bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Renal Production of Transforming Growth Factor-β1 in Patients with Type II Diabetes
Authors: Sharma Kumar , Ziyadeh Fuad N , Alzahabi Bashar , McGowan Tracy A , Kapoor Shiv , Kurnik Brenda R C , Kurnik Peter B , Weisberg Lawernce S
Published: September 2013

Diabetic nephropathy is a common complication in patients with either type I or type II diabetes. The pathogenesis of diabetic nephropathy is thought to involve both metabolic and vascular factors leading to chronic accumulation of glomerular mesangial matrix. In this context, both transforming growth factor-β (TGF-β) and endothelin may contribute to these processes. To determine if diabetic patients demonstrate increased renal production of TGF-β and endothelin, aortic, renal vein, and urinary levels of these factors were measured in 14 type II diabetic patients and 11 nondiabetic patients who were undergoing elective cardiac catheterization. Renal blood flow was measured in all patients to calculate net mass balance across the kidney. Diabetic patients demonstrated net renal production of immunoreactive TGF-β1 (830 ± 429 ng/min [mean ± SE]), whereas nondiabetic patients demonstrated net renal extraction of circulating TGF-β1 (−3479 ± 1010 ng/min, P < 0.001). Urinary levels of bioassayable TGF-β were also significantly increased in diabetic patients compared with nondiabetic patients (2.435 ± 0.385 vs. 0.569 ± 0.190 ng/mg creatinine, respectively; P < 0.001). Renal production of immunoreactive endothelin was not significantly increased in diabetic patients. In summary, type II diabetes is associated with enhanced net renal production of TGF-β1, whereas nondiabetic patients exhibit net renal extraction of circulating TGF-β1. Increased renal TGF-β production may be an important manifestation of diabetic kidney disease.
Progression of Nephropathy in Spontaneous Diabetic Rats Is Prevented by OPB-9195, a Novel Inhibitor of Advanced Glycation
Authors: Nakamura Sakurako , Makita Zenji , Ishikawa Shintaro , Yasumura Koichi , Fujii Wataru , Yanagisawa Katsuyuki , Kawata Tetsuya , Koike Takao
Published: September 2013

Levels of tissue advanced glycation end products (AGEs) that result from nonenzymatic reactions of glucose and proteins are high in both diabetic and aging people. Irreversible AGE formation is based on increases in AGE-derived protein-to-protein cross-linking and is considered to be a factor contributing to the complications of diabetes. A novel inhibitor of advanced glycation, OPB-9195, belongs to a group of thiazolidine derivatives, known as hypoglycemic drugs; however, they do not lower blood glucose levels. We did studies to determine if OPB-9195 would prevent the progression of nephropathy in spontaneous diabetic rats. In vitro inhibitory effects of OPB-9195 on AGE formation and AGE-derived cross-linking were examined by enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE, respectively. Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats, a model of NIDDM, were used to evaluate the therapeutic effect of OPB-9195. Light microscopic findings by periodic acid-Schiff (PAS) staining, the extent of AGE accumulation detected by immunohistochemical staining in the kidneys, the levels of serum AGEs by AGE-specific ELISA, and urinary albumin excretion were examined. OPB-9195 effectively inhibited both AGE-derived cross-linking and the formation of AGEs, in a dose-dependent manner in vitro. In addition, the administration of OPB-9195 prevented the progression of glomerular sclerosis and AGE deposition in glomeruli. Elevation of circulating AGE levels and urinary albumin excretion were dramatically prevented in rats, even at 56 weeks of age and with persistent hyperglycemia. We concluded that a novel thiazolidine derivative, OPB-9195, prevented the progression of diabetic glomerular sclerosis in OLETF rats by lowering serum levels of AGEs and attenuating AGE deposition in the glomeruli.
Forearm Nitric Oxide Balance, Vascular Relaxation, and Glucose Metabolism in NIDDM Patients
Authors: Avogaro Angelo , Piarulli Francesco , Valerio Anna , Miola Marina , Calveri Massimo , Pavan Paola , Vicini Paolo , Cobelli Claudio , Tiengo Antonio , Calò Lorenzo , Del Prato Stefano
Published: September 2013

Endothelium-dependent and -independent vascular responses were assessed in 10 NIDDM patients and 6 normal subjects with no evidence of atherosclerotic disease. Changes in forearm blood flow and arteriovenous (AV) serum nitrite/nitrate (NO2−/NO3-) concentrations were measured in response to intra-arterial infusion of acetylcholine (ACh) (7.5, 15, 30 μg/min, endothelium-dependent response) and sodium nitroprusside (SNP) (0.3, 3, 10 μg/min, endothelium-independent response). Insulin sensitivity (determined by minimal model intravenous glucose tolerance test) was lower in NIDDM patients (0.82 ± 0.20 vs. 2.97 ± 0.29 104 min · μU−1 · ml−1; P < 0.01). Baseline forearm blood flow (4.8 ± 0.3 vs. 4.4 ± 0.3 ml · 100 ml−1 tissue · min−1; NS), mean blood pressure (100 ± 4 vs. 92 ± 4 mmHg; NS), and vascular resistance (21 ± 1 vs. 21 ± 1 units; NS), as well as their increments during ACh and SNP, infusion were similar in both groups. No difference existed in baseline NO2−/NO3− concentrations (4.09 ± 0.33 ]NIDDM patients] vs. 5.00 ± 0.48 μmol/1 ]control subjects]; NS), their forearm net balance (0.31 ± 0.08 ]NIDDM patients] vs. 0.26 ± 0.08 μmol/1 · 100 ml−1 tissue · min−1; NS), and baseline forearm glucose uptake. During ACh infusion, both NO2− and NO3− concentrations and net balance significantly increased in both groups, whereas glucose uptake increased only in control subjects. When data from NIDDM and control groups were pooled together, a correlation was found between the forearm AV NO2− and NO3− differences and blood flow (r = 0.494, P = 0.024). On the contrary, no correlation was evident between NO2− and NO3− concentrations or net balance and insulin sensitivity. In summary, 1) no difference existed in basal and ACh-stimulated NO generation and endothelium-dependent relaxation between uncomplicated NIDDM patients and control subjects; 2) in both NIDDM and control groups, forearm NO2− and NO3− net balance following ACh stimulation was related to changes in the forearm blood flow; and 3) ACh-induced increase in forearm blood flow was associated with an increase in glucose uptake only in control subjects but not in NIDDM patients. In conclusion, our results argue against a role of impaired NO generation and blood flow regulation in determining the insulin resistance of uncomplicated NIDDM patients; rather, it supports an independent insulin regulation of hemodynamic and metabolic effects.
Impact of Natural IRS-1 Mutations on Insulin Signals: Mutations of IRS-1 in the PTB Domain and Near SH2 Protein Binding Sites Result in Impaired Function at Different Steps of IRS-1 Signaling
Authors: Yoshimura Ryohei , Araki Eiichi , Ura Sachiko , Todaka Mikio , Tsuruzoe Kaku , Noboru Furukawa , Motoshima Hiroyuki , Yoshizato Kazuaki , Kaneko Kengo , Matsuda Kohji , Kishikawa Hideki , Shichiri Motoaki
Published: September 2013

Insulin receptor substrate-1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates various insulin signals downstream. In this study, we have examined the impact of three natural IRS-1 mutations identified in NIDDM patients (G971R, P170R, and m209T) on insulin signaling. G971R is located near src homology 2 protein binding sites, and P170R and m209T are located in the phosphotyrosine binding domain of IRS-1. 32D-IR cells, stably overexpressing human insulin receptor, were transfected with wild-type human IRS-1 cDNA (WT) or three mutant IRS-1 cDNAs and analyzed. All the cell lines expressing mutant IRS-1 showed a significant reduction in ]3H]thymidine incorporation compared with WT. Upon insulin stimulation, cells expressing G971R showed a 39% decrease (P < 0.005) in phosphatidylinositol 3-kinase (PI 3-kinase) activity, a 43% decrease (P < 0.01) in binding of the 85-kDa regulatory subunit of PI 3-kinase, and a 22% decrease (P < 0.05) in mitogen-activated protein kinase activity compared with those expressing WT. Cells expressing P170R and m209T showed slight but significant decreases in PI 3-kinase activity (17 and 14%, respectively; both P < 0.05) and in binding of p85 (22 and 16%, respectively; both P < 0.05) and a greater decrease in mitogen-activated protein kinase activity (41 and 43%, respectively; both P < 0.005) compared with WT. After insulin stimulation, cells expressing P170R and m209T showed significant decreases in IRS-1 phosphorylation (37 and 42%, respectively; both P < 0.05) and in IRS-1 binding to the insulin receptor (48 and 53%, respectively; P < 0.01) compared with WT. G971R showed no changes in IRS-1 phosphorylation and in IRS-1 binding to the insulin receptor compared with WT. These data suggest that the impaired mitogenic response of P170R and m209T was mainly due to reduced binding to the insulin receptor, whereas the impaired response of G971R was mainly due to reduced association with PI 3-kinase p85.
Improved Human Islet Isolation Using a New Enzyme Blend, Liberase
Authors: Linetsky Elina , Bottino Rita , Lehmann Roger , Alejandro Rodolfo , Inverardi Luca , Ricordi Camillo
Published: September 2013

Enzymatic digestion of donor pancreases is a vital step in human and large mammalian islet isolation. The variable enzymatic activities of different batches of commercially available collagenase is a major obstacle in achieving reproducibility in islet isolation procedures. In the present work, the effectiveness of Liberase, a standardized mixture of highly purified enzymes recently developed for the separation of human islets, was compared with that of a traditional collagenase preparation (type P). The results of 50 islet isolations using Liberase enzyme were compared with those of 36 isolations with collagenase, type P. No significant differences in donor age, cold ischemia time, digestion time, or weight of the pancreases were observed between the two groups. Islet yield was significantly higher in the group where the Liberase enzyme was used. All parameters examined (islet number, islet number per gram of tissue, islet equivalent number, and islet equivalent number per gram of tissue) were significantly improved when Liberase enzyme was used. Different lots of Liberase enzyme were tested, and no difference was observed. Islets isolated with Liberase enzyme were also of larger size and were much less fragmented, suggesting a gentler enzymatic action and better preservation of anatomical integrity. Islets isolated with Liberase enzyme, assessed both in vitro and in vivo, revealed a functional profile similar to that of islets separated with collagenase. Liberase enzyme appears, therefore, to represent a new powerful tool for improving the quality of human islet isolation.
Dietary Cow's Milk Protein Does Not Alter the Frequency of Diabetes in the BB Rat
Authors: Malkani S , Nompleggi D , Hansen J W , Greiner D L , Mordes J P , Rossini A A
Published: September 2013

One theory of the pathogenesis of IDDM proposes that exposure to cow's milk proteins triggers the disease in genetically susceptible individuals. We tested this hypothesis in the BB/Wor rat model of human IDDM. Diabetes-prone (DP) BB/Wor rats spontaneously develop IDDM. Coisogenic diabetes-resistant (DR) BB/Wor rats do not develop diabetes spontaneously, but IDDM can readily be induced by treatment with polyinosinicrpolycytidylic acid and depletion of RT6+ Tcells. Pregnant BB/Wor rats were fed one of four experimental diets or a standard Purina commercial rat chow (5010) that was certified to be free of cow's milk protein. Offspring were maintained on the maternal diet after weaning. DP-BB/Wor rats, fed either of two experimental diets based on hydrolyzed casein and free of intact milk protein (Nutramigen or D11236), developed diabetes at only half the rate of animals fed Purina 5010 chow. Neither the addition of bovine serum albumin (BSA) to Nutramigen nor the substitution of total milk protein for the hydrolyzed casein in the D11236 diet increased the frequency of spontaneous diabetes. In contrast, there was no relationship between diet and susceptibility of DR-BB/Wor rats to IDDM induction. However, the methods used to induce IDDM in DRBB/ Wor animals were found to induce antibodies against BSA. We conclude the following: 1) Dietary modification can reduce spontaneous IDDM expression in DP-BB/Wor rats, but the agent of protection is not elimination of cow's milk protein. 2) The addition of BSA or intact milk protein does not abrogate the effectiveness of a protective diet. 3) The genetic susceptibility of the DR-BB/Wor rat to autoimmune diabetes is unaffected by any of the tested diets, but a role of anti-BSA–like autoreactivity in IDDM expression cannot be excluded.
Stimulation of Islet Protein Kinase C Translocation by Palmitate Requires Metabolism of the Fatty Acid
Authors: Alcáazar O , Qiu-yue Zhu , Giné Elena , Tamarit-Rodriguez Jorge
Published: September 2013

The secretory, metabolic, and signaling aspects of glucose/ palmitate interaction on β-cell function have been studied on rat islets. Palmitate potentiated the glucose-induced insulin response of perifused islets at suprathreshold (>3 mmol/1) sugar concentrations. This potentiating effect could be suppressed by 8-bromocGMP, which also blocks palmitate metabolism. Palmitate did not modify glucose utilization, but it slightly reduced glucose oxidation and concomitantly increased lactate production. The very low rate of palmitate oxidation (80-fold lower than that of 20 mmol/l glucose) might explain its lack of effect on glycolysis and hence that the glucose/fatty acid cycle is inoperative in islet cells. However, glucose determines the metabolic fate of exogenous palmitate, which is mainly diverted toward lipid synthesis at high sugar concentrations and might then generate lipid messengers for cell signaling. Palmitate did not increase glucose-induced production of inositol-l,4,5-trisphosphate, but it stimulated the translocation of protein kinase C activity from a cytosolic to a particulate fraction at 20 but not at 3 mmol/l glucose. This increased translocation was partially or completely blocked by hydroxycitrate or 8-bromo-cGMP, respectively, which are agents interfering with palmitate metabolism (inhibiting lipid synthesis). The metabolic interaction between glucose and palmitate might generate lipid messengers (diacylglycerol, phosphatidylserine) necessary for the activation of islet protein kinase C, which would in turn result in a potentiation of glucose-induced insulin secretion.
Early, but Not Advanced, Glomerulopathy Is Reversed by Pancreatic Islet Transplants in Experimental Diabetic Rats: Correlation With Glomerular Extracellular Matrix mRNA Levels
Authors: Pugliese Giuseppe , Pricci Flavia , Pesce Carlo , Romeo Giulio , Lenti Elisabetta , Caltabiano Vera , Vetri Mario , Purrello Francesco , Di Mario Umberto
Published: September 2013

In this study, we investigated 1) whether long-term restoration of euglycemia by means of pancreatic islet transplants is capable of preventing and/or reversing renal functional and structural alterations in an experimental model of insulin-deficient diabetes, and 2) whether changes in extracellular matrix (ECM) and cell turnover at the glomerular level and biochemical abnormalities associated with hyperglycemia correlate with the renal outcome after transplantation. Male Lewis rats, rendered diabetic by intravenous injection of streptozotocin, underwent homologous islet transplantation via the portal vein at 2 weeks (study A), at 4 months (study B), and at 8 months (study C) after the induction of diabetes and killed 12 months after transplantation in study A and 4 months after transplantation in studies B and C. Age-matched nondiabetic and untreated diabetic rats were used as control animals and were studied at 4, 8, and 12 months. In the untreated diabetic animals, metabolic derangement was associated with increased erythrocyte polyol and fructose levels, tail-tendon content of advanced glycation end products (AGEs), total proteinuria, albuminuria, kidney weight, and mean glomerular volume as well as with marked glomerular and extraglomerular lesions. Glomerular gene expression for the ECM components fibronectin and collagen IV and for TGF-β was also increased, whereas glomerular cell proliferation was unaffected by diabetes. In study A, changes in renal function and structure observed in diabetic rats at 12 months were completely prevented by successful islet transplants. In study B, all functional and structural abnormalities detected in diabetic rats at 4 months of disease duration were virtually reversed by 4 months of euglycemia in transplanted animals, whereas they progressed further in untreated diabetic rats. In study C, the course of functional and structural changes observed in untreated diabetic rats was not reversed by islet transplantation. Likewise, tissue AGE accumulation and particularly upregulation of glomerular ECM and transforming growth factor (TGF)-β gene expression, which are believed to play a role in the pathogenesis of altered renal function and structure in diabetes, were normalized in transplanted rats from study A and study B, but not in those from study C. These experiments show that restoration of euglycemia by islet transplants is capable of preventing experimental diabetic glomerulopathy and reversing early changes in renal function and structure induced by diabetes. In a later phase of the disease, when glomerular matrix gene expression becomes independent of hyperglycemia, possibly because of the persistent increase in tissue AGE accumulation, metabolic control is not capable of reversing renal abnormalities.
PPAR-γ Gene Expression Is Elevated in Skeletal Muscle of Obese and Type II Diabetic Subjects
Authors: Park Kyong Soo , Ciaraldi Theodore P , Abrams-Carter Leslie , Mudaliar Sunder , Nikoulina Svetlana E , Henry Robert R
Published: September 2013

The peroxisome proliferator activated receptor PPAR-γ has been identified as a nuclear receptor for thiazolidenediones, which are compounds with insulin-sensitizing properties in several tissues, including skeletal muscle. To determine whether this receptor is expressed and possibly involved in insulin action/resistance in skeletal muscle, PPAR-γ mRNA abundance and its regulation by insulin were quantified in muscle tissue and cultures from lean and obese nondiabetic and type II diabetic subjects using competitive reverse transcription–polymerase chain reaction (RT-PCR). In muscle biopsy specimens, PPAR-γ mRNA was elevated in obese nondiabetic and type II diabetic subjects (23.4 ± 4.2 and 28.0 ± 5.69 × 103 copies/µg total RNA, respectively; both P < 0.05) compared with lean nondiabetic control subjects (9.4 ± 2.3 × 103 copies/µg total RNA). Significant positive correlations were present among skeletal muscle PPAR-γ mRNA levels, BMI (r = 0.67, P < 0.01), and fasting insulin concentration (r = 0.76, P < 0.001). PPAR-γ mRNA levels were also elevated in muscle cultures from type II diabetic subjects compared with lean nondiabetic control subjects (330.1 ± 52.9 vs. 192.1 ± 27.0 × 103 copies/µg total RNA, P < 0.05). Insulin stimulation of muscle tissue (by hyperinsulinemic- euglycemic clamp for 3–4 h) or muscle cultures (30 nmol/1 for 120 min) stimulated PPAR-γ mRNA expression up to fourfold (10.0 ± 2.7 to 41.3 ± 7.4 × 103 copies/µg total RNA, P < 0.05, and 174.9 ± 56.9 to 268.2 ± 78.6 × 103 copies/µg total RNA, P < 0.05, respectively). In summary, PPAR-γ mRNA expression in human skeletal muscle is acutely regulated by insulin and is increased in both obese nondiabetic and type II diabetic subjects in direct relation to BMI and fasting insulinemia. We conclude that abnormalities of PPAR-γ may be involved in skeletal muscle insulin resistance of obesity and type II diabetes.
Tissue Distribution and Quantification of the Expression of mRNAs of Peroxisome Proliferator–Activated Receptors and Liver X Receptor-α in Humans: No Alteration in Adipose Tissue of Obese and NIDDM Patients
Authors: Auboeuf Didier , Rieusset Jennifer , Fajas Lluis , Vallier Paulette , Frering Vincent , Riou Jean Paul , Staels Bart , Auwerx Johan , Laville Martine , Vidal Hubert
Published: September 2013

Members of the peroxisome proliferator–activated receptor (PPAR) family might be involved in pathologies with altered lipid metabolism. They participate in the control of the expression of genes involved in lipid metabolism and adipocyte differentiation. In addition, thiazolidinediones improve insulin resistance in vivo by activating PPARγ. However, little is known regarding their tissue distribution and relative expression in humans. Using a quantitative and sensitive reverse transcription (RT)-competitive polymerase chain reaction (PCR) assay, we determined the distribution and relative mRNA expression of the four PPARs (α, β, γ1, and γ2) and liver X receptor-α (LXRα) in the main tissues implicated in lipid metabolism. PPARα and LXRα were mainly expressed in liver, while PPARγ1 predominated in adipose tissue and large intestine. We found that PPARγ2 mRNA was a minor isoform, even in adipose tissue, thus causing question of its role in humans. PPARβ mRNA was present in all the tissues tested at low levels. In addition, PPARγ mRNA was barely detectable in skeletal muscle, suggesting that improvement of insulin resistance with thiazolidinediones may not result from a direct effect of these agents on PPARγ in muscle. Obesity and NIDDM were not associated with change in PPARs and LXRα expression in adipose tissue. The mRNA levels of PPARγ1, the predominant form in adipocytes, did not correlate with BMI, leptin mRNA levels, or fasting insulinemia in 29 subjects with various degrees of obesity. These results indicated that obesity is not associated with alteration in PPAR gene expression in abdominal subcutaneous adipose tissue in humans.
Are Metabolic Oscillations Responsible for Normal Oscillatory Insulin Secretion?
Authors: Tornheim Keith
Published: September 2013

Normal insulin secretion is oscillatory in vivo and in vitro, with a period of ∼5–10 min. The mechanism of generating these oscillations is not yet established, but a metabolic basis seems most likely for glucose-stimulated secretion. The rationale is that 1) spontaneous oscillatory operation of glycolysis is a well-established phenomenon; 2) oscillatory behavior of glycolysis involves oscillations in the ATP/ADP ratio, which can cause alternating opening and closing of ATP-sensitive K+ channels, leading to the observed oscillations in membrane potential and Ca2+ influx in pancreatic β-cells, and may also have downstream effects on exocy-tosis; 3) spontaneous Ca2+ oscillations are an unlikely basis in this case, since intracellular stores are not of primary importance in the stimulus-secretion coupling, and furthermore, insulin oscillations occur under conditions when intracellular Ca2+ levels are not changing; 4) a neural basis cannot account for insulin oscillations from perifused islets and clonai β-cells or from transplanted islets or pancreas in vivo; 5) observed oscillations in metabolite levels and fluxes further support a metabolic basis, as does the presence in β-cells of the oscillatory isoform of phosphofructokinase (PFK-M). The fact that normal oscillatory secretion is impaired in patients with NIDDM and in their near relatives suggests that such derangement may be involved in the development of the disease; furthermore, this probably reflects an early defect in the regulation and operation of the fuel metabolizing/sensing pathways of the pancreatic β-cell.
Impaired Glucose-Induced Insulin Response in Transgenic Mice Overexpressing the L-phosphofructokinase Gene
Authors: Knobler Hilla , Weiss Yael , Peled Mira , Groner Yoram
Published: September 2013

The selective impairment of glucose-induced insulin secretion in NIDDM can be attributed to defects in the glucose-signaling system. An alteration in the activity of phosphofructokinase (PFK), a key enzyme in the glycolytic pathway, may play a role in the abnormal glucose-induced insulin secretion. In this study, we evaluated insulin secretion in transgenic (Tg) mice overexpressing the liver-type subunit of phosphofructokinase (PFKL). Three independently derived Tg-PFKL lines showed random and postprandial hyperglycemia with diminished acute insulin response following intravenous glucose tolerance load. Isolated islets of Tg-PFKL mice exhibited a shift to the right of the glucose insulin dose curve. However, the maximal insulin secretory capacity, as well as the potentiation effect by arginine, were retained. PFK activity in Tg-PFKL islets was increased by 30–70%, because of the overexpression of PFKL. Conceivably, a selective overexpression of the PFKL isoform in Tg-PFKL mice altered the enzymatic properties of the tetrameric PFK and thereby affected glucose metabolism. A similar phenomenon was previously observed in transfected PC12-PFKL cells. The data show that overexpression of PFKL in transgenic mice was associated with diminished glucose-induced insulin response and suggest a mechanism to explain the role of β-cell PFK activity in glucose-induced insulin secretion.
Identification of Muscarinic Receptor Subtypes in RINm5F Cells by Means of Polymerase Chain Reaction, Subcloning, and DNA Sequencing
Authors: Tang Shao Hua , Sharp Geoffrey W G
Published: September 2013

Carbachol can stimulate insulin release in RINm5F cells by a mechanism that does not involve the elevation of cytosolic free Ca2+ concentrations or the activation of conventional protein kinase Cs (Mol Pharmacol 47:863–870, 1995). Thus, a novel signal transduction pathway links the muscarinic activation of the cells to increased insulin secretion. The question arises as to whether the pathway results from a novel receptor, different from the five established muscarinic receptors, or whether a “normal” receptor in the RINm5F cell activates a novel pathway. To distinguish between these two possibilities, the muscarinic receptors in the RINm5F cell were identified. Using polymerase chain reaction, combined with subcloning and DNA sequencing techniques, the cDNAs that encode the established M3 and M4 receptors were identified. The cDNAs for the M1, M2, and M5 receptors were not found. Pharmacological studies showed a rank order of potency for muscarinic receptor subtype antagonists to inhibit carba-chol-induced insulin release (half-maximal inhibitory concentration [pIC50] values given in parentheses): atropine (nonselective, 9.0) > 4-diphenyl-acetoxy-iV-methyl piperidine methiodide (M3/M1, 8.6) > para-fluoro-hexahydrosiladiphenidol (M3, 8.1) > hexahydrosiladiphenidol (M3, 8.0) > tropicamide (M4, 6.4) > pirenzepine (M1, 6.1) > methoctramine (M2, 5.9). This antagonist profile suggests that it is the M3 receptor that mediates carbachol-induced insulin release. In this case, the novel signaling involved in the unusual carbachol response would not be due to a novel receptor but to the well-characterized M3 receptor. It appears, therefore, that the novel portion of the signaling pathway lies downstream of the M3 receptor and may consist of products of phosphatidylinositol hydrolysis, other than inositol triphosphate and diacylglycerol, resulting from the activation of phospholipase C. While a contributory role of the M4 receptor cannot be ruled out, there is no evidence in its favor other than its presence in the cell.
Changes in Pancreatic Islet Glucokinase and Hexokinase Activities With Increasing Age, Obesity, and the Onset of Diabetes
Authors: Cockburn Brian N , Ostrega Diane M , Sturis Jeppe , Kubstrup Christian , Polonsky Kenneth S , Bell Graeme I
Published: September 2013

We examined changes in high- and low-Km glucose phos-phorylating activity in pancreatic islet extracts from the prediabetic Zucker diabetic fatty (ZDF) rat between 5–6 weeks and 12 weeks of age (after the onset of diabetes). Comparisons were made between the activity observed in the ZDF rat and that seen in the ZDF lean control (ZLC) rat and the obese nondiabetic Zucker fatty (ZF) rat. At 5–6 weeks of age, insulin resistant ZDF and ZF rats were hyperinsulinemic, compared with the ZLC rat, but had normal plasma glucose levels. Kinetic parameters (Vmax and Km for glucose) of hex-okinase (HK) and Km of glucokinase (GCK) did not differ between groups. Islet GCK activity for ZDF and ZF rats was 1.7-fold greater than in ZLC rats (P < 0.02 and P < 0.001, respectively). By 12 weeks of age, hyper-secretion of insulin at 5.0 mmol/1 glucose was observed in perifused islets from both obese groups relative to the ZLC rat. Islets from ZDF rats failed to increase insulin secretion in response to increased glucose concentration. Group differences in the kinetic parameters for GCK or in the Km values for HK were not significant. Islet HK activity for ZDF and ZF rats was 1.9-fold (P < 0.05) and 1.7-fold (P < 0.05) greater, respectively, than for ZLC rats. Compared with the 5- to 6-week-old animals, HK activity increased 3.1-fold (P < 0.001), 2.5-fold (P < 0.002), and 2.0-fold (P < 0.05) for ZDF, ZF, and ZLC rats, respectively. Differences in GCK activity between 5- to 6- and 12-week-old rats were not significant for any of the groups. We conclude: 1) increased islet glucose phosphorylating activity is present in insulin resistant and hyperinsulinemic ZF and ZDF rats, relative to the ZLC rat; 2) at 12 weeks of age, hyperinsulinemic ZDF and ZF rats demonstrated significant increases in HK activity, compared with lean controls; and 3) deficiency in GCK activity does not explain failure of diabetic ZDF islets to respond to glucose, since differences between diabetic ZDF and non-diabetic ZF rats were not statistically significant. Increases in pancreatic islet phosphorylating activity seem to be important in maintaining basal hyperinsulinemia in insulin-resistant animals, but do not appear to play a role in the progression to glucose intolerance and diabetes.
rhIGF-I Administration Reduces Insulin Requirements, Decreases Growth Hormone Secretion, and Improves the Lipid Profile in Adults With IDDM
Authors: Carroll Paul V , Umpleby Margot , Ward Gill S , Imuere Stephen , Alexander Elaine , Dunger David , Sönksen Peter H , Russell-Jones David L
Published: September 2013

IDDM is associated with elevated circulating levels of growth hormone (GH) and reduced insulin-like growth factor I (IGF-I). GH antagonizes the action of insulin-increasing insulin requirements in IDDM. The effects of subcutaneously administered rhIGF-I on glycemie control, insulin requirements, and GH secretion were studied in eight adults with IDDM. Patients received either placebo or rhIGF-I (50 pg/kg b.i.d.) for 19 days in a randomized, double-blind, parallel-design, placebo-controlled trial. Overnight GH, plasma glucose, free insulin, IGF-I, fructosamine, and lipid profiles were assessed during this period. rhIGF-I therapy increased IGF-I concentration from 117.1 ± 14.2 (mean ± SE) ng/ml (baseline) to 310.5 ± 40.6 and 257.1 ± 41.2 ng/ml on day 5 (P < 0.01 vs. baseline) and day 20 (P < 0.01 vs. baseline), respectively. After 19 days of rhIGF-I treatment, fructosamine concentrations were unchanged compared with baseline (439 ± 32 vs. 429 ± 35 μmol/l, day –1 vs. day 20, respectively), yet insulin requirements were decreased by ∼45% (0.67 ± 0.08 vs. 0.36 ± 0.07 U · kg−1 · day−1, day –1 vs. day 19, respectively, P < 0.005). After 4 days of rhIGF-I therapy, there was a decrease in free insulin levels (8.38 ± 1.47 vs. 4.98 ± 0.84 mU/l, P < 0.05), mean overnight GH concentration (12.6 ± 3.3 vs. 3.8 ± 2.1 mU/l, P = 0.05), and total cholesterol and triglycerides (4.68 ± 0.31 vs. 4.25 ± 0.35 mmol/l, P < 0.05, 1.27 ± 0.19 vs. 0.95 ± 0.21 mmol/l, P < 0.001, respectively). There was no change in any variable in the placebo-treated patients. This study demonstrates that subcutaneous administration of rhIGF-I decreases insulin requirements and improves the plasma lipid profile while maintaining glycemie control in adults with IDDM. The excess nocturnal release of GH, characteristic of IDDM, is also decreased by rhIGF-I therapy. Exogenous rhIGF-I therapy may have a role in the treatment of adults with IDDM, particularly in the setting of abnormal lipids and a high insulin requirement.
Vascular Endothelial Growth Factor–Induced Retinal Permeability Is Mediated by Protein Kinase C In Vivo and Suppressed by an Orally Effective β-Isoform–Selective Inhibitor
Authors: Aiello Lloyd Paul , Bursell Sven-Erik , Clermont Allen , Duh Elia , Ishii Hidehiro , Takagi Chikako , Mori Fumi , Ciulla Thomas A , Ways Kirk , Jirousek Michael , Smith Lois E H , King George L
Published: September 2013

Increased vascular permeability and excessive neovas-cularization are the hallmarks of endothelial dysfunction, which can lead to diabetic macular edema and proliferative diabetic retinopathy in the eye. Vascular endothelial growth factor (VEGF) is an important mediator of ocular neovascularization and a known vasopermeability factor in nonocular tissues. In these studies, we demonstrate that intravitreal injection of VEGF rapidly activates protein kinase C (PKC) in the retina at concentrations observed clinically, inducing membrane translocation of PKC isoforms α, βII, and δ and > threefold increases in retinal vasopermeability in vivo. The effect of VEGF on retinal vascular permeability appears to be mediated predominantly by the β-isoform of PKC with >95% inhibition of VEGF-induced permeability by intravitreal or oral administration of a PKC β-isoform-selective inhibitor that did not inhibit histamine-mediated effects. These studies represent the first direct demonstration that VEGF can increase intraocular vascular permeability through activation of PKC in vivo and suggest that oral pharmacological therapies involving PKC β-isoform-selective inhibitors may prove efficacious for the treatment of VEGF-asso-ciated ocular disorders such as diabetic retinopathy.
Glucose-Induced Protein Kinase C Activation Regulates Vascular Permeability Factor mRNA Expression and Peptide Production by Human Vascular Smooth Muscle Cells In Vitro
Authors: Williams Bryan , Gallacher Barbara , Patel Hashmukh , Orme Carole
Published: September 2013

Hyperglycemia is an independent risk factor for the development of diabetic microvascular disease. Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) is a potent cytokine family that induces angiogenesis and markedly increases endothelial permeability. VPF is produced by many cell types, including vascular smooth muscle (VSM) cells, and has been implicated in the pathogenesis of neovascularization and endothelial dysfunction in diabetes. This study used cultured human VSM cells to study the regulation of VPF production and determine whether elevated glucose concentrations, per se, are a sufficient stimulus for increased VPF production by human cells. In human VSM cells, high extracellular glucose concentrations (20 mmol/l) increased VPF mRNA expression within 3 h (3-fold vs. glucose 5 mmol/l) and significantly increased VPF peptide production within 24 h (1.5-fold) in a time- and glucose concentration-dependent manner. The high glucose-induced increase in VPF mRNA expression was rapidly reversed after normalizing the extracellular glucose concentration and was specific for a high D-glucose concentration, as these effects were not reproduced by osmotic control media containing elevated concentrations of mannitol or L-glucose. High glucose concentrations activate protein kinase C (PKC) in human VSM cells, and PKC inhibitors (H-7 or chelerythrine chloride) or PKC downregulation each prevented the glucose-induced increases in VPF mRNA expression by human VSM cells. In conclusion, high glucose concentrations directly increase VPF mRNA expression and peptide production by human VSM cells via a PKC-dependent mechanism. These results demonstrate a cellular mechanism, whereby hyperglycemia could directly contribute to the development of endothelial dysfunction and neovascularization in diabetes.
Low Plasma Leptin in Response to Dietary Fat in Diabetes- and Obesity-Prone Mice
Authors: Surwit Richard S , Petro Ann E , Parekh Priti , Collins Sheila
Published: September 2013

Despite the fact that mutations resulting in the absence of leptin or its receptor have been associated with severe obesity and diabetes, such mutations do not appear to be responsible for most human obesity. Indeed, diet-induced obesity in animals and humans has been characterized by hyperleptinemia. This has been interpreted as evidence for leptin resistance. However, no careful longitudinal studies evaluating the role of leptin in the development of obesity exist. We report a series of studies in A/J and C57BL/6J (B/6) mice that demonstrate a direct relationship between the ability to increase plasma leptin levels in response to a high-fat diet and resistance to the subsequent development of obesity and diabetes. While leptin levels are similar in lean, low-fat-fed A/J and B/6 mice, the effects of a high-fat diet on plasma leptin differ dramatically between the two strains. After 4 weeks of high-fat feeding, leptin levels in A/J mice increased 10-fold, and this elevated level was maintained independent of weight gain throughout a 14-week feeding period. However, in B/6 mice, leptin levels remained at least twofold lower and only rose very gradually along with a significant increase in adiposity, hyperglycemia, and hyperinsulinemia. These differences in the response of leptin to diet are independent of food intake and plasma insulin levels during the 1st month of feeding. Further, we demonstrated that leptin administration did not influence the expression of the novel uncoupling protein UCP2, which also responds to dietary fat. From these results, we suggest that the response of leptin to fat feeding may be an important predictor of the development of subsequent obesity.
Altered Expression Levels and Impaired Steps in the Pathway to Phosphatidylinositol 3-Kinase Activation via Insulin Receptor Substrates 1 and 2 in Zucker Fatty Rats
Authors: Anai Motonobu , Funaki Makoto , Ogihara Takehide , Terasaki Jungo , Inukai Kouichi , Katagiri Hideki , Fukushima Yasushi , Yazaki Yoshio , Kikuchi Masatoshi , Oka Yoshitomo , Asano Tomoichiro
Published: September 2013

To elucidate the mechanism of obesity-related insulin resistance, we investigated the impaired steps in the processes of phosphatidylinositol (PI) 3-kinase activation through binding with insulin receptor substrates 1and 2 (IRS-1 and IRS-2) in liver and muscle of Zucker fatty rats. The expressions of IRS-1 and IRS-2 were shown to be downregulated in both liver and muscle in fatty rats (hepatic IRS-1, 83%; hepatic IRS-2, 45%; muscle IRS-1, 60%; muscle IRS-2, 78%), resulting in decreased tyrosine phosphorylation in response to insulin stimulation. Despite the decrease in the tyrosine phosphorylation levels of hepatic IRS-1 and IRS-2 being mild to moderate, associated PI 3-kinase activities were dramatically decreased in fatty rats (IRS-1, 14%; IRS-2, 10%), which may suggest alteration in the sites of phosphorylated tyrosine residues of hepatic IRS-1 and IRS-2. In addition, we demonstrated that the expressions of p85α and p55α regulatory subunits of PI 3-kinase were reduced (p85α, 67%; p55α, 54%), and that the p50α regulatory subunit was markedly upregulated (176%) in the livers of fatty rats without apparent alterations in expressions of the catalytic subunits p110α and p110β. These alterations may reflect the obesity-related insulin resistance commonly observed in human NIDDM.
Troglitazone Reduces LDL Oxidation and Lowers Plasma E-selectin Concentration in NIDDM Patients
Authors: Cominacini Luciano , Garbin Ulisse , Pasini Anna Fratta , Campagnola Mario , Davoli Anna , Foot Elizabeth , Sighieri Giuseppe , Sironi Anna M , Cascio Vincenzo Lo , Ferrannini Ele
Published: September 2013

Troglitazone, an oral antidiabetic agent with antioxidant properties, has previously been shown to increase the resistance of LDL to oxidation in vitro and in vivo in healthy volunteers. In a randomized, placebo-controlled, parallel-group study in 29 patients with NIDDM, we tested the effect of troglitazone (200 mg once daily) on the resistance of LDL to oxidation and on circulating levels of preformed lipid hydroperoxides and the adhesion molecule Eselectin. Resistance of LDL to oxidation was assessed by measuring 1) fluorescence development induced by copper treatment (lag phase), and 2) amount of thiobarbituric acid-reactive substances (TBARS) generated by incubation with umbilical vein endothelial cells. At 8 weeks, the lag phase was increased by 23% (P < 0.01 by analysis of covariance [ANCOVA]) in the patients receiving troglitazone (n = 18) compared with the group receiving placebo (n = 11). At the same time, TBARS were 3.63 ± 0.10 nmol/1 (vs. 5.32 ± 0.10 nmol/1 in the placebo group, P = 0.009), LDL hydroperoxide concentration was reduced from 1.48 ± 0.03 to 1.19 ± 0.03 ng/mg (no change in the placebo group, P < 0.01), and plasma E-selectin levels decreased from 56.5 ± 2.33 to 43.7 ± 1.77 μg/l (no change in the placebo group, P < 0.01). In NIDDM, troglitazone may slow down the development of atherosclerosis by modifying LDLrelated atherogenic events.
GLUT2 in Pancreatic Islets: Crucial Target Molecule in Diabetes Induced With Multiple Low Doses of Streptozotocin in Mice
Authors: Wang Zhiyong , Gleichmann Helga
Published: September 2013

In mice, diabetes can be induced by multiple low doses of streptozotocin (MLD-STZ), i.e., 40 mg/kg body wt on each of 5 consecutive days. In this model, diabetes develops only when STZ induces both β-cell toxicity and T-cell- dependent immune reactions. The target molecule(s) of MLD-STZ-induced β-cell toxicity are not known, however. In this study, we report that GLUT2 is a target molecule for MLD-STZ toxicity. Ex vivo, a gradual decrement of both GLUT2 protein and mRNA expression was found in pancreatic islets isolated from MLDSTZ-treated C57BL/6 male mice, whereas mRNA expression of β-actin, glucokinase, and proinsulin remained unaffected. Significant reduction of both GLUT2 protein and mRNA expression was first noted 1 day after the third STZ injection, clearly preceding the onset of hyperglycemia. The extent of reduction increased with the number of STZ injections administered and increased over time, after the last, i.e., fifth, STZ injection. The STZ-induced reduction of GLUT2 protein and mRNA was not due to an essential loss of β-cells, because ex vivo, not only the total RNA yield and protein content in isolated islets, but also proinsulin mRNA expression, failed to differ significantly in the differently treated groups. Furthermore, islets isolated from MLD-STZ-treated donors responded to the nonglucose secretagogue arginine in a pattern similar to that of solvent-treated donors. Interestingly, the MLD-STZ-induced reduction of both GLUT2 protein and mRNA was prevented by preinjecting mice with 5-thio-D-glucose before each STZ injection. Apparently, GLUT2 is a crucial target molecule of MLD-STZ toxicity, and this toxicity seems to precede the immune reactions against β-cells.
Glucagon-Like Peptide 1(7-36) Amide Stimulates Exocytosis in Human Pancreatic β-Cells by Both Proximal and Distal Regulatory Steps in Stimulus-Secretion Coupling
Authors: Gromada Jesper , Bokvist Krister , Ding Wei-Guang , Holst Jens Juul , Nielsen Jens Høiriis , Rorsman Patrik
Published: September 2013

The effect of glucagon-like peptide 1(7–36) amide [GLP-l(7–36) amide] on membrane potential, wholecell ATP-sensitive potassium channel (KATP) and Ca2+ currents, cytoplasmic Ca2+ concentration, and exocytosis was explored in single human β-cells. GLP-1(7–36) amide induced membrane depolarization that was associated with inhibition of whole-cell KATP current. In addition, GLP-l(7–36) amide (and forskolin) produced greater than fourfold potentiation of Ca2+-dependent exocytosis. The latter effect resulted in part (40%) from acceleration of Ca2+ influx through voltage-dependent (L-type) Ca2+ channels. More importantly, GLP- 1(7–36) amide (via generation of cyclic AMP and activation of protein kinase A) potentiated exocytosis at a site distal to a rise in the cytoplasmic Ca2+ concentration. Photorelease of caged cAMP produced a two- to threefold potentiation of exocytosis when the cytoplasmic Ca2+ concentrations were clamped at >170 nmol/1. The effect of GLP-1(7–36) amide was antagonized by the islet hormone somatostatin. Similar effects on membrane potential, ion conductances, and exocytosis were observed with glucose-dependent insulinotropic polypeptide (GIP), the second major incretin. The present data suggest that the strong insulinotropic action of GLP-1(7–36) amide and GIP in humans results from its interaction with several proximal as well as distal important regulatory steps in the stimulus-secretion coupling.
Dual Glucagon Recognition by Pancreatic β-Cells via Glucagon and Glucagon-Like Peptide 1 Receptors
Authors: Moens Karen , Flamez Daisy , Schravendijk Chris Van , Ling Zhidong , Pipeleers Daniel , Schuit Frans
Published: September 2013

cAMP is required for normal glucose-induced insulin release by pancreatic β-cells. In a previous study, we showed that cAMP production in β-cells depends on the expression of receptors for glucagon, glucagon-like peptide 1(7-36) amide [GLP-1(7-36) amide], and glucose- dependent insulinotropic polypeptide. Although the latter two peptides are thought to amplify mealinduced insulin release (incretin effect), the role of glucagon in the regulation of insulin release remains elusive. In the present study, we analyzed the interaction of glucagon with its own receptor and with the glucagon-like peptide 1 (GLP-1) receptor using purified rat β-cells. Glucagon binding was partially displaced by 1 μmol/1 des-His1-[Glu9]glucagon-amide, a glucagon receptor antagonist, and by 1 μmol/1 GLP-1. Conversely, GLP-1 binding was competitively inhibited by high glucagon concentrations (Ki = 0.3 μmol/1). Glucagoninduced cAMP production in β-cells was inhibited both by 1 μmol/1 des-His1-[Glu9]glucagon-amide and exendin-(9-39)-amide, a specific GLP-1 receptor antagonist, whereas GLP-1-induced cAMP formation was suppressed only by exendin-(9-39)-amide. Finally, addition of 1 βmol/1 exendin-(9-39)-amide to 20 mmol/1 glucosestimulated β-cells did not antagonize the potentiating effect of 1 nmol/1 glucagon, although it prevented 45% of glucagon potentiation when the peptide was administered at 10 nmol/1. Our data suggest that glucagon recognition via two distinct receptors allows pancreatic β-cells to detect this peptide both when diluted in the systemic circulation and when concentrated as local signal in the islet interstitium.
Hypoglycemia Overrides Amylin-Mediated Regulation of Gastric Emptying in Rats
Authors: Gedulin Bronislava R , Young Andrew A
Published: September 2013

Amylin, a 37-amino acid peptide hormone co-secreted with insulin, potently governs the rate of gastric emptying. Hypoglycemia, in the absence of agents such as amylin, is reported to accelerate gastric emptying. We asked whether hypoglycemia had a similar action on gastric emptying in the presence of amylin. In preliminary experiments using a phenol red gavage technique in fasted SD rats, we showed that insulin administration accelerated gastric emptying in a dosage-dependent manner. This acceleration was totally prevented by coadministration of glucose in dosages that prevented a change in plasma glucose, indicating that insulin per se did not affect gastric emptying. The effect on gastric emptying of hypoglycemia induced by a 5 mU/min insulin infusion (t = 5–90 min) was assessed in conscious rats continuously infused with amylin (50 pmol · kg−1 · min−1; t = −30 to 90 min). Gastric emptying was indicated by the appearance in plasma of label from 3-O-methyl-[3H]glucose gavaged at t = 0 min. Label appearance was markedly inhibited in rats preinfused with amylin (84% reduced vs. saline controls at t = 30min), indicating amylin inhibition of gastric emptying. In amylin-treated rats that were subsequently infused with insulin, gavaged label abruptly appeared in plasma when plasma glucose had fallen to 2.1 ± 0.1 mmol/1 (at t ≅ 45 min), consistent with a reversal by hypoglycemia of amylin's inhibition of gastric emptying. These data support the idea of a central “fail-safe” mechanism whereby hypoglycemia can override the slowing of gastric emptying by amylin.