bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Free Fatty Acids on Glucose Uptake and Utilization in Healthy Women
Authors: Homko Carol J. , Cheung Peter , Boden Guenther
Published: March 2007

To study effects of sex on free fatty acid (FFA)-induced insulin resistance, we have examined the effects of acute elevations of plasma FFA levels on insulin-stimulated total body glucose uptake in nine healthy young women. Euglycemic-hyperinsulinemic (∼500 pmol/l) clamps were performed for 4 h with coinfusion of either lipid/heparin (L/H) to acutely raise plasma FFA levels (from ∼600 to ∼1,200 μmol/l) or saline/glycerol to lower fatty acids (from ∼600 to ∼50 μmol/l). L/H infusion inhibited insulin-stimulated glucose uptake (determined with [3-3H]glucose) and glycogen synthesis by 31 and 40%, respectively (P < 0.01), almost completely abolished insulin suppression of endogenous glucose production (EGP) (13.6 vs. 10.0 μmol · kg−1 · min−1, NS), prevented the insulin induced increase in carbohydrate oxidation (8.1 vs. 7.4 μmol · kg−1 · min−1, NS), and stimulated fat oxidation (from 3.6 to 5.1 μmol · kg−1 · min−1, P < 0.01). These data showed that acute increases in plasma FFA levels inhibited the actions of insulin on glucose uptake, glycogen synthesis, and EGP in women to a degree similar to that previously reported in men. We conclude that at insulin and FFA levels in the postprandial range, women and men were susceptible to FFA-induced peripheral and hepatic insulin resistance.
Protein Kinase C β Inhibition Attenuates the Progression of Experimental Diabetic Nephropathy in the Presence of Continued Hypertension
Authors: Kelly Darren J. , Zhang Yuan , Hepper Claire , Gow Renae M. , Jaworski Kassie , Kemp Bruce E. , Wilkinson-Berka Jennifer L. , Gilbert Richard E.
Published: March 2007

In addition to hyperglycemia, hypertension and the renin-angiotensin system have been consistently implicated in the pathogenesis of diabetic nephropathy. Each of these pathogenetic factors may induce changes in cellular function by a common intracellular signaling pathway, the activation of protein kinase C (PKC) β. The present study thus sought to determine the in vivo effect of PKC β inhibition in experimental diabetic nephropathy in the setting of continued hyperglycemia, hypertension, and activation of the RAS. Studies were conducted in the (mRen-2)27 rat, a rodent that is transgenic for the entire mouse renin gene (Ren-2) and develops many of the structural, functional, and molecular characteristics of human diabetic nephropathy when experimental diabetes is induced with streptozotocin (STZ). Six-week-old female Ren-2 rats received an injection of STZ or vehicle and were maintained for 6 months. Within 24 h, diabetic rats were further randomized to receive treatment with the specific PKC β inhibitor, LY333531, admixed in diet (10 mg · kg−1 · d−1) or no treatment (n = 8/group). Diabetic rats developed albuminuria, glomerulosclerosis, and tubulointerstitial fibrosis with a concomitant increase in transforming growth factor-β (TGF-β). Western blot analysis demonstrated increased PKC β in diabetic animals, localized by immunofluorescence to the glomerular mesangium. In vivo inhibition of PKC β with LY333531 led to a reduction in albuminuria, structural injury, and TGF-β expression, despite continued hypertension and hyperglycemia.
Contribution of Fatty Acids Released From Lipolysis of Plasma Triglycerides to Total Plasma Fatty Acid Flux and Tissue-Specific Fatty Acid Uptake
Authors: Teusink Bas , Voshol Peter J. , Dahlmans Vivian E.H. , Rensen Patrick C.N. , Pijl Hanno , Romijn Johannes A. , Havekes Louis M.
Published: March 2007

There is controversy over the extent to which fatty acids (FAs) derived from plasma free FAs (FFAs) or from hydrolysis of plasma triglycerides (TGFAs) form communal or separate pools and what the contribution of each FA source is to cellular FA metabolism. Chylomicrons and lipid emulsions were labeled with [3H]triolein, injected into mice, and appearance in plasma of [3H]oleic acid was estimated, either through a steady-state approach or by compartmental modeling. [14C]oleic acid was included to trace plasma FFA. Eighty to 90% of triglyceride (TG) label was recovered in plasma, irrespective of tracer method or TG source. The contribution of TG lipolysis to total plasma FA turnover was 10–20%. After infusion of [3H]TG and [14C]FA, the retention of these labels varied substantially among liver, adipose tissue, and skeletal and heart muscle. Retention of TG label changed during fasting in the same direction as lipoprotein lipase (LPL) activity is regulated. We propose a model that reconciles the paradoxical 80–90% loss of TG label into plasma with LPL-directed differential uptake of TGFA in tissues. In this model, TGFAs mix locally at the capillaries with plasma FFAs, where they would lead to an increase in the local FA concentration, and hence, FA uptake. Our data indicate that a distinction between TG-derived FA and plasma FFA cannot be made.
Regulation of PGC-1 Promoter Activity by Protein Kinase B and the Forkhead Transcription Factor FKHR
Authors: Daitoku Hiroaki , Yamagata Kazuyuki , Matsuzaki Hitomi , Hatta Mitsutoki , Fukamizu Akiyoshi
Published: March 2007

Peroxisome proliferator–activated receptor-γ coactivator-1 (PGC-1) plays a major role in mediating hepatic gluconeogenesis in response to starvation, during which PGC-1 is induced by the cyclic AMP response element binding protein. Although it is observed that insulin counteracts PGC-1 transcription, the mechanism by which insulin suppresses the transcription of PGC-1 is still unclear. Here, we show that forkhead transcription factor FKHR contributes to mediating the effects of insulin on PGC-1 promoter activity. Reporter assays demonstrate that insulin suppresses the basal PGC-1 promoter activity and that coexpression of protein kinase (PK)-B mimics the effect of insulin in HepG2 cells. Insulin response sequences (IRSs) are addressed in the PGC-1 promoter as the direct target for FKHR in vivo. Coexpression of FKHR stimulates the PGC-1 promoter activity via interaction with the IRSs, while coexpression of FKHR (3A), in which the three putative PKB sites in FKHR are mutated, mainly abolishes the suppressive effect of PKB. Whereas deletion of the IRSs prevents the promoter stimulation by FKHR, that activity is still partially inhibited by insulin. These results indicate that signaling via PKB to FKHR can partly account for the effect of insulin to regulate the PGC-1 promoter activity via the IRSs.
Effect of Short-Term Fasting and Refeeding on Transcriptional Regulation of Metabolic Genes in Human Skeletal Muscle
Authors: Pilegaard Henriette , Saltin Bengt , Neufer P. Darrell
Published: March 2007

During short-term fasting, substrate utilization in skeletal muscle shifts from predominantly carbohydrate to fat as a means of conserving glucose. To examine the potential influence of short-term fasting and refeeding on transcriptional regulation in skeletal muscle, muscle biopsies were obtained from nine male subjects at rest, after 20 h of fasting, and 1 h after consuming either a high-carbohydrate (CHO trial) or a low-carbohydrate (FAT trial) meal. Fasting induced an increase in transcription of the pyruvate dehydrogenase kinase 4 (PDK4) (10-fold), lipoprotein lipase (LPL) (∼2-fold), uncoupling protein 3 (UCP3) (∼5-fold), and carnitine palmitoyltransferase I (CPT I) (∼2.5-fold) genes. Surprisingly, transcription of PDK4 and LPL increased further in response to refeeding (both trials) to more than 50-fold and 6- to 10-fold, respectively, over prefasting levels. However, responses varied among subjects with two subjects in particular displaying far greater activation of PDK4 (>100-fold) and LPL (>20-fold) than the other subjects (mean ∼8-fold and ∼2-fold, respectively). Transcription of UCP3 decreased to basal levels after the CHO meal but remained elevated after the FAT meal, whereas CPT I remained elevated after both refeeding meals. The present findings demonstrate that short-term fasting/refeeding in humans alters the transcription of several genes in skeletal muscle related to lipid metabolism. Marked heterogeneity in the transcriptional response to the fasting/refeeding protocol suggests that individual differences in genetic profile may play an important role in adaptive molecular responses to metabolic challenges.
Nocturnal and Postprandial Free Fatty Acid Kinetics in Normal and Type 2 Diabetic Subjects
Authors: Miles John M. , Wooldridge David , Grellner Wayne J. , Windsor Sheryl , Isley William L. , Klein Samuel , Harris William S.
Published: March 2007

Whether free fatty acid (FFA) rate of appearance (Ra) is increased in type 2 diabetes is controversial. To characterize nocturnal and postprandial abnormalities in FFA kinetics and to determine the effects of treatment with insulin sensitizers on lipolysis, we measured palmitate Ra in control subjects (n = 6) and individuals with poorly controlled, sulfonylurea-treated type 2 diabetes (HbA1c = 8.7 ± 0.2%, n = 20), the latter before and at the end of 12 weeks of treatment with troglitazone (600 mg/day, n = 4), metformin (∼2,000 mg/day, n = 8), or placebo (n = 8). Subjects consumed a standard breakfast at 0800 h. Results in control subjects and type 2 diabetic subjects were compared at baseline. Integrated nocturnal FFA Ra (AUC1:00–8:00 a.m.) was ∼50% higher in type 2 diabetic subjects than in control subjects (29.4 ± 3.0 vs. 19.4 ± 3.9 mmol · m−2 · 7 h−1, respectively, P < 0.05), whereas postprandial palmitate Ra (AUC0–240 min) was almost threefold higher in type 2 diabetic subjects than in control subjects (14.2 ± 1.7 vs. 5.3 ± 1.0 mmol · m−2 · 4 h−1, respectively, P < 0.01). After troglitazone treatment, nocturnal palmitate Ra did not change, but postprandial palmitate Ra decreased by ∼30% (P < 0.05). Palmitate kinetics did not change with metformin or placebo treatment. In summary, nocturnal and postprandial FFA Ra is increased in type 2 diabetes. Postprandial lipolysis appears to be preferentially improved by thiazolidinediones compared with nocturnal lipolysis.
Differential Sensitivity to Central Leptin and Insulin in Male and Female Rats
Authors: Clegg Deborah J. , Riedy Christine A. , Smith Kathleen A. Blake , Benoit Stephen C. , Woods Stephen C.
Published: March 2007

The distribution of fat in the body differs between the male and female sexes and is associated with the relative secretion of the two “adiposity” hormones leptin and insulin. We now report that the brains of male and female rats are differentially sensitive to the catabolic actions of small doses of these two hormones. Leptin (1 or 3.5 μg/2 μl) or saline (2 μl) was administered into the third cerebral ventricle of age- and weight-matched male and female rats. Leptin significantly reduced food intake in female and male rats over 4 h; however, leptin reduced 24-h intake in female but not in male rats. When the same rats were administered insulin (1 or 4 mU/2 μl) or saline (2 μl), male but not female rats had a robust reduction in food intake over 24 h. Previous research demonstrates the melanocortins are a central mediator of the effects of both leptin and insulin. However, we found no sex differences in sensitivity to the melanocortin agonist MTII (0.01, 0.1, 0.3, and 1.0 nmol/2 μl). These results suggest that the sex differences in sensitivity to leptin and insulin at the doses that we injected occur upstream of the melanocortin receptors. Because insulin and leptin reflect different fat beds and are differentially distributed in the male and female sexes, the implication is that the male and female sexes regulate adiposity-relevant parameters differently.
Processing and Presentation of the Islet Autoantigen GAD by Vascular Endothelial Cells Promotes Transmigration of Autoreactive T-Cells
Authors: Greening James E. , Tree Timothy I.M. , Kotowicz Karolena T. , van Halteren Astrid G. , Roep Bart O. , Klein Nigel J. , Peakman Mark
Published: March 2007

Type 1 diabetes is characterized by T-cell infiltration of the islets of Langerhans and abundant HLA class II molecule expression on islet endothelial cells (ECs). The specificity of infiltrating T-cells for islet autoantigens has been amply demonstrated in animal models, and is implicit in human diabetes, but the processes regulating endothelial transmigration of islet autoantigen-specific T-cells into islets are not known. We examined the ability of ECs expressing HLA class II molecules to process and present the islet autoantigen GAD65 and examined the effects of presentation on transmigration of GAD65-specific T-cells. Primary cultures of human vascular ECs expressing the DRB1*0401 (VEC1) and DRB1*0301 (VEC2) genotypes were established and de novo expression of HLA class II molecules induced with interferon-γ. Under these conditions, VEC1 efficiently processed and presented whole GAD65 to the HLA-DR4–restricted murine T-cell hybridoma T33.1 that recognizes the 274-286 epitope of GAD65. Using a transwell system, we examined the effect of GAD65 presentation on migration of GAD65-specific T-cells across EC monolayers. Migration of T33.1 hybridoma cells and of the human T-cell clone, PM1#11 (recognizes GAD65 epitope 339-352 presented by HLA-DR3) across VEC1 and VEC2, respectively, were greatly enhanced in the presence of GAD65, commencing more rapidly and achieving a higher peak migration at 3 h. Migrated PM1#11 cells retained full proliferative capacity. These results support the hypothesis that presentation of autoantigens by islet endothelium in vivo could promote transmigration of circulating islet autoantigen-specific T-cells primed in regional lymph nodes against islet autoantigens.
Neonatal Exendin-4 Prevents the Development of Diabetes in the Intrauterine Growth Retarded Rat
Authors: Stoffers Doris A. , Desai Biva M. , DeLeon Diva D. , Simmons Rebecca A.
Published: March 2007

Uteroplacental insufficiency resulting in fetal growth retardation is a common complication of pregnancy and a significant cause of perinatal morbidity and mortality. Epidemiological studies show an increased incidence of type 2 diabetes in humans who were growth retarded at birth. The mechanisms by which an abnormal intrauterine milieu leads to the development of diabetes in adulthood are not known. Therefore, a rat model of uteroplacental insufficiency was developed; intrauterine growth-retarded (IUGR) rats develop diabetes with a phenotype similar to that observed in the human with type 2 diabetes. We show here that administration of a pancreatic β-cell trophic factor, exendin-4 (Ex-4), during the prediabetic neonatal period dramatically prevents the development of diabetes in this model. This occurs because neonatal Ex-4 prevents the progressive reduction in insulin-producing β-cell mass that is observed in IUGR rats over time. Expression of PDX, a critical regulator of pancreas development and islet differentiation, is restored to normal levels, and islet β-cell proliferation rates are normalized by the neonatal Ex-4 treatment. These results indicate that exposure to Ex-4 in the newborn period reverses the adverse consequences of fetal programming and prevents the development of diabetes in adulthood.
Dipeptidyl Peptidase IV Inhibitor Treatment Stimulates β-Cell Survival and Islet Neogenesis in Streptozotocin-Induced Diabetic Rats
Authors: Pospisilik J. Andrew , Martin Jennifer , Doty Timothy , Ehses Jan A. , Pamir Nathalie , Lynn Francis C. , Piteau Shalea , Demuth Hans-Ulrich , McIntosh Christopher H.S. , Pederson Raymond A.
Published: March 2007

Recent studies into the physiology of the incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) have added stimulation of β-cell growth, differentiation, and cell survival to well-documented, potent insulinotropic effects. Unfortunately, the therapeutic potential of these hormones is limited by their rapid enzymatic inactivation in vivo by dipeptidyl peptidase IV (DP IV). Inhibition of DP IV, so as to enhance circulating incretin levels, has proved effective in the treatment of type 2 diabetes both in humans and in animal models, stimulating improvements in glucose tolerance, insulin sensitivity, and β-cell function. We hypothesized that enhancement of the cytoprotective and β-cell regenerative effects of GIP and GLP-1 might extend the therapeutic potential of DP IV inhibitors to include type 1 diabetes. For testing this hypothesis, male Wistar rats, exposed to a single dose of streptozotocin (STZ; 50 mg/kg), were treated twice daily with the DP IV inhibitor P32/98 for 7 weeks. Relative to STZ-injected controls, P32/98-treated animals displayed increased weight gain (230%) and nutrient intake, decreased fed blood glucose (∼26 vs. ∼20 mmol/l, respectively), and a return of plasma insulin values toward normal (0.07 vs. 0.12 nmol/l, respectively). Marked improvements in oral glucose tolerance, suggesting enhanced insulin secretory capacity, were corroborated by pancreas perfusion and insulin content measurements that revealed two- to eightfold increases in both secretory function and insulin content after 7 weeks of treatment. Immunohistochemical analyses of pancreatic sections showed marked increases in the number of small islets (+35%) and total β-cells (+120%) and in the islet β-cell fraction (12% control vs. 24% treated) in the treated animals, suggesting that DP IV inhibitor treatment enhanced islet neogenesis, β-cell survival, and insulin biosynthesis. In vitro studies using a β-(INS-1) cell line showed a dose-dependent prevention of STZ-induced apoptotic cell-death by both GIP and GLP-1, supporting a role for the incretins in eliciting the in vivo results. These novel findings provide evidence to support the potential utility of DP IV inhibitors in the treatment of type 1 and possibly late-stage type 2 diabetes.
Development and Characterization of a Glucagon-Like Peptide 1-Albumin Conjugate
Authors: Kim Jung-Guk , Baggio Laurie L. , Bridon Dominique P. , Castaigne Jean-Paul , Robitaille Martin F. , Jetté Lucie , Benquet Corinne , Drucker Daniel J.
Published: March 2007

The rapid degradation of native glucagon-like peptide 1 (GLP-1) by dipeptidyl peptidase-IV (DPP-IV) has fostered new approaches for generation of degradation-resistant GLP-1 analogues. We examined the biological activity of CJC-1131, a DPP-IV-resistant drug affinity complex (DAC) GLP-1 compound that conjugates to albumin in vivo. The CJC-1131 albumin conjugate bound to the GLP-1 receptor (GLP-1R) and activated cAMP formation in heterologous fibroblasts expressing a GLP-1R. CJC-1131 lowered glucose in wild-type mice, but not in GLP-1R−/− mice. Basal glucose and glycemic excursion following glucose challenge remained significantly reduced 10–12 h following a single injection of CJC-1131. Twice daily administration of CJC-1131 to db/db mice significantly reduced glycemic excursion following oral and IP glucose challenge (P < 0.01 to 0.05) but did not significantly lower body weight during the 4-week study period. Levels of random fed glucose were significantly lower in CJC-1131-treated +/+ and db/db mice and remained significantly lower even 1 week following discontinuation of CJC-1131 administration. CJC-1131 increased levels of pancreatic proinsulin mRNA transcripts, percent islet area, and the number of bromodeoxyuridine-positive islet cells. These findings demonstrate that an albumin-conjugated DAC:GLP-1 mimics the action of native GLP-1 and represents a new approach for prolonged activation of GLP-1R signaling.
Inflammatory Cytokines and the Risk to Develop Type 2 Diabetes
Authors: Spranger Joachim , Kroke Anja , Möhlig Matthias , Hoffmann Kurt , Bergmann Manuela M. , Ristow Michael , Boeing Heiner , Pfeiffer Andreas F.H.
Published: March 2007

A subclinical inflammatory reaction has been shown to precede the onset of type 2 (non-insulin-dependent) diabetes. We therefore examined prospectively the effects of the central inflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) on the development of type 2 diabetes. We designed a nested case-control study within the prospective population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study including 27,548 individuals. Case subjects were defined to be those who were free of type 2 diabetes at baseline and subsequently developed type 2 diabetes during a 2.3-year follow-up period. A total of 192 cases of incident type 2 diabetes were identified and matched with 384 non-disease-developing control subjects. IL-6 and TNF-α levels were found to be elevated in participants with incident type 2 diabetes, whereas IL-1β plasma levels did not differ between the groups. Analysis of single cytokines revealed IL-6 as an independent predictor of type 2 diabetes after adjustment for age, sex, BMI, waist-to-hip ratio (WHR), sports, smoking status, educational attainment, alcohol consumption, and HbA1c (4th vs. the 1st quartile: odds ratio [OR] 2.6, 95% CI 1.2–5.5). The association between TNF-α and future type 2 diabetes was no longer significant after adjustment for BMI or WHR. Interestingly, combined analysis of the cytokines revealed a significant interaction between IL-1β and IL-6. In the fully adjusted model, participants with detectable levels of IL-1β and elevated levels of IL-6 had an independently increased risk to develop type 2 diabetes (3.3, 1.7–6.8), whereas individuals with increased concentrations of IL-6 but undetectable levels of IL-1β had no significantly increased risk, both compared with the low-level reference group. These results were confirmed in an analysis including only individuals with HbA1c <5.8% at baseline. Our data suggest that the pattern of circulating inflammatory cytokines modifies the risk for type 2 diabetes. In particular, a combined elevation of IL-1β and IL-6, rather than the isolated elevation of IL-6 alone, independently increases the risk of type 2 diabetes. These data strongly support the hypothesis that a subclinical inflammatory reaction has a role in the pathogenesis of type 2 diabetes.
Effect of Reinstitution of Good Glycemic Control on Retinal Oxidative Stress and Nitrative Stress in Diabetic Rats
Authors: Kowluru Renu A.
Published: March 2007

Clinical and experimental studies have shown that reinstitution of good glycemic control (GC) after a period of poor glycemic control (PC) does not produce immediate benefits on the progression of retinopathy, and hyperglycemia is sufficient to initiate the development of diabetic retinopathy. In this study, the effect of reinstitution of GC on hyperglycemia-induced increased oxidative stress and nitrative stress was evaluated in the retina of rats maintained in PC before initiation of GC. In diabetic rats, 2 or 6 months of PC (GHb >11.0%) was followed by 7 months of GC (GHb <5.5%). Reinstitution of GC after 2 months of PC inhibited elevations in retinal lipid peroxides and NO levels by ∼50%, but failed to have any beneficial effects on nitrotyrosine formation. However, reversal of hyperglycemia after 6 months of PC had no significant effect on retinal oxidative stress and NO levels (P < 0.02 vs. normal). In the same rats, inducible nitric oxide synthase expression and nitrotyrosine levels remained elevated by >80% compared with normal rats or rats kept in GC for the duration. This suggests that oxidative and nitrative modifications in retina occur early in the course of development of retinopathy in diabetes. These abnormalities are not easily reversed by reinstitution of GC, and the duration of PC before initiation of GC influences the outcome of the reversal. Characterization of the abnormalities responsible for the resistance of retinopathy to arrest after reinstitution of GC will help identify potential future therapies to inhibit progression of diabetic retinopathy.
Latent Transforming Growth Factor-β Binding Protein-1, a Component of Latent Transforming Growth Factor-β Complex, Accelerates the Migration of Aortic Smooth Muscle Cells in Diabetic Rats Through Integrin-β3
Authors: Kanzaki Tetsuto , Otabe Masako
Published: March 2007

Aortic smooth muscle cells (SMCs) of diabetic animals have unique properties, including the overexpression of transforming growth factor-β (TGF-β) type II receptor, fibronectin, and platelet-derived growth factor β-receptor. TGF-β1 is produced and secreted as latent high–molecular weight complex consisting of mature TGF-β1, latency-associated peptide (LAP), and a latent TGF-β1 binding protein (LTBP-1). LAP has an important function in the latency of TGF-β complex, but the role of LTBP-1 is not known in diabetic angiopathy. SMC migration from the medial layer to the intimal layer of an artery is an initial major process of the formation of intimal thickening of an artery. Migration activities of SMCs from diabetic rat with 1–500 pg/ml of LTBP-1 increased significantly compared with that without LTBP-1. LTBP-1 at 10–500 pg/ml stimulated the migration of diabetic SMCs more than SMCs from control rat. An anti–integrin-β3 antibody reduced LTBP-1–stimulated migration of diabetic SMCs to 51% compared with no antibody, but it did not reduce that of control SMCs. Furthermore, cross-linking experiments show that LTBP-1 binds integrin-β3 in diabetic SMCs much more than in control SMCs in coincidence with the increase of integrin-β3 in diabetic aorta by immunohistochemistry. Taken together, these observations suggest that LTBP-1 plays a critical role in intimal thickening of diabetic artery through the acceleration of SMC migration via integrin-β3.
Characterization of Retinal Leukostasis and Hemodynamics in Insulin Resistance and Diabetes
Authors: Abiko Toru , Abiko Atsuko , Clermont Allen C. , Shoelson Brett , Horio Naoichi , Takahashi Junichi , Adamis Anthony P. , King George L. , Bursell Sven-Erik
Published: March 2007

Increases in leukostasis/monocyte adhesion to the capillary endothelium (leukostasis) and decreases in retinal blood flow may be causally associated and are implicated in the pathogenesis of diabetic retinopathy. In this study, we demonstrate that increases in leukostasis are observed in insulin-resistant states without diabetes, whereas decreases in retinal blood flow require diabetes and hyperglycemia. Microimpaction studies using beads mimicking retinal capillary obstruction by leukocytes did not affect retinal blood flow. In diabetic rats, treatment with the antioxidant α-lipoic acid normalized the amount of leukostasis but not retinal blood flow. In contrast, treatment with d-α-tocopherol and protein kinase-C β-isoform inhibition (LY333531) prevented the increases in leukostasis and decreases in retinal blood flow in diabetic rats. Serum hydroxyperoxide, a marker of oxidative stress, was increased in diabetic rats, but normalized by treatment with antioxidants α-lipoic acid and d-α-tocopherol and, surprisingly, PKC β-isoform inhibition. These findings suggest that leukostasis is associated with endothelial dysfunction, insulin resistance, and oxidative stress but is not related to retinal blood flow and is not sufficient to cause diabetic-like retinopathy. Moreover, treatment with PKC β inhibition is effective to normalize diabetes or hyperglycemia-induced PKC β-isoform activation and oxidative stress.
Role of Protein Kinase C on the Expression of Platelet-Derived Growth Factor and Endothelin-1 in the Retina of Diabetic Rats and Cultured Retinal Capillary Pericytes
Authors: Yokota Tamotsu , Ma Ronald C. , Park Joong-Yeol , Isshiki Keiji , Sotiropoulos Konstantinos B. , Rauniyar Ravi K. , Bornfeldt Karin E. , King George L.
Published: March 2007

Increased expression of endothelin-1 (ET-1) is associated with diabetic retinopathy and vasculopathy, although the molecular explanation has not been defined. The effects of high glucose and protein kinase C (PKC) activation on platelet-derived growth factor (PDGF)-BB and of ET-1 expression in the retina of streptozotocin (STZ)-induced diabetic rats and bovine retinal pericytes (BRPC) were examined. In 4-week diabetic rats, PDGF-B and prepro-ET-1 (ppET-1) mRNA levels increased significantly by 2.8- and 1.9-fold, respectively, as quantified by RT-PCR. Treatment with PKC-β isoform–specific inhibitor (LY333531) or insulin normalized retinal ET-1 and PDGF-B expression. In BRPC, high glucose levels increased ppET-1 and PDGF-B mRNA expression by 1.7- and 1.9-fold, respectively. The addition of PDGF-BB but not PDGF-AA increased expression of ppET-1 and vascular endothelial growth factor mRNA by 1.6- and 2.1-fold, respectively, with both inhibited by AG1296, a selective PDGF receptor kinase inhibitor. A general PKC inhibitor, GF109203X, suppressed PDGF-BB’s induction of ET-1 mRNA. Thus, increased ET-1 expression in diabetic retina could be due to increased expression of PDGF-BB, mediated via PDGF-β receptors in part by PKC activation. The novel demonstration of elevated expression of PDGF-B and its induction by PKC activation identifies a potential new molecular step in the pathogenesis of diabetic retinopathy.
Increased Serum IGF-I During Pregnancy Is Associated With Progression of Diabetic Retinopathy
Authors: Lauszus Finn F. , Klebe Joachim G. , Bek Toke , Flyvbjerg Allan
Published: March 2007

The IGF system has been associated with development and progression of diabetic retinopathy. We examined whether a simple measurement of the IGF system (serum total IGF-I) correlated with progression of diabetic retinopathy in pregnancy in type 1 diabetes. A prospective observational study was performed in 103 pregnant women with type 1 diabetes. Serum IGF-I was measured in maternal serum from week 14, every fourth week until week 30, and every second week until delivery. Twenty-four–hour blood pressure was measured with a portable oscillometry monitor. The women had visual acuity testing and fundus photography before pregnancy, once in each trimester, and 4 months after birth. Each eye was assigned an overall retinopathy grade on a scale from 1 to 6 independently by two experienced graders. During pregnancy, serum IGF-I increased with increasing gestational age until a plateau was reached in week 32. Progression of retinopathy was significantly associated with a higher level of IGF-I (P < 0.01). Serum IGF-I increased with increasing progression of retinopathy. Change of retinopathy was significantly associated with level of IGF-I (P < 0.01). During pregnancy, serum IGF-I increased with increasing birth weight until a plateau was reached in week 32. Birth weight was significantly associated with a higher level of serum IGF-I (P < 0.01).
A Genome-Wide Scan in Families With Maturity-Onset Diabetes of the Young
Authors: Frayling Timothy M. , Lindgren Cecilia M. , Chevre Jean Claude , Menzel Stephan , Wishart Marie , Benmezroua Yamina , Brown Alison , Evans Julie C. , Rao Pamidghantam Subba , Dina Christian , Lecoeur Cécile , Kanninen Timo , Almgren Peter , Bulman Michael P. , Wang Youxiang , Mills James , Wright-Pascoe Rosemarie , Mahtani Melanie M. , Prisco Francesco , Costa Angels , Cognet Ignacio , Hansen Torben , Pedersen Oluf , Ellard Sian , Tuomi Tiinamaija , Groop Leif C. , Froguel Philippe , Hattersley Andrew T. , Vaxillaire Martine
Published: March 2007

Maturity-onset diabetes of the young (MODY) is a heterogeneous single gene disorder characterized by non–insulin-dependent diabetes, an early onset and autosomal dominant inheritance. Mutations in six genes have been shown to cause MODY. Approximately 15–20% of families fitting MODY criteria do not have mutations in any of the known genes. These families provide a rich resource for the identification of new MODY genes. This will potentially enable further dissection of clinical heterogeneity and bring new insights into mechanisms of β-cell dysfunction. To facilitate the identification of novel MODY loci, we combined the results from three genome-wide scans on a total of 23 families fitting MODY criteria. We used both a strict parametric model of inheritance with heterogeneity and a model-free analysis. We did not identify any single novel locus but provided putative evidence for linkage to chromosomes 6 (nonparametric linkage [NPL]score 2.12 at 71 cM) and 10 (NPL score 1.88 at 169–175 cM), and to chromosomes 3 (heterogeneity LOD [HLOD] score 1.27 at 124 cM) and 5 (HLOD score 1.22 at 175 cM) in 14 more strictly defined families. Our results provide evidence for further heterogeneity in MODY.
Human Metabolic Syndrome Resulting From Dominant-Negative Mutations in the Nuclear Receptor Peroxisome Proliferator-Activated Receptor-γ
Authors: Savage David B. , Tan Garry D. , Acerini Carlo L. , Jebb Susan A. , Agostini Maura , Gurnell Mark , Williams Rachel L. , Umpleby A. Margot , Thomas E. Louise , Bell Jimmy D. , Dixon Adrian K. , Dunne Fidelma , Boiani Romina , Cinti Saverio , Vidal-Puig Antonio , Karpe Fredrik , Chatterjee V. Krishna K. , O’Rahilly Stephen
Published: March 2007

We previously reported a syndrome of severe hyperinsulinemia and early-onset hypertension in three patients with dominant-negative mutations in the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR)-γ. We now report the results of further detailed pathophysiological evaluation of these subjects, the identification of affected prepubertal children within one of the original families, and the effects of thiazolidinedione therapy in two subjects. These studies 1) definitively demonstrate the presence of severe peripheral and hepatic insulin resistance in the affected subjects; 2) describe a stereotyped pattern of partial lipodystrophy associated with all the features of the metabolic syndrome and nonalcoholic steatohepatitis; 3) document abnormalities in the in vivo function of remaining adipose tissue, including the inability of subcutaneous abdominal adipose tissue to trap and store free fatty acids postprandially and the presence of very low circulating levels of adiponectin; 4) document the presence of severe hyperinsulinemia in prepubertal carriers of the proline-467-leucine (P467L) PPAR-γ mutation; 5) provide the first direct evidence of cellular resistance to PPAR-γ agonists in mononuclear cells derived from the patients; and 6) report on the metabolic response to thiazolidinedione therapy in two affected subjects. Although the condition is rare, the study of humans with dominant-negative mutations in PPAR-γ can provide important insight into the roles of this nuclear receptor in human metabolism.
Ghrelin Directly Interacts With Neuropeptide-Y-Containing Neurons in the Rat Arcuate Nucleus
Authors: Kohno Daisuke , Gao Hong-Zhi , Muroya Shinji , Kikuyama Sakae , Yada Toshihiko
Published: March 2007

Ghrelin is a newly discovered peptide that is released from the stomach and from neurons in the hypothalamic arcuate nucleus (ARC) and potently stimulates growth hormone release and food intake. Neuropeptide-Y (NPY) neurons in the ARC play an important role in the stimulation of food intake. The present study aimed to determine whether ghrelin directly activates NPY neurons and, if so, to explore its signaling mechanisms. Whether the neurons that respond to ghrelin could be regulated by orexin and leptin was also examined. We isolated single neurons from the ARC of rats and measured the cytosolic Ca2+ concentration ([Ca2+]i) with fura-2 fluorescence imaging. Ghrelin (10−12 to 10−8 mol/l) concentration-dependently increased [Ca2+]i, which occurred in 35% of the ARC neurons. Approximately 80% of these ghrelin-responsive neurons were proved to be NPY-containing by immunocytochemical staining, and 58% of them were glucose-sensitive neurons as judged by their responses to lowering glucose concentrations. The [Ca2+]i responses to ghrelin were markedly attenuated by inhibitors of protein kinase A (PKA) but not protein kinase C and by a blocker of N-type but not L-type Ca2+ channels. Orexin increased [Ca2+]i and leptin attenuated ghrelin-induced [Ca2+]i increases in the majority (80%) of ghrelin-responsive NPY neurons. These results demonstrate that ghrelin directly interacts with NPY neurons in the ARC to induce Ca2+ signaling via PKA and N-type Ca2+ channel-dependent mechanisms. The integration of stimulatory effects of ghrelin and orexin and inhibitory effect of leptin may play an important role in the regulation of the activity of NPY neurons and thereby feeding.
Inhibition of Mitochondrial Na+-Ca2+ Exchanger Increases Mitochondrial Metabolism and Potentiates Glucose-Stimulated Insulin Secretion in Rat Pancreatic Islets
Authors: Lee Bumsup , Miles Philip D. , Vargas Leonardo , Luan Peng , Glasco Susan , Kushnareva Yulia , Kornbrust Elisabeth S. , Grako Kathryn A. , Wollheim Claes B. , Maechler Pierre , Olefsky Jerrold M. , Anderson Christen M.
Published: March 2007

The mitochondrial Na+-Ca2+ exchanger (mNCE) mediates efflux of Ca2+ from mitochondria in exchange for influx of Na+. We show that inhibition of the mNCE enhances mitochondrial oxidative metabolism and increases glucose-stimulated insulin secretion in rat islets and INS-1 cells. The benzothiazepine CGP37157 inhibited mNCE activity in INS-1 cells (50% inhibition at IC50 = 1.5 μmol/l) and increased the glucose-induced rise in mitochondrial Ca2+ ([Ca2+]m) 2.1 times. Cellular ATP content was increased by 13% in INS-1 cells and by 49% in rat islets by CGP37157 (1 μmol/l). Krebs cycle flux was also stimulated by CGP37157 when glucose was present. Insulin secretion was increased in a glucose-dependent manner by CGP37157 in both INS-1 cells and islets. In islets, CGP37157 increased insulin secretion dose dependently (half-maximal efficacy at EC50 = 0.06 μmol/l) at 8 mmol/l glucose and shifted the glucose dose response curve to the left. In perifused islets, mNCE inhibition had no effect on insulin secretion at 2.8 mmol/l glucose but increased insulin secretion by 46% at 11 mmol/l glucose. The effects of CGP37157 could not be attributed to interactions with the plasma membrane sodium calcium exchanger, l-type calcium channels, ATP-sensitive K+ channels, or [Ca2+]m uniporter. In hyperglycemic clamp studies of Wistar rats, CGP37157 increased plasma insulin and C-peptide levels only during the hyperglycemic phase of the study. These results illustrate the potential utility of agents that affect mitochondrial metabolism as novel insulin secretagogues.
Protein Kinase C δ Activation and Translocation to the Nucleus Are Required for Fatty Acid-Induced Apoptosis of Insulin-Secreting Cells
Authors: Eitel Katrin , Staiger Harald , Rieger Johannes , Mischak Harald , Brandhorst Heide , Brendel Mathias D. , Bretzel Reinhard G. , Häring Hans-Ulrich , Kellerer Monika
Published: March 2007

Insulin resistance as well as pancreatic β-cell failure can be induced by elevated free fatty acid (FFA) levels. We studied the mechanisms of FFA-induced apoptosis in rat and human β-cells. Chronic treatment with high physiological levels of saturated fatty acids (palmitate and stearate), but not with monounsaturated (palmitoleate and oleate) or polyunsaturated fatty acids (linoleate), triggers apoptosis in ∼20% of cultured RIN1046-38 cells. Apoptosis restricted to saturated FFAs was also observed in primary cultured human β-cells, suggesting that this mechanism is potentially relevant in vivo in humans. To further analyze FFA-induced signaling pathways leading to apoptosis, we used RIN1046-38 cells. Apoptosis was accompanied by a rapid (within 15 min) nuclear translocation of protein kinase C (PKC)-δ and subsequent lamin B1 disassembly. This translocation was impaired by the phospholipase C inhibitor U-73122, which also substantially reduced apoptosis. Furthermore, lamin B1 disassembly and apoptosis were decreased by cell transfection with a dominant-negative mutant form of PKC-δ. These data suggest that nuclear translocation and kinase activity of PKC-δ are both necessary for saturated fatty acid-induced apoptosis.
Adipose Tissue Is a Major Source of Interleukin-1 Receptor Antagonist
Authors: Juge-Aubry Cristiana E. , Somm Emmanuel , Giusti Vittorio , Pernin Agnès , Chicheportiche Rachel , Verdumo Chantal , Rohner-Jeanrenaud Françoise , Burger Danielle , Dayer Jean-Michel , Meier Christoph A.
Published: March 2007

The secreted form of the interleukin-1 receptor antagonist (IL-1Ra) is an acute-phase protein intervening in the counterregulation of inflammatory processes. We previously showed that this cytokine antagonist is upregulated in the serum of obese patients, correlating with BMI and insulin resistance. In this study, we examined the expression pattern of IL-1Ra and showed that it is highly expressed not only in liver and spleen, but also in white adipose tissue (WAT), where it is upregulated in obesity. In WAT of obese humans, IL-1Ra was also markedly increased. Moreover, human WAT explants secreted IL-1Ra into the medium, a process that could be stimulated fivefold by interferon-β. Finally, lipopolysaccharide administration induced a long-lasting expression of IL-1Ra in mouse WAT, suggesting that adipose tissue is an important source of IL-1Ra in both obesity and inflammation. In summary, we demonstrated that WAT is one of the most important sources of IL-1Ra quantitatively, suggesting that this tissue could represent a novel target for anti-inflammatory treatment. Moreover, it can be speculated that IL-1Ra, whose production is markedly increased in WAT in obese individuals, contributes further to weight gain because of its endocrine and paracrine effects on the hypothalamus and adipocytes, respectively.
Diabetes Antibody Standardization Program: First Assay Proficiency Evaluation
Authors: Bingley Polly J. , Bonifacio Ezio , Mueller Patricia W. ,   Participating Laboratories
Published: March 2007

The aims of the first proficiency evaluation of the Diabetes Antibody Standardization Program (DASP) were to assess general implementation of assay methods and to evaluate the new World Health Organization (WHO) reference reagent for autoantibodies to GAD and IA-2. Forty-six laboratories in 13 countries received coded sera from 50 patients with newly diagnosed type 1 diabetes and 50 blood donor control subjects, together with the WHO reference reagent and diluent serum. Results were analyzed using receiver operator characteristic (ROC) curves. Sensitivity was adjusted to 90% specificity in workshop controls. The median adjusted sensitivity for GADA (45 laboratories) was 84% (range 62–96%), for IA-2A (43 laboratories) was 58% (50–74%), and for insulin autoantibody (IAA; 23 laboratories) was 36% (13–66%). ROC curve analysis showed all GADA and IA-2A assays, and 18/23 IAA assays found significant differences between patients and control subjects. There was good concordance between laboratories in ranking of samples by GADA and IA-2A levels or if results were expressed in relation to the WHO reference reagent. Assays that achieved the highest sensitivity for IAA were also concordant in ranking samples, but overall concordance for IAA was poor. Differences in assay protocols between laboratories must be addressed so that all centers and kit manufacturers can perform to the same high standard.
A Novel Glucose-Sensing Mechanism Contributing to Glucagon-Like Peptide-1 Secretion From the GLUTag Cell Line
Authors: Gribble Fiona M. , Williams Leanne , Simpson Anna K. , Reimann Frank
Published: March 2007

Glucagon-like peptide 1 (GLP-1) secretion from intestinal l-cells is triggered by luminal nutrients. We reported previously that glucose-triggered GLP-1 release from the l-cell model GLUTag involves closure of ATP-sensitive K+ (KATP) channels. We show here that GLP-1 secretion and electrical activity of GLUTag cells is triggered not only by metabolizable sugars (glucose or fructose) but also by the nonmetabolizable monosaccharide methyl-α-glucopyranoside. Responses to glucose and methyl-α-glucopyranoside were impaired by the sodium-glucose cotransporter (SGLT) inhibitor phloridzin. SLGT1 and 3 were detected in GLUTag cells by RT-PCR. Whereas fructose closed KATP channels, methyl-α-glucopyranoside increased the membrane conductance and generated an inward current. Low concentrations of glucose and methyl-α-glucopyranoside also triggered small inward currents and enhanced the action potential frequency. We conclude that whereas low concentrations of metabolizable sugars trigger GLP-1 secretion via KATP channel closure, SGLT substrates generate small inward currents as a result of the electrogenic action of the transporter. This transporter-associated current can trigger electrical activity and secretion when the concentration of substrate is high or when outward currents are reduced by metabolic closure of the KATP channels. Electrogenic sugar entry via SGLTs provides a novel mechanism for glucose sensing by neuroendocrine cells.
NCEP-Defined Metabolic Syndrome, Diabetes, and Prevalence of Coronary Heart Disease Among NHANES III Participants Age 50 Years and Older
Authors: Alexander Charles M. , Landsman Pamela B. , Teutsch Steven M. , Haffner Steven M.
Published: March 2007

Although the individual components of the metabolic syndrome are clearly associated with increased risk for coronary heart disease (CHD), we wanted to quantify the increased prevalence of CHD among people with metabolic syndrome. The Third National Health and Nutrition Examination Survey (NHANES III) was used to categorize adults over 50 years of age by presence of metabolic syndrome (National Cholesterol Education Program [NCEP] definition) with or without diabetes. Demographic and risk factor information was determined for each group, as well as the proportion of each group meeting specific criteria for metabolic syndrome. The prevalence of CHD for each group was then determined. Metabolic syndrome is very common, with ∼44% of the U.S. population over 50 years of age meeting the NCEP criteria. In contrast, diabetes without metabolic syndrome is uncommon (13% of those with diabetes). Older Americans over 50 years of age without metabolic syndrome regardless of diabetes status had the lowest CHD prevalence (8.7% without diabetes, 7.5% with diabetes). Compared with those with metabolic syndrome, people with diabetes without metabolic syndrome did not have an increase in CHD prevalence. Those with metabolic syndrome without diabetes had higher CHD prevalence (13.9%), and those with both metabolic syndrome and diabetes had the highest prevalence of CHD (19.2%) compared with those with neither. Metabolic syndrome was a significant univariate predictor of prevalent CHD (OR 2.07, 95% CI 1.66–2.59). However, blood pressure, HDL cholesterol, and diabetes, but not presence of metabolic syndrome, were significant multivariate predictors of prevalent CHD. The prevalence of CHD markedly increased with the presence of metabolic syndrome. Among people with diabetes, the prevalence of metabolic syndrome was very high, and those with diabetes and metabolic syndrome had the highest prevalence of CHD. Among all individuals with diabetes, prevalence of CHD was increased compared with those with metabolic syndrome without diabetes. However, individuals with diabetes without metabolic syndrome had no greater prevalence of CHD compared with those with neither.