bluuebunny/crossref_metadata_2025 huggingface dataset:
Prolonged oxidative stress impairs insulin-induced GLUT4 translocation in 3T3-L1 adipocytes.
Authors: Rudich A , Tirosh A , Potashnik R , Hemi R , Kanety H , Bashan N
Published: March 2007

Prolonged exposure of 3T3-L1 adipocytes to micromolar concentrations of H2O2 results in an impaired response to the acute metabolic effects of insulin. In this study, we further characterized the mechanisms by which oxidative stress impairs insulin stimulation of glucose transport activity. Although insulin induced a 2.5-fold increase in plasma membrane GLUT4 content and a 50% reduction in its abundance in the low-density microsomal (LDM) fraction in control cells, oxidation completely prevented these responses. The net effect of insulin on 2-deoxyglucose uptake activity was reduced in oxidized cells and could be attributed to GLUT1 translocation. Insulin stimulation of insulin receptor substrate (IRS) 1 tyrosine phosphorylation and the association of IRS-1 with phosphatidylinositol (PI) 3-kinase were not impaired by oxidative stress. However, a 1.9-fold increase in the LDM content of the p85 subunit of PI 3-kinase after insulin stimulation was observed in control, but not in oxidized, cells. Moreover, although insulin induced an increase in IRS-1-associated PI 3-kinase activity in the LDM in control cells, this effect was prevented by oxidation. These findings suggest that prolonged low-grade oxidative stress impairs insulin-stimulated GLUT4 translocation, potentially by interfering with compartment-specific activation of PI 3-kinase.
Ciliary neurotrophic factor potentiates the beta-cell inhibitory effect of IL-1beta in rat pancreatic islets associated with increased nitric oxide synthesis and increased expression of inducible nitric oxide synthase.
Authors: Wadt K A , Larsen C M , Andersen H U , Nielsen K , Karlsen A E , Mandrup-Poulsen T
Published: March 2007

Proinflammatory cytokines are implicated as effector molecules in the pathogenesis of IDDM. Interleukin-6 (IL-6) alone or in combination with IL-1beta inhibits glucose-stimulated insulin release from isolated rat pancreatic islets by unknown mechanisms. Here we investigated 1) if the effects of IL-6 are mimicked by ciliary neurotrophic factor (CNTF), another member of the IL-6 family of cytokines signaling via gp130, 2) the possible cellular mechanisms for these effects, and 3) if islet endocrine cells are a source of CNTF. CNTF (20 ng/ml) potentiated IL-1beta-mediated (5-150 pg/ml) nitric oxide (NO) synthesis from neonatal Wistar rat islets by 31-116%, inhibition of accumulated insulin release by 34-49%, and inhibition insulin response to a 2-h glucose challenge by 31-36%. CNTF potentiated IL-1beta-mediated NO synthesis from RIN-5AH cells by 83%, and IL-1beta induced islet inducible NO-synthase (iNOS) mRNA expression fourfold. IL-6 (10 ng/ml) also potentiated IL-1beta-mediated NO synthesis and inhibition of insulin release, whereas beta-nerve growth factor (NGF) (5 or 50 ng/ml) had no effect. mRNA for CNTF was expressed in rat islets and in islet cell lines. In conclusion, CNTF is constitutively expressed in pancreatic beta-cells and potentiates the beta-cell inhibitory effect of IL-1beta in association with increased iNOS expression and NO synthesis, an effect shared by IL-6 but not by beta-NGF. These findings indicate that signaling via gp130 influences islet NO synthesis associated with iNOS expression. We hypothesize that CNTF released from destroyed beta-cells during the inflammatory islet lesion leading to IDDM may potentiate IL-1beta action on the beta-cells.
Acute lowering of plasma fatty acids lowers basal insulin secretion in diabetic and nondiabetic subjects.
Authors: Boden G , Chen X , Iqbal N
Published: March 2007

The objective of this study was to determine whether basal plasma free fatty acid (FFA) concentrations affect basal insulin secretion rates (ISRs). Effects of FFA levels on basal ISRs were evaluated by lowering basal plasma FFA levels with nicotinic acid (NA) (100-150 mg p.o., q 30 min x 4 h) in type 2 diabetic patients and in normal volunteers. Lowering of FFAs (from approximately 600 to approximately 100 micromol/l) lowered ISRs in type 2 diabetic patients during isoglycemic clamping (from 139 to 101 pmol/min; -23%; P < 0.02) and euglycemic clamping (from 99 to 63 pmol/min; -36%; P < 0.03) and in normal subjects during euglycemic clamping (from 127 to 96 pmol/min; -25%; P < 0.03). In addition, peripheral insulin concentrations decreased by approximately 30% in diabetic and nondiabetic subjects. NA had no direct effect on ISRs; that is, NA did not change ISRs when plasma FFAs were prevented from decreasing with a lipid/heparin infusion. We concluded that 1) basal plasma FFAs exerted physiologically important, long-lasting effects supporting 25-33% of basal insulin secretion in nondiabetic and diabetic subjects; 2) basal plasma FFAs were responsible for some of the hyperinsulinemia in normoglycemic obese subjects; and 3) NA had no direct effect on insulin secretion.
Peripheral neuropathy in transgenic diabetic mice: restoration of C-fiber function with human recombinant nerve growth factor.
Authors: Elias K A , Cronin M J , Stewart T A , Carlsen R C
Published: March 2007

Mice (Ins.Dd1) with hypoinsulinemic diabetes were created by increased expression of syngeneic major histocompatibility complex (MHC) class I protein in pancreatic beta-cells. The diabetic state was characterized in these mice by high glucose concentrations and islet pathology. To determine whether a neuropathy would develop, motor and sensory conduction velocities (CV) were determined in the sciatic nerves of 2-, 4-, and 7-month-old control and diabetic littermate male mice. Recording bipolar electrodes were placed in the plantar muscles of the hind foot of anesthetized (ketamine/xylazine) mice. Bipolar stimulating electrodes were positioned near the sciatic nerve at the sciatic notch or near the tibial nerve at the ankle. Motor CV from alpha-motor fibers and sensory CV from proprioceptive Aalpha nerves were measured and expressed as meters per second (m/s). Group data are reported as mean +/- SE and compared by analysis of variance. The CVs from nondiabetic mice (controls) were not different across the three ages and averaged 41.3 +/- 1.7 m/s for motor and 38.7 +/- 1.7 m/s for sensory. The motor CVs from diabetic mice at 2 and 4 months were similar to controls. Sensory CVs were unchanged at 2 months but were lower at 4 months (18.9 +/- 2.4 m/s). Both sensory (23.9 +/- 2.1 m/s) and motor (18.9 +/- 1.8 m/s) CVs were significantly reduced at 7 months, which is indicative of a polyneuropathy. NGF has well-known trophic effects on sympathetic and small sensory neurons. To determine whether NGF could influence this neuropathy, 6-month-old control and diabetic mice were divided into the following groups: 1) control + vehicle, 2) diabetic + vehicle, and 3) diabetic + NGF (1 mg/kg, 3x week, s.c.). After 1 month of treatment, motor and sensory CVs were determined. In some mice, the branches of the sciatic nerve were exposed and in situ recordings from the sural nerve were performed to determine compound C-fiber CV, integral, and amplitude. Sensory CV, determined via Hoffmann's reflex (H-reflex) (A-fiber), was decreased in diabetic compared with control animals as expected (P < 0.05), and NGF did not alter this parameter. Continuing diabetes reduced the amplitude (0.9 +/- 0.2 vs. 3.2 +/- 0.7 mV x 10(-2); P < 0.05) and integral (6.9 +/- 1.9 mV/ms vs. 18.8 +/- 4.4 mV/ms; P < 0.05) of the C-fiber response versus control, suggesting fiber loss. NGF treatment normalized C-fiber amplitude (2.9 +/- 0.8 mV x 10(-2)) and integral (21.2 +/- 6.5 mV/ms) in animals with established diabetes, with no effect on blood glucose. The C-fiber CV was similar in all groups, indicating that the animals had some normally conducting small fiber sensory nerves. These studies characterized a motor and sensory polyneuropathy in transgenic diabetic mice and are the first to demonstrate directly that NGF treatment can protect or restore abnormal sensory C-fiber function.
Prevalence of insulin resistance in metabolic disorders: the Bruneck Study.
Authors: Bonora E , Kiechl S , Willeit J , Oberhollenzer F , Egger G , Targher G , Alberiche M , Bonadonna R C , Muggeo M
Published: March 2007

The prevalence of insulin resistance in the most common metabolic disorders is still an undefined issue. We assessed the prevalence rates of insulin resistance in subjects with impaired glucose tolerance (IGT), NIDDM, dyslipidemia, hyperuricemia, and hypertension as identified within the frame of the Bruneck Study. The study comprised an age- and sex-stratified random sample of the general population (n = 888; aged 40-79 years). Insulin resistance was estimated by homeostasis model assessment (HOMA(IR)), preliminarily validated against a euglycemic-hyperinsulinemic clamp in 85 subjects. The lower limit of the top quintile of HOMA(IR) distribution (i.e., 2.77) in nonobese subjects with no metabolic disorders (n = 225) was chosen as the threshold for insulin resistance. The prevalence of insulin resistance was 65.9% in IGT subjects, 83.9% in NIDDM subjects, 53.5% in hypercholesterolemia subjects, 84.2% in hypertriglyceridemia subjects, 88.1% in subjects with low HDL cholesterol, 62.8% in hyperuricemia subjects, and 58.0% in hypertension subjects. The prevalence of insulin resistance in subjects with the combination of glucose intolerance (IGT or NIDDM), dyslipidemia (hypercholesterolemia and/or hypertriglyceridemia and/or low HDL cholesterol), hyperuricemia, and hypertension (n = 21) was 95.2%. In isolated hypercholesterolemia, hypertension, or hyperuricemia, prevalence rates of insulin resistance were not higher than that in nonobese normal subjects. An appreciable number of subjects (n = 85, 9.6% of the whole population) was insulin resistant but free of IGT, NIDDM, dyslipidemia, hyperuricemia, and hypertension. These results from a population-based study documented that 1) in hypertriglyceridemia and a low HDL cholesterol state, insulin resistance is as common as in NIDDM, whereas it is less frequent in hypercholesterolemia, hyperuricemia, and hypertension; 2) the vast majority of subjects with multiple metabolic disorders are insulin resistant; 3) in isolated hypercholesterolemia, hyperuricemia, or hypertension, insulin resistance is not more frequent than can be expected by chance alone; and 4) in the general population, insulin resistance can be found even in the absence of any major metabolic disorders.
Removal of adenosine decreases the responsiveness of muscle glucose transport to insulin and contractions.
Authors: Han D H , Hansen P A , Nolte L A , Holloszy J O
Published: March 2007

Adenosine in the extracellular space modulates stimulated glucose transport in striated muscle. In the heart and in adipocytes, adenosine potentiates insulin-stimulated glucose transport. There is controversy regarding the effect of adenosine in skeletal muscle, with reports of both an inhibitory effect and no effect, on insulin-stimulated glucose transport. We found that, in rat epitrochlearis and soleus muscles, removing adenosine with adenosine deaminase or blocking its action with the adenosine receptor blocker CPDPX markedly reduces the responsiveness of glucose transport to stimulation by 1) insulin alone, 2) contractions alone, and 3) insulin and contractions in combination. Measurement of the increase in GLUT4 at the cell surface in response to a maximally effective insulin stimulus in the epitrochlearis muscle, using the exofacial label ATB-[3H]BMPA, showed that adenosine deaminase treatment markedly reduces cell-surface GLUT4 labeling. The reduction in cell-surface GLUT4 labeling was similar in magnitude to the decrease in maximally insulin-stimulated glucose transport activity in adenosine deaminase-treated muscles. These results show that adenosine potentiates insulin- and contraction-stimulated glucose transport in skeletal muscle by enhancing the increase in GLUT4 at the cell surface and raise the possibility that decreased adenosine production or action could play a causative role in insulin resistance.
Acetylcholine activates intracellular movement of insulin granules in pancreatic beta-cells via inositol trisphosphate-dependent [correction of triphosphate-dependent] mobilization of intracellular Ca2+.
Authors: Niwa T , Matsukawa Y , Senda T , Nimura Y , Hidaka H , Niki I
Published: March 2007

Intracellular movement of secretory granules is a proximal stage in the secretory cascade that ends in the release product from cells. We investigated mechanisms underlying the control of this movement by acetylcholine using an insulinoma cell line, MIN6, in which acetylcholine increases both insulin secretion and granule movement. The peak activation of movement was observed 3 min after an acetylcholine challenge. The effects were nullified by the muscarinic inhibitor atropine, phospholipase C (PLC) inhibitors (D 609 and compound 48/80), and pretreatment with the Ca2+ pump inhibitor, thapsigargin. Inhibitors of Ca2+-dependent phospholipase A2 (arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphate) also partially inhibited the movement caused by acetylcholine, but downregulation of protein kinase C by overnight incubation with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate failed to exert any influence. Acetylcholine stimulation of granule movement was not reproduced by membrane depolarization with high K+. Phosphorylation of the endogenous myosin light chain in MIN6 cells was increased by addition of acetylcholine and decreased by the Ca2+ chelator BAPTA (1,2-bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid). The calmodulin inhibitor W-7 and the myosin light-chain kinase inhibitor ML-9 decreased the motile events in the beta-cells under both nonstimulated and acetylcholine-stimulated conditions. These findings led us to conclude that inositol trisphosphate [corrected] causes Ca2+ mobilization by muscarinic activation of PLC, leading to intracellular translocation of insulin granules to the ready-releasable pool in pancreatic beta-cells via Ca2+/calmodulin-dependent phosphorylation of myosin light chains.
Increased levels of free fatty acids in fasted mice stimulate in vivo beta-cell electrical activity.
Authors: Fernandez J , Valdeolmillos M
Published: March 2007

The electrical activity of pancreatic beta-cells in 48-h fasted mice has been recorded in vivo. Their electrical activity is exceedingly high at low levels of blood glucose when compared with control animals. For example, at a blood glucose concentration of 4.5 mmol/l, at which beta-cells are permanently hyperpolarized in control animals, fasted animals show continuous spiking activity. In fasted animals, hyperpolarization only occurs at glycemias below 2.2 mmol/l. As in fed animals, the electrical activity in fasted mice can be decreased or suppressed by the injection of diazoxide, indicating the participation of K(ATP) channels. The treatment of fasted animals with nicotinic acid, an inhibitor of lipolysis, produces a decrease in the levels of free fatty acids (FFAs) and a decrease in electrical activity, thereby restoring the dose-response curve for glucose in fasted animals to values close to those found in fed animals. Conversely, the injection of palmitic acid produces an increase in electrical activity without a change in blood glucose. These results point to FFAs as important regulators of electrical activity during fasting in vivo. They also indicate a dissociation of electrical activity and insulin release in fasted animals, since an increase in electrical activity is not associated with increased insulin secretion.
Diadenosine polyphosphates in insulin-secreting cells: interaction with specific receptors and degradation.
Authors: Verspohl E J , Johannwille B
Published: March 2007

A role of diadenosine polyphosphates as second messengers was suggested for insulin-secreting cells. It has not yet been investigated whether specific receptors for these compounds exist and how these extracellular compounds and their degradation products may contribute to insulin release. Specific saturable binding sites for diadenosine polyphosphates exist in INS-1 cells and rat pancreatic islets. In INS-1 cells, the rank order of diadenosine polyphosphates displacing [3H]Ap4A from binding sites was Ap4A = Ap5A >Ap3A = Ap6A. Binding was specific, since suramin was not able to displace the binding; adenosine, ATP, UTP, alpha,beta-methylene ATP, beta,gamma-methylene ATP, ADP-betaS, 2-methylthio ATP, and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) were able to displace [3H]Ap4A from its binding sites. Insulin release was investigated in INS-1 cells. Perifusion experiments showed an increase in insulin release stimulated by the diadenosine polyphosphates in the presence of 8.3 mmol/l glucose; in static incubations (90 min), however, insulin release was inhibited dose dependently by the four diadenosine polyphosphates. This discrepancy might be due to the instability of the compounds. [3H]Ap4A was degraded in the extracellular medium to mainly adenosine and low concentrations of ATP, ADP, AMP, and inosine (half-maximal degradation after 25 min). The insulin stimulatory effect is due to the original compounds (acute perifusion experiments), and the insulin inhibitory effect (static incubation experiments) is due to the production of inhibitory compounds, such as adenosine, in the medium. Small amounts of intact [3H]Ap4A, but mainly [3H]ATP, accumulated in the cells within 20 min. The uptake of labeled compounds is dependent on an intact metabolism and intact receptor internalization. This data indicates that 1) specific bindings sites for diadenosine polyphosphates exist in INS-1 cells and rat pancreatic islets mediating insulin release; 2) the receptors involved in INS-1 cells may be diadenosine polyphosphate receptors, albeit others, such as P2X-receptors, cannot be ruled out; and 3) diadenosine polyphosphates, and mainly their degradation products in the extracellular space, are to a high degree accumulated within cells with unknown function. Thus, diadenosine polyphosphates are worth being investigated more closely in physiological and pathophysiological terms.
Plasma levels of the soluble fraction of tumor necrosis factor receptor 2 and insulin resistance.
Authors: Fernández-Real J M , Broch M , Ricart W , Casamitjana R , Gutierrez C , Vendrell J , Richart C
Published: March 2007

Recent studies have shown that the tumor necrosis factor (TNF) system is implicated in the insulin resistance of human obesity. Plasma concentrations of the soluble fraction of the TNF receptors 1 and 2 (sTNFR1 and sTNFR2) are thought to reflect the degree of activation of the TNF system. The purpose of this study was to explore whether this activation, as measured by the levels of circulating sTNFR1 and sTNFR2, is associated with insulin resistance. A total of 19 men (mean age 36.2 +/- 1.9; BMI 28.8 +/- 1.2, range 22.2-35.7) and 17 premenopausal women (age 34.9 +/- 1.4; BMI 28.1 +/- 0.8, range 19-37.9) were studied. Men showed higher levels of plasma sTNFR1 and sTNFR2 than women. However, obese men showed increased levels of sTNFR2 but similar levels of sTNFR1 in comparison with obese women. In fact, sTNFR2 levels correlated with BMI (r = 0.50, P = 0.002), fat-free mass (FFM) (r = 0.61, P < 0.0001), and waist-to-hip ratio (WHR) (r = 0.39, P = 0.02), but not with fat mass or percent fat mass. sTNFR2 levels correlated with basal glucose levels (r = 0.45, P = 0.007), area under the curve (AUC) for glucose during an oral glucose tolerance test (r = 0.42, P = 0.013), and with the quotient AUC glucose/log AUC insulin (r = 0.41, P = 0.015). sTNFR2 also correlated negatively with insulin sensitivity (S(I)), evaluated using the frequently sampled intravenous glucose tolerance test with minimal model analysis (r = -0.38, P = 0.02). Plasma sTNFR1 levels were not associated with any of these variables. Because WHR influenced both S(I) and sTNFR2 levels, we constructed a multiple linear regression to predict S(I), with WHR and sTNFR2 as independent variables. In this model, both WHR (P = 0.0078) and sTNFR2 levels (P = 0.025) contributed to 47% of the variance in S(I). In parallel with higher FFM, lean and obese men showed a lower S(I) (2.9 +/- 0.9 vs. 5.2 +/- 1.3 min(-1) x mU x l(-1), P = 0.001; and 1.15 +/- 1.1 vs. 1.8 +/- 0.8 min(-1) x mU x l(-1), P = 0.035, respectively) and higher sTNFR2 levels in comparison with lean and obese women, respectively. After controlling for FFM, the correlation between S(I) and sTNFR2 levels disappeared, indicating that FFM was significantly influencing these associations. In summary, plasma sTNFR2 levels, but not sTNFR1, were proportional to BMI, WHR, FFM (a well-known confounder in the evaluation of insulin sensitivity), basal and postload glucose levels, and insulin resistance. These findings support TNF-alpha as a system regulating insulin action in human obesity.
Effect of nerve growth factor treatment on p75NTR gene expression in lumbar dorsal root ganglia of streptozocin-induced diabetic rats.
Authors: Delcroix J D , Michael G J , Priestley J V , Tomlinson D R , Fernyhough P
Published: March 2007

Previous work shows that gene expression for p75NTR in lumbar dorsal root ganglia (DRG) is deficient in streptozocin-induced diabetic rats, while expression of trkA protein is unaffected. This is the first study on the effect of diabetes on immunohistochemical staining and axonal transport of p75NTR in sensory neurons. We also investigated the novel effect of nerve growth factor (NGF) treatment on the levels of mRNA and protein of the NGF receptors, trkA and p75NTR, in normal and diabetic rats. Immunohistochemical staining for p75NTR was significantly reduced in DRG of 12-week streptozocin-induced diabetic rats, confirming the previous work. Anterograde and retrograde axonal transport of p75NTR within the sciatic nerve was similarly decreased in diabetic rats, while levels of transport of trkA were unaffected. Treatment with systemic NGF reversed the diabetes-induced deficit in p75NTR transcripts and protein within the DRG and left expression levels of trkA unchanged. We propose that in sensory neurons of diabetic rats, the ability to capture and retrogradely transport NGF may be impaired because of suboptimal production of p75NTR receptors and that NGF therapy may overcome this deficiency.
Novel susceptibility gene for late-onset NIDDM is localized to human chromosome 12q.
Authors: Shaw J T , Lovelock P K , Kesting J B , Cardinal J , Duffy D , Wainwright B , Cameron D P
Published: March 2007

NIDDM has a substantial genetic component, but the nature of the genetic susceptibility is largely unknown. Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous monogenic form of NIDDM characterized by an early age of onset and autosomal dominant inheritance, and linkage studies have identified genes that are mutated in different MODY pedigrees on chromosome 20 (MODY1 locus, hepatocyte nuclear factor-4alpha [HNF-4alpha] gene), chromosome 7 (MODY2 locus, glucokinase gene), and chromosome 12 (MODY3 locus, HNF-1alpha gene). We studied an extended pedigree in which multiple members are affected by late-onset NIDDM associated with insulin resistance and performed linkage analysis with four microsatellite markers in the MODY3 region of chromosome 12q. We found significant evidence for linkage between NIDDM and the MODY3 locus (logarithm of odds score 3.65 at theta = 0.008 telomeric to marker D12S321), but sequencing of the 10 exons and promoter of HNF-1alpha did not identify any causative mutation in this gene. Our results indicate that the region of chromosome 12q close to MODY3 harbors a novel susceptibility gene or genes for NIDDM.
Increased adipose expression of the uncoupling protein-3 gene by thiazolidinediones in Wistar fatty rats and in cultured adipocytes.
Authors: Matsuda J , Hosoda K , Itoh H , Son C , Doi K , Hanaoka I , Inoue G , Nishimura H , Yoshimasa Y , Yamori Y , Odaka H , Nakao K
Published: March 2007

Uncoupling protein (UCP) 3 and UCP2, mitochondrial carrier proteins dissipating electrochemical gradient across the mitochondrial inner membrane, have been implicated in the regulation of energy metabolism. The UCP3 gene is expressed abundantly in the skeletal muscle, while the UCP2 gene is detected in the white adipose tissue (WAT) with diffuse localization throughout the body. Uncoupling of electron transport and ATP synthesis has been reported to increase glucose uptake, suggesting that UCP may be involved in glucose metabolism. Thiazolidinediones (TZDs), which are insulin-sensitizing agents for NIDDM, have been reported to increase energy expenditure. To elucidate the pathophysiologic significance of UCP3 and UCP2 in the effect of TZDs on glucose metabolism and energy expenditure, we examined their basal mRNA levels in the WAT, brown adipose tissue (BAT), and skeletal muscle from Wistar fatty rats, a rat model of NIDDM and obesity with leptin receptor defect, and investigated expression of the genes encoding UCP3 and UCP2 in Wistar fatty rats and in Wistar lean rats with 2-week oral administration of 3 mg x kg(-1) x day(-1) pioglitazone, a TZD derivative. Basal UCP3 mRNA levels were significantly lower (38 +/- 8, 45 +/- 13, and 76 +/- 6%) in the retroperitoneal WAT, BAT, and skeletal muscle from Wistar fatty rats than in those from Wistar lean rats, while basal UCP2 mRNA levels were significantly higher by 2.1-, 1.8-, and 2.5-fold in the subcutaneous WAT, retroperitoneal WAT, and BAT from Wistar fatty rats, respectively, than in those from Wistar lean rats. In pioglitazone-treated Wistar fatty rats, UCP3 mRNA levels were significantly increased by 2.1-, 2.0-, and 1.6-fold in the epididymal WAT, retroperitoneal WAT, and BAT, respectively, as compared with those in nontreated fatty rats. In pioglitazone-treated lean rats, UCP3 mRNA levels were significantly increased by 1.3-fold in the BAT as compared with those in nontreated lean rats. No significant change of UCP2 mRNA levels was observed in pioglitazone-treated fatty and lean rats. In addition, to examine the direct effect of TZDs on adipocytes, we examined the regulation of UCP3 and UCP2 gene expression using the primary culture of rat mature adipocytes from Sprague-Dawley rats. In rat cultured mature adipocytes, UCP3 mRNA levels were increased in a dose-responsive manner by 10(-5) to 10(-4) mol/l pioglitazone, while there was no significant change of UCP2 mRNA levels. These results clearly demonstrate that UCP3 gene expression is upregulated by TZDs in the WAT and BAT in Wistar fatty rats, an obese model with leptin receptor defect, and that adipose UCP3 gene expression is increased in response to TZDs in vitro. The present study suggests the involvement of UCP3 in the effects of TZDs on energy and glucose metabolism.
Shared biochemical properties of glucotoxicity and lipotoxicity in islets decrease citrate synthase activity and increase phosphofructokinase activity.
Authors: Liu Y Q , Tornheim K , Leahy J L
Published: March 2007

Diabetic states are characterized by a raised serum/islet level of triglycerides and a lowered EC50 (concentration at half-maximal stimulation) for glucose-induced insulin secretion. Culturing islets with long-chain fatty acids (FAs) replicates the basal insulin hypersecretion. In a previous study, we showed that the mechanism involved deinhibition of hexokinase by a 60% decrease in glucose-6-phosphate (G-6-P). The key event was proposed to be an increased phosphofructokinase (PFK) Vmax secondary to an upregulatory effect of the FA metabolite, long-chain acyl-coenzyme A (LC-CoA). We now show another contributory factor, a lowered content of the PFK inhibitor citrate. Citrate synthase Vmax and citrate levels were lowered 45% in rat islets cultured with 250 micromol/l oleate for 24 h. Both effects were reversed by triacsin C, an inhibitor of fatty acyl-CoA synthetase, the enzyme that generates LC-CoA. Culturing islets with high doses of glucose (16.7 mmol/l) for 48 h should also raise cytosolic LC-CoA. As predicted, citrate synthase Vmax was lowered and PFK Vmax was increased, both in a triacsin C-reversible fashion. These results show shared selected functional and biochemical properties in beta-cells of so-called glucotoxicity and lipotoxicity.
Hypoxia induces vascular endothelial growth factor gene and protein expression in cultured rat islet cells.
Authors: Vasir B , Aiello L P , Yoon K H , Quickel R R , Bonner-Weir S , Weir G C
Published: March 2007

The formation of new microvasculature by capillary sprouting at the site of islet transplantation is crucial for the long-term survival and function of the graft. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, may be a key factor in modulating the revascularization of islets after transplantation. In this study, we examined the gene expression of VEGF mRNA in three tumor cell lines and in isolated whole and dispersed rat islets in vitro by Northern blot hybridization in normoxic (5% CO2, 95% humidified air) and hypoxic (1% O2, 5% CO2, 94% N2) culture conditions. Increased expression of VEGF mRNA was observed in beta-TC3, RAW 264.7, and IC-21 tumor cell lines when subjected to hypoxia. With isolated whole islets in normoxic culture, a threefold increase in VEGF mRNA (P < 0.001) was seen at 48 h as compared with freshly isolated islets. This response was similar to the 3.8-fold increase observed with islets subjected to hypoxia. Dispersed rat islet cell clusters cultured on Matrigel for 24 h under hypoxic conditions showed a 3.4-fold increase (P < 0.01) in VEGF mRNA compared with those cultured in normoxia. This correlated with increased VEGF secretion as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies revealed the presence of increased expression of VEGF protein near the center of islets after 24 h of normoxic culture. Islet cell clusters on Matrigel showed intense cellular localization of VEGF in both beta-cells and non-beta-cells. These findings suggest that rat islet cells, when subjected to hypoxia during the first few days after transplantation, may act as a major source of VEGF, thereby initiating revascularization and maintaining the vascular permeability of the grafted islets.
Previous episodes of hypoglycemic coma are not associated with permanent cognitive brain dysfunction in IDDM patients on intensive insulin treatment.
Authors: Kramer L , Fasching P , Madl C , Schneider B , Damjancic P , Waldhäusl W , Irsigler K , Grimm G
Published: March 2007

Intensive insulin treatment of IDDM is associated with increased frequency of hypoglycemic coma. The extent of possible cerebral sequelae after recovery is still unknown. We studied the impact of previous hypoglycemic coma on neurophysiological measures of cognitive brain function in 108 patients with adult-onset IDDM receiving intensive insulin treatment. In the study, 55 IDDM patients (age 38 +/- 14 years, mean +/- SD) who had a history of > or =1 (median 3, range 1-35) comatose hypoglycemic event were compared with 53 IDDM patients (age 34 +/- 12 years) with no history of hypoglycemic events using P300 event-related potentials and psychometric tests (the Mini-Mental State Exam and trailmaking test, part A). Findings on these patients were compared with those from 108 matched healthy control subjects. No difference was observed in P300 latencies and psychometric tests between patients with and without a history of hypoglycemic coma (P300 latency, 346 vs. 342 ms; trailmaking test, 31 vs. 30 s; Mini-Mental State Exam, 29.5 vs. 29.6; NS). In diabetic patients, however, P300 latencies were delayed compared with those of healthy control subjects (344 vs. 332 ms; P < 0.001) and were correlated to diabetes duration but not to total hypoglycemic episodes. Scores on the Mini-Mental State Exam (29.5 vs. 29.6; P = 0.59) and trailmaking test (31 vs. 28 s; P = 0.10) were not different between patients and control subjects. In conclusion, previous episodes of hypoglycemic coma are not associated with permanent impairment of cognitive brain function in patients with adult-onset IDDM receiving intensive insulin treatment compared with patients without such episodes. Cognitive brain function, however, is subclinically impaired in relation to duration of diabetes.
Pancreatic beta-cell glucokinase: closing the gap between theoretical concepts and experimental realities.
Authors: Matschinsky F M , Glaser B , Magnuson M A
Published: March 2007

There remains a wide gap between theoretical concepts and experimental realities in the enzyme kinetics and biochemical genetics of the pancreatic beta-cell glucokinase-glucose sensor. It is the goal of present efforts in many laboratories to bridge this gap. This perspective intends to provide a timely review of this crucial aspect of research in glucose homeostasis. It deals briefly with some fundamentals of glucokinase enzyme kinetics, offers some pertinent biochemical genetic considerations, takes stock of the current experimental database of the field by emphasizing human studies and referring to recent mouse studies, and ventures a few extrapolations into the future of this endeavor.
Tolbutamide and diazoxide influence insulin secretion by changing the concentration but not the action of cytoplasmic Ca2+ in beta-cells.
Authors: Mariot P , Gilon P , Nenquin M , Henquin J C
Published: March 2007

Sulfonylureas stimulate insulin secretion by blocking ATP-sensitive K+ channels (K+-ATP channels) of the beta-cell membrane, thereby causing depolarization, Ca2+ influx, and rise in cytoplasmic Ca2+ concentration ([Ca2+]i), whereas diazoxide inhibits insulin secretion by opening K+-ATP channels. It has been suggested recently that these drugs also respectively increase and decrease the efficacy of Ca2+ on exocytosis. This hypothesis was tested here with intact islets or single beta-cells from normal mice. Depolarizing islet cells by raising extracellular K+ from 4.8 to 15, 30, and 60 mmol/l progressively raised [Ca2+]i and stimulated insulin secretion. The magnitude of the [Ca2+]i rise produced by a subsequent addition of 100 micromol/l tolbutamide decreased as the concentration of K+ was increased. The effect on insulin secretion paralleled that on [Ca2+]i. Similarly, the magnitudes of the [Ca2+]i drop and of the inhibition of insulin secretion produced by 250 micromol/l diazoxide were inversely related to the concentration of K+. Either drug was effective on secretion only when it increased or decreased [Ca2+]i. Exocytosis of insulin granules from single, voltage-clamped beta-cells was also studied by measuring cell capacitance changes. In the perforated patch configuration, exocytosis was evoked by depolarizing pulses. Addition of tolbutamide to the extracellular medium did not affect the Ca2+ current and the resulting change in cell capacitance. In the whole-cell configuration, cell capacitance increased with the concentration of free Ca2+ in the solution diffusing from the pipette into the cell. It was markedly potentiated by cAMP, was inhibited by activation of alpha2-adrenoceptors with clonidine, and was strongly augmented by acetylcholine. In contrast, tolbutamide was ineffective whether applied intra- or extracellularly, at low or high free Ca2+, and with or without cAMP. Diazoxide also failed to interfere directly with exocytosis. These results indicate that tolbutamide and diazoxide affect insulin secretion by changing the concentration, not the action, of Ca2+ in beta-cells.
Upregulation of the vascular endothelial growth factor/vascular endothelial growth factor receptor system in experimental background diabetic retinopathy of the rat.
Authors: Hammes H P , Lin J , Bretzel R G , Brownlee M , Breier G
Published: March 2007

Vascular endothelial growth factor (VEGF) is a major contributor to retinal neovascularization. The possible participation of VEGF and its high-affinity tyrosine kinase receptors, flk-1 and flt-1, in early background diabetic retinopathy was studied in the streptozotocin-induced diabetic rat model of experimental retinopathy using in situ hybridization, blotting techniques, and immunohistochemistry. Diabetic retinopathy was assessed by quantitative morphometry of retinal digest preparations. The number of acellular capillaries increased 2.7-fold in diabetic animals with diabetes' duration of 6 months compared with nondiabetic controls. VEGF expression was not detectable by in situ hybridization in nondiabetic rats but was highly increased in the ganglion cell layer and in the inner and outer nuclear layers of retinas from diabetic animals. VEGF protein was extractable only from diabetic retinas, and a strong immunolabeling was detected in vascular and perivascular structures. Increased flk-1 and flt-1 mRNA levels were also found in the ganglion cell and both nuclear layers of diabetic samples only. Dot blot and Western blot analyses confirmed the increase in flk-1 mRNA and protein in diabetic retinas. Also, flk-1 immunoreactivity was associated with vascular and nonvascular structures of the inner retinas from diabetic animals. These data obtained from a rodent model in which retinal neovascularization does not occur support the concept that the VEGF/VEGF receptor system is upregulated in early diabetic retinopathy.
Expression of transforming growth factor-beta1 and type IV collagen in the renal tubulointerstitium in experimental diabetes: effects of ACE inhibition.
Authors: Gilbert R E , Cox A , Wu L L , Allen T J , Hulthen U L , Jerums G , Cooper M E
Published: March 2007

Transforming growth factor-beta (TGF-beta) and the renin-angiotensin system (RAS) have both been implicated in the pathogenesis of glomerulosclerosis in diabetic kidney disease. However, tubulointerstitial pathology may also be an important determinant of progressive renal dysfunction in diabetic nephropathy. In the present study, we investigated tubulointerstitial injury, TGF-beta1 expression, and the effect of blocking the RAS by inhibition of ACE. We randomized 36 male SD rats to control and diabetic groups. Diabetes was induced in 24 rats by administration of streptozotocin; 12 diabetic rats were further randomized to receive the ACE inhibitor ramipril (3 mg/l drinking water). At 6 months, experimental diabetes was associated with tubulointerstitial damage, a 70% increase in expression of TGF-beta1 (P < 0.05 vs. control), and a 120% increase in alpha1 (IV) collagen gene expression (P < 0.01 vs. control). In situ hybridization demonstrated a diffuse increase in both TGF-beta1 and alpha1 (IV) collagen mRNA in renal tubules. In addition, intense expression of both transcripts was noted in regions of focal tubular dilatation. Administration of the ACE inhibitor ramipril prevented tubulointerstitial injury and the overexpression of TGF-beta1 and alpha1 (IV) collagen mRNA. Changes in gene expression were accompanied by parallel changes in immunostaining for TGF-beta1 and type IV collagen. The observed beneficial effects of ramipril on the tubulointerstitium in experimental diabetes suggest that this mechanism may contribute to the therapeutic effect of ACE inhibitors in diabetic nephropathy.
Predisposition to essential hypertension and development of diabetic nephropathy in IDDM patients.
Authors: Fagerudd J A , Tarnow L , Jacobsen P , Stenman S , Nielsen F S , Pettersson-Fernholm K J , Grönhagen-Riska C , Parving H H , Groop P H
Published: March 2007

Conflicting results have been reported on the relationship between familial predisposition to hypertension and development of diabetic nephropathy in IDDM. In our case-control study, we assessed the prevalence of hypertension among parents of 73 IDDM patients with diabetic nephropathy (DN+; persistent albuminuria > 200 microg/min or > 300 mg/24 h) and 73 IDDM patients without diabetic nephropathy (DN-; urinary albumin excretion < 20 microg/min or < 30 mg/24 h). Arterial hypertension, defined as antihypertensive therapy or a 24-h ambulatory blood pressure (SpaceLabs 90207) > or = 135/85 mmHg, was present in 57% of parents of DN+ patients compared with 41% of parents of DN- patients (P = 0.034; difference 16% [95% CI 1.3-29.6%]). In addition, the cumulative incidence of hypertension was higher among parents of DN+ patients (log-rank test P < 0.001), with a shift toward younger age at onset of hypertension in this group. However, the difference in prevalence of parental hypertension was not evident using office blood pressure measurements (64 vs. 57%; NS; difference 7% [-5.8-20%). Furthermore, patients with DN+ and with antihypertensive therapy in both parents were themselves more frequently treated for hypertension than were patients with DN+ and without parental treatment for hypertension (100 vs. 61%; P = 0.034; difference 39% [21-57%]). In conclusion, familial predisposition to essential hypertension increases the risk of diabetic nephropathy and may also contribute to the development of systemic hypertension in patients with IDDM and diabetic nephropathy.
Angiotensin I-converting enzyme gene polymorphism modulates the consequences of in utero growth retardation on plasma insulin in young adults.
Authors: Cambien F , Léger J , Mallet C , Lévy-Marchal C , Collin D , Czernichow P
Published: March 2007

In utero growth retardation has been linked to a reduced rate of cell division in the fetal organs that undergo rapid growth and to permanent changes and adaptations (programming) that may affect the physiology in adult life. In particular, in utero growth retardation as reflected by a low birth weight for gestational age has been shown to be associated with a relative insulin resistance in adults. How programming may influence glucose metabolism is not completely understood, and the possible role of genetic factors has not been explored. The angiotensin I-converting enzyme gene insertion/deletion (ACE I/D) polymorphism may predispose to insulin resistance and modulate the expression of several common cardiovascular and renal disorders, especially in people with diabetes. The possible impact of this polymorphism on plasma glucose and insulin levels was investigated in a group of young adults born at term whose length or weight at birth were in the lowest 3% of the sex and gestational age-adjusted distribution (SGA, n = 172) and a group of control individuals born with an appropriate birth weight for gestational age (AGA, n = 207). In this study, we have previously demonstrated an association between SGA and relative insulin resistance, especially in those with shorter gestational age. In the SGA group, fasting plasma glucose and insulin levels were significantly correlated (R = 0.196, P < 0.015), with this association being significant only in ACE II individuals (R = 0.539, P < 0.0009). In the AGA group, fasting plasma glucose and insulin levels were not significantly correlated. Consistent with this observation, the relationship between the ACE polymorphism and the insulin response to a glucose load was significantly heterogeneous between the AGA and SGA groups (P < 0.05); this was due to a tendency for ACE II individuals in the SGA group to exhibit increased 30-min plasma insulin levels (P < 0.05). In the SGA group, there was a significant interaction between gestational age and genotype on the insulin area (P < 0.0004); this index was inversely associated with gestational age in ACE II (P < 0.0005) and ACE ID (P < 0.005) subjects, but not in DD homozygotes (P > 0.05). The ACE D allele may thus attenuate the additive consequences of SGA and relatively short duration of gestation on insulin resistance in young adults.
Identification and functional analysis of sulfonylurea receptor 1 variants in Japanese patients with NIDDM.
Authors: Ohta Y , Tanizawa Y , Inoue H , Hosaka T , Ueda K , Matsutani A , Repunte V P , Yamada M , Kurachi Y , Bryan J , Aguilar-Bryan L , Permutt M A , Oka Y
Published: March 2007

The sulfonylurea receptor 1 (SUR1) is an essential regulatory subunit of the beta-cell ATP-sensitive K+ channel (K[ATP]). The possible role of SUR1 gene mutation(s) in the development of NIDDM remains controversial as both a positive association and negative linkage results have been reported. Therefore, we examined the SUR1 gene at the single nucleotide level with single strand conformation polymorphism analysis in 100 Japanese NIDDM patients. We identified a total of five amino acid substitutions and 17 silent mutations by examining all 39 exons of this gene. Two rare novel mutations, D811N in exon 20 and R835C in exon 21, were identified in the first nucleotide-binding fold (NBF), a functionally important region of SUR1, in one patient each, both heterozygotes. To analyze possible functional alterations, we reconstituted the mutant K(ATP) by coexpressing beta-cell inward rectifier (BIR) (Kir 6.2), a channel subunit of K(ATP), and mutant SUR1 in HEK293T and COS-7 cells. As demonstrated by the patch clamp technique and rubidium (Rb+) efflux studies, neither mutation alters the properties of channel activities. Two other rare missense mutations, R275Q in exon 6 and V560M in exon 12, were also identified. The R275Q substitution was not found in 67 control subjects, and V560M was present in three control subjects. Neither of these substitutions appeared to cosegregate with NIDDM in the probands' families. A previously reported S1370A substitution located in the second NBF was also common in the Japanese subjects (allelic frequency 0.37), and was found at an equal frequency in nondiabetic control subjects. In conclusion, SUR1 mutations impairing K(ATP) function do not appear to be major determinants of NIDDM susceptibility in Japanese.
PPAR-gamma: adipogenic regulator and thiazolidinedione receptor.
Authors: Spiegelman B M
Published: March 2007

The past several years have seen an explosive increase in our understanding of the transcriptional basis of adipose cell differentiation. In particular, a key role has been illustrated for PPAR-gamma, a member of the nuclear hormone receptor superfamily. PPAR-gamma has also been recently identified as the major functional receptor for the thiazolidinedione class of insulin-sensitizing drugs. This review examines the evidence that has implicated this transcription factor in the processes of adipogenesis and systemic insulin action. In addition, several models are discussed that may explain how a single protein can be involved in these related but distinct physiological actions. I also point out several important areas where our knowledge is incomplete and more research is needed. Finally, I discuss how advances in our understanding of nuclear receptor function, particularly the docking of cofactors in a ligand-dependent fashion, should lead to improved drugs that utilize the PPAR-gamma system for the treatment of insulin resistance.