observed (P < 0.001). However, when the cannula was advanced adjacent to the liver, by comparison with PER, there was no suppression in the sympathoadrenal response (P = 0.73). While both PORUPS and PORADJ yielded elevated liver glycemia in the face of systemic hypoglycemia, only POR(UPS yielded an elevated portal vein glucose concentration. That only PORUPS resulted in a significant suppression of the sympathoadrenal response is consistent with the localization of the glucosensors to the portal vein.
Effects of Acute Glucose Overload on Histamine H2 Receptor–Mediated Ca2+ Mobilization in Bovine Cerebral Endothelial Cells
Authors: Kimura Chiwaka , Oike Masahiro , Kashiwagi Seizaburo , Ito Yushi
Published: September 2013

[Ca2+]i and whole-cell membrane current were measured in microvascular endothelial cells from bovine brain. The effects of histamine on [Ca2+]i were examined, and the acute effect of changing extracellular glucose concentration on Ca2+ homeostasis was investigated. Application of 10 μmol/l histamine evoked an initially transient and then sustained increase in [Ca2+]i in normal Krebs solution, but only the transient component in Ca2+-free solution, thereby indicating that histamine mobilizes Ca2+ both from intracellular store sites and extracellular space. The effects of histamine on [Ca2+]i were inhibited by the H2 antagonists, ranitidine and cimetidine, but not by the H1 antagonist, pyrilamine. Incubation of the cells for 2 h in solutions containing low (1.1 and 2.3 mmol/l) or high (23 mmol/l) concentrations of glucose did not influence the resting level of [Ca2+]i. Treatment with low concentrations of glucose did not impair histamine-induced Ca2+ mobilization. On the other hand, when histamine was applied to the cells pretreated with 23 mmol/l glucose, it failed to mobilize Ca2+ from both intracellular store sites and extracellular space. The effect of histamine was mimicked by dibutyryl cyclic AMP, but glucose overload failed to inhibit this, suggesting that glucose overload inhibits H2 receptor-mediated cyclic AMP production. Glucose overload-induced impairment of histamine action was reversed by pretreatment with staurosporine and calphostin C and mimicked by phorbol- 12,13-dibutyrate, thereby suggesting the involvement of protein kinase C in the high glucose-induced inhibition of Ca2+ mobilization. Whole-cell membrane current measurement showed that there was no difference in the membrane currents between control and high glucose- treated cells. These results indicate that in bovine brain microvascular endothelial cells, histamine induces Ca2+ release from intracellular store sites and subsequent entry from the extracellular space through the activation of H2 receptors. Glucose overload acutely inhibits histamine-induced Ca2+ mobilization by the activation of protein kinase C.
Hyperglycemia Rather Than Insulin Resistance Is Related to Reduced Coronary Flow Reserve in NIDDM
Authors: Yokoyama Ikuo , Ohtake Tohru , Momomura Shin-ichi , Yonekura Katsunori , Woo-Soo Shin , Nishikawa Junichi , Sasaki Yasuhito , Omata Masao
Published: September 2013

To clarify if coronary flow reserve (CFR) is related to insulin resistance or hyperglycemia in normotensive NIDDM, myocardial blood flow (MBF) at baseline and during dipyridamole loading were measured with 13N-ammonia positron-emission tomography. CFR was significantly reduced in NIDDM patients compared with agematched control subjects. CFR in patients with well-controlled NIDDM was significantly higher than in those with poorly controlled NIDDM, whereas insulin resistance was comparable between the two groups. CFR in NIDDM patients was not related to the degree of insulin resistance. CFR correlated significantly with average fasting glucose concentration and average HbA1c, but not with insulin resistance, age, lipid parameters, or blood pressure. In conclusion, control of blood glucose concentration rather than insulin resistance is most likely related to the reduced CFR in NIDDM.
Thiazolidinedione Exposure Increases the Expression of Uncoupling Protein 1 in Cultured Human Preadipocytes
Authors: Digby Janet E , Montague Carl T , Sewter Ciaran P , Sanders Louise , Wilkison William O , O'Rahilly Stephen , Prins Johannes B
Published: September 2013

Thiazolidinediones (TZDs) are a novel class of insulinsensitizing agents used in the treatment of NIDDM and are potent agonists for the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ). The thiazolidinedione BRL 49653 has been shown to promote the differentiation of the HIB-1B brown preadipocyte cell line and to increase rat interscapular brown adipose tissue (BAT) mass. Given the importance of brown fat in the control of energy metabolism in rodents, this may represent an important therapeutic effect of this class of compound. To date, however, no studies examining the effects of TZDs on human brown fat have been reported. In the present study, we have measured uncoupling protein 1 (UCP-1) mRNA, a specific marker for BAT, in isolated adipocytes and subcultured preadipocytes prepared from different adult human adipose tissue depots. Consistent with previous studies of adult human whole adipose tissue, UCP-1 mRNA was detectable in isolated human adipocytes prepared from all depots studied with a rank order of perirenal, omental, and subcutaneous. BRL 49653 treatment of subcultured human pre-adipocytes prepared from all depots resulted in increased levels of UCP-1 mRNA, compared with those of the vehicletreatedcells. When exposed to BRL 49653 for 5 days, preadipocytes from the human perirenal depot accumulated lipid, and a proportion of cells showed clear mitochondrial staining for UCP-1 protein by confocal microscopy. Thus, cells of the brown fat lineage were detectable in all human adipose depots studied, and cultured human pre-adipocytes, particularly from the perirenal depot, showed a marked increase in UCP-1 expression in response to thiazolidinediones. Given the role of brown adipocytes in the enhancement of energy expenditure, promotion of brown fat adipogenesis by thiazolidinediones could contribute to the beneficial effects of these drugs on insulin resistance in humans.
Effects of Cell-Permeable Ceramides and Tumor Necrosis Factor-α on Insulin Signaling and Glucose Uptake in 3T3-L1 Adipocytes
Authors: Wang Chuen-Neu , O'Brien Lori , Brindley David N
Published: September 2013

Incubation of 3T3-L1 adipocytes with C2- and C6- ceramides (N-acetyl- and N-hexanoylsphingosines) but not dihydro-C2-ceramide increased 2-deoxyglucose uptake in the absence of insulin. This effect was inhibited by PD 98059, LY 294002, and rapamycin, which block the activation of mitogen-activated protein kinase, phosphatidylinositol (PI) 3-kinase, and ribosomal S6 kinase, respectively. Long-term increases in PI 3-kinase activity associated with insulin receptor substrate 1 (IRS-1) increased GLUT1 and GLUT4 concentrations in plasma membranes. This together with increased GLUT1 (but not GLUT4) synthesis explains the increase in non-insulin-dependent glucose uptake. C2-ceramide inhibited insulin-stimulated glucose uptake after 2 h by decreasing insulin-induced translocation of GLUT1 and GLUT4 to plasma membranes. This occurred when there was no increase in basal glucose uptake or decrease in activation of IRS-1 or PI 3-kinase. Incubation for 24 h with tumor necrosis factorα (TNF-α) but not C2-ceramide decreased the concentration and insulin-induced tyrosine phosphorylation of IRS-1 in this experimental system. Cell-permeable ceramides mimic some effects of TNF-α, especially in stimulating basal glucose uptake. We identified a site for inhibiting insulin-stimulated glucose uptake that is downstream of PI 3-kinase. Our work provides further mechanisms for the effects of TNF-α and ceramides in increasing non-insulin-dependent glucose uptake and decreasing insulin-stimulated uptake in vivo.
Paradoxical Antidiabetogenic Effect of γ-Interferon in DP-BB Rats
Authors: Nicoletti Ferdinando , Zaccone Paola , Marco Roberto Di , Magro Gaetano , Grasso Sebastiano , Stivala Franca , Calori Giliola , Mughini Lanfranco , Meroni Pier Luigi , Garotta Gianni
Published: September 2013

Previous studies have shown that anti-γ-interferon (IFN-γ) antibody reduces the frequency of autoimmune IDDM in the DP-BB rat. We tested the effects of systemically administered recombinant rat IFN-γ in both DP-BB and DR-BB rats. Unexpectedly, IFN-γ markedly reduced the incidence of IDDM as compared with control rats when administered six times per week at a dosage of 280,000 U between ages 30-35 to 105 days or ages 60-64 to 105 days. A lower dosage (28,000 U on alternate days) was also protective when administered to DP-BB rats between birth and age 60 days. However, long-lasting protection against IDDM development over the 1-year study period was achieved only by the highest dosage of IFN-γ administered from age 30 to 105 days. Ex vivo production of tumor necrosis factor-a from splenic lymphoid cells (SLCs) and peritoneal macrophages of the rats treated with IFN-γ was comparable with that of controls; however, SLCs from the IFN-γ-treated animals secreted lower amounts of IFN-γ after stimulation with concanavalin A. IFN-γ treatment also markedly reduced the frequency of phenotypically activated SLC-expressing class II antigens and interleukin-2 receptor. Finally, in agreement with the observed antidiabetogenic effects, exogenously administered IFN-γ induced neither insulitis nor IDDM development in DR-BB rats, a subline of DP-BB rats in which autoimmune diabetes rarely occurs spontaneously but can be induced by administration of polyinosinic-polycytidilic acid.
Protection Against Autoimmune Diabetes With Oral Insulin Is Associated With the Presence of IL-4 Type 2 T-Cells in the Pancreas and Pancreatic lymph Nodes
Authors: Ploix Corinne , Bergerot Isabelle , Fabien Nicole , Perche Stéphanie , Moulin Valerie , Thivolet Charles
Published: September 2013

Oral administration of antigens has been proposed in the prevention and treatment of autoimmune diseases. We reported that oral administration of 0.8 mg of recombinant human insulin to 6-week-old NOD mice every other day for a month generated regulatory T-cells that were able to reduce the severity of insulitis and the percentage of clinical diabetes in naive irradiated recipients when co-injected with diabetogenic T-cells. In the present study, immunohistochemical analysis of the pancreatic glands revealed that injection of T-cells from insulin-fed mice upregulated the number of interleukin (IL)-4-secreting cells within the islets. Using two strains of NOD mice congenic at the Theta, or Thyl, locus, we observed a higher proportion of T-cells from insulin-fed mice in both the spleen (7.73 ± 0.3 vs. 5.57 ± 0.2%; P < 0.001) and the pancreatic lymph nodes (10.1 ± 0.8 vs. 7.2 ± 0.7%; P < 0.05) of cotransferred mice. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, mice reconstituted with T-cells from insulin-fed animals had detectable amounts of IL- 4 mRNA, specifically in the pancreatic lymph nodes (8 of 9 experimental mice vs. 1 of 9 control mice) and the pancreas (3 of 3 experimental mice vs. 0 of 3 control mice). γ-Interferon mRNA was detectable in all cotransferred animals, but IL-10 mRNA and transforming growth factor β mRNA were undetectable. These results suggested a shift from a T-helper 1 (Thl) to a Th2 pattern of cytokine expression and underlined the role of pancreatic lymph nodes in the protection. Repeated injections of 500 ug s.c. of anti-IL-4 monoclonal antibody led to an accentuation of the severity of islet infiltration and to the development of clinical diabetes. We concluded that oral administration of insulin can induce the presence of regulatory T-cells in the pancreas and the corresponding draining lymph nodes, initiate the secretion of IL-4 in this microenvironment sufficiently to suppress the activity of Thl autoreactive T-cell clones, and ultimately provide protection against autoimmune diabetes.
Electrophysiological and Metabolic Characterization of Single β-Cells and Islets From Diabetic GK Rats
Authors: Hughes S J , Faehling M , Thorneley C W , Proks P , Ashcroft F M , Smith P A
Published: September 2013

We have used the whole-cell recording technique to determine whether ATP-sensitive potassium (KATP) currents, voltage-dependent Ca2+ currents, and exocytosis are different in single β-cells from pancreatic islets of Goto-Kakizaki (GK) rats, a novel model of NIDDM, and normal rats. In addition, we have also measured the insulin secretory responses, ATP content, and the rate of glucose metabolism in intact islets. Although the glucose sensitivity of the KATP current was similar between GK rats and controls, in the absence of glucose, KATP current density was larger in GK rats, which resulted in a more hyperpolarized membrane potential. Whole-cell Ca2+ currents were similar. By monitoring the cell capacitance with a fixed intracellular solution, no difference was detected in the exocytotic responses of β-cells from normal and GK rats. In islets from GK rats, the rates of glucose utilization ([3H]H2O production from 5-[3H]glucose) and oxidation ([14C]CO2 production from U-[14C]glucose) were not significantly different from controls. Insulin secretion, however, was impaired (by 50%), and this was paralleled by a smaller increase in ATP content in response to stimulation by 10 mmol/1 glucose in islets from GK rats when compared with controls. Under conditions in which KATP channels were held open and the effects of glucose were independent of membrane potential, insulin release was still significantly lower in GK rat islets than in controls. These findings suggest that the impaired insulin secretion in islets from GK rats does not simply result from a failure to close KATP channels, nor does it result from an impairment in calcium secretion coupling.
Expression of Insulin/IGF-I Hybrid Receptors Is Increased in Skeletal Muscle of Patients With Chronic Primary Hyperinsulinemia
Authors: Federici Massimo , Lauro Davide , D'Adamo Monica , Giovannone Barbara , Porzio Ottavia , Mellozzi Monica , Tamburrano Guido , Sbraccia Paolo , Sesti Giorgio
Published: September 2013

The insulin receptor (IR) shares structural and functional homology with the IGF-I receptor (IGF-IR). Hybrid receptors composed of an IR αβ-heterodimer and an IGF-IR αβ-heterodimer are formed in tissues expressing both molecules. Hybrids behave as IGF-IR rather than IR with respect to ligand binding affinity, receptor autophosphorylation, and hormone internalization and degradation. Factors regulating hybrid formation in vivo are unknown. We recently reported that in skeletal muscle of NIDDM patients, expression of hybrids is increased and correlated with a decrease in IR number and an increase in fasting insulin levels. However, it is not clear whether increased expression of hybrid receptors is a primary defect specifically associated with NIDDM or a secondary event caused by hyperinsulinemia. To address this issue, we used a quantitative microwell-based immunoassay to measure hybrid receptor abundance in skeletal muscle of 11 normal subjects and 12 patients with insulinoma, a state of primary nongenetically determined hyperinsulinemia. Total insulin binding was lower in insulinoma patients than in normal subjects (0.70 ± 0.18 vs. 4.59 ± 0.77; P < 0.0001). Total IGF-I binding did not differ between the two groups (0.81 ± 0.27 and 0.85 ± 0.10, respectively). The amount of hybrids, expressed as bound/total (B/T), was higher in patients with insulinoma than in normal subjects (0.57 ± 0.19 vs. 0.36 ± 0.03; P < 0.0006) and was inversely correlated with total insulin binding (r = −0.64, P < 0.0004). Increased abundance of hybrid receptors was positively correlated with insulin levels (r = −0.82, P < 0.0009) and inversely correlated with insulin-mediated glucose uptake (r = −0.80, P < 0.01). No correlations were observed between insulin-mediated glucose uptake and maximal specific insulin binding (r = 0.19, P = 0.64). These results indicate that insulin-induced IR downregulation may lead to the formation of a higher proportion of hybrid receptors, whose abundance is negatively correlated with in vivo insulin sensitivity. These results, therefore, support a role for insulin in the regulation of hybrid receptors formation and suggest that increased expression of hybrids in NIDDM may be a secondary event caused by hyperinsulinemia rather than a primary defect.
Expression of Glutamine:Fructose-6-Phosphate Amidotransferase in Human Tissues: Evidence for High Variability and Distinct Regulation in Diabetes
Authors: Nerlich Andreas G , Sauer Ulrich , Kolm-Litty Verena , Wagner Eva , Koch Matthias , Schleicher Erwin D
Published: September 2013

Recent in vitro and in vivo studies suggested that the increased flux of glucose through the hexosamine biosynthetic pathway may contribute to glucoseinduced insulin resistance and to the induction of the synthesis of growth factors. Because glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the first and rate-limiting step in the formation of hexosamine products, this enzyme is the key regulator in this pathway and is therefore possibly also involved in the alterations occurring in preclinical or manifest diabetic patients. To study the expression of GFAT in human tissues, we produced and characterized a peptic antiserum specifically recognizing GFAT protein and a riboprobe for the detection of GFAT mRNA. Immunohistochemical and nonradioactive in situ hybridization analysis revealed high levels of expression of GFAT protein and mRNA in adipocytes and skeletal muscle. Furthermore, a marked GFAT expression was found in vascular smooth muscle cells with unexpectedly high variability and lower levels in other cells, e.g., peripheral nerve sheath cells or endocrine-active cells, including the pancreatic islet cell. GFAT protein expression was below detection level in endothelium, osteocytes, lymphocytes, granulocytes, and in most quiescent fibroblasts. In renal tissue, GFAT was expressed in tubular epithelial cells, while glomerular cells remained essentially unstained. Renal sections obtained from patients with diabetic nephropathy showed significant GFAT expression in some glomerular epithelial and mesangial cells, indicating that GFAT expression may be induced by manifest diabetes. Our data indicate that GFAT is expressed in most tissues involved in the development of diabetic late complications. Furthermore, the results suggest that GFAT gene expression is highly regulated
Excessive Production of Nitric Oxide by Macrophages From DP-BB Rats Is Secondary to the T-Lymphopenic State of These Animals
Authors: Lau Albert , Ramanathan Sheela , Poussier Philippe
Published: September 2013

Activated macrophages are the first mononuclear cells to migrate to the pancreas of DP-BB rats at the initiation of insulitis. These cells produce an excess of NO, which has been implicated as a mediator of both (β-cell damage and inhibition of T-cell proliferation in this rat strain. Genetic studies have shown that the impaired proliferative response of T-cells segregates with one of the diabetes-susceptibility genes of the DP-BB rat, lyp, which is responsible for a peripheral T-lymphopenia. This observation suggests that the dysregulated expression of inducible NO synthase (iNOS) is under the control of lyp itself or a gene in linkage disequilibrium with lyp. Using two models of hemopoietic chimeras—DP-BB rats reconstituted with isocongenic Tcells and irradiated (WF × DP-BB)F1 animals reconstituted with bone marrow of both parental strains—we demonstrated that the production of NO by DP-BB macrophages is normal when these cells originate from a non–T-lymphopenic environment. Consequently, these macrophages no longer inhibit the stimulation of DNA synthesis in activated T-cells. Macrophages of young WF rats were found to produce high levels of NO, which inhibited T-cell proliferation in vitro. This observation strongly suggests that upregulation of NO synthesis in DP-BB macrophages represents the abnormal persistence of a phenomenon restricted to the first few weeks of life in non-diabetes-prone rats. Taken together, these results demonstrate that the elevated production of NO by DP-BB macrophages results from the lyp mutation and represents a crucial mechanism through which T-lymphopenia contributes to the development of diabetes.
Induction of Hyperinsulinemia Combined With Hyperglycemia and Hypertriglyceridemia Increases Plasminogen Activator Inhibitor 1 in Blood in Normal Human Subjects
Authors: Calles-Escandon Jorge , Mirza Shirwan A , Sobel Burton E , Schneider David J
Published: September 2013

Hypofibrinolysis caused by increased plasminogen activator inhibitor 1 (PAI-1) has been implicated in the vasculopathy of type 2 diabetes, typified by increased insulin, glucose, and triglycerides. However, short-term infusions of insulin have not increased PAI-1 in normal subjects. We hypothesized that induction of increased insulin accompanied by increased glucose and triglycerides would increase PAI-1. Accordingly, 30% glucose and 10% Intralipid were infused for 6 h in ten normal lean individuals (54 ± 3 years) resulting in increased insulin (42 ± 5 μU/dl), glucose (200 ± 24 mg/dl), and triglycerides (425 ± 45 mg/dl), simulating changes in type 2 diabetes. In contrast to results with infusion of saline alone (n = 16) and euglycemic-hyperinsulinemic clamps (n = 10, serum insulin = 89 ± 7 μU/dl), PAI-1 in blood increased significantly 6 h after the onset of infusion (15 ± 5 ng/ml, P <0.05 vs. baseline = 7.4± 1.1, saline 6 h = 3.4 ± 1.1, and insulin alone 6 h = 3.7 ± 0.8) and remained elevated for an additional 6 h (combined infusion = 13.8 ± 3.8 ng/ml, saline = 6.7 ± 2 ng/ml, insulin alone = 7.8 ± 1.7 ng/ml, P = 0.06). Our data suggest that combined hyperinsulinemia, hypertriglyceridemia, and hyperglycemia are likely to contribute to hypofibrinolysis of type 2 diabetes by increasing the blood levels of PAI-1. Moreover, these results underscore the potential importance of modifying insulin resistance as well as achieving glycemic and lipidemic control in individuals with type 2 diabetes.
Hypothalamic Pro-Opiomelanocortin mRNA Is Reduced By Fasting in ob/ob and db/db Mice, but Is Stimulated by Leptin
Authors: Mizuno Tooru M , Kleopoulos Steven P , Bergen Hugo T , Roberts James L , Priest Catherine A , Mobbs Charles V
Published: September 2013

Reduction in the activity of the α-melanocyte-stimulating hormone (α-MSH) system causes obesity, and infusions of α-MSH can produce satiety, raising the possibility that α-MSH may mediate physiological satiety signals. Since α-MSH is coded for by the pro-opiomelanocortin (POMC) gene, we examined if POMC gene expression would be inhibited by fasting in normal mice or in models of obesity characterized by leptin insufficiency (ob/ob) or leptin insensitivity (db/db). In wild-type mice, hypothalamic POMC mRNA was decreased >60% after a 2-day fast and was positively correlated with leptin mRNA. Similarly, compared with controls, POMC mRNA was decreased by at least 60% in both db/db and ob/ob mice. POMC mRNA was negatively correlated with both neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) mRNA. Finally, treatment of both male and female ob/ob mice with leptin stimulated hypothalamic POMC mRNA by about threefold. These results suggest that impairment in production, processing, or responsiveness to α-MSH may be a common feature of obesity and that hypothalamic POMC neurons, stimulated by leptin, may constitute a link between leptin and the melanocortin system.