bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of Normal and Mutant Recombinant Human Ketohexokinases and Implications for the Pathogenesis of Essential Fructosuria
Authors: Asipu Aruna , Hayward Bruce E. , O’Reilly John , Bonthron David T.
Published: March 2007

Alternative splicing of the ketohexokinase (fructokinase) gene generates a “central” predominantly hepatic isoform (ketohexokinase-C) and a more widely distributed ketohexokinase-A. Only the abundant hepatic isoform is known to possess activity, and no function is defined for the lower levels of ketohexokinase-A in peripheral tissues. Hepatic ketohexokinase deficiency causes the benign disorder essential fructosuria. The molecular basis of this has been defined in one family (compound heterozygosity for mutations Gly40Arg and Ala43Thr). Here we show that both ketohexokinase isoforms are indeed active. Ketohexokinase-A has much poorer substrate affinity than ketohexokinase-C for fructose but is considerably more thermostable. The Gly40Arg mutation seems null, rendering both ketohexokinase-A and ketohexokinase-C inactive and largely insoluble. The Ala43Thr mutant retains activity, but this mutation decreases the thermal stability of both ketohexokinase-A and ketohexokinase-C. At physiologic temperature, this results in significant loss of ketohexokinase-C activity but not of ketohexokinase-A. Affected individuals who carry both mutations therefore probably have a selective deficiency of hepatic ketohexokinase, with peripheral ketohexokinase-A being preserved. These findings raise the possibility that ketohexokinase-A serves an unknown physiologic function that remains intact in essential fructosuria. Further mutation analysis in this rare disorder could illuminate the question of whether ketohexokinase-A activity is, unlike that of ketohexokinase-C, physiologically indispensable.
Genetic Variation of the GLUT10 Glucose Transporter (SLC2A10) and Relationships to Type 2 Diabetes and Intermediary Traits
Authors: Andersen Gitte , Rose Christian Schack , Hamid Yasmin Hassan , Drivsholm Thomas , Borch-Johnsen Knut , Hansen Torben , Pedersen Oluf
Published: March 2007

The SLC2A10 gene encodes the GLUT10 facilitative glucose transporter, which is expressed in high amounts in liver and pancreas. The gene is mapped to chromosome 20q12-q13.1, a region that has been shown to be linked to type 2 diabetes. The gene was examined in 61 Danish type 2 diabetic patients, and a total of six variants (−27C→T, Ala206Thr, Ala272Ala, IVS2 + 10G→A, IVS4 + 18T→G, and IVS4 + 26G→A) were identified and investigated in an association study, which included 503 type 2 diabetic patients and 510 glucose-tolerant control subjects. None of the variants were associated with type 2 diabetes. Interestingly, carriers of the codon 206 Thr allele had 18% lower fasting serum insulin levels (P = 0.002) and 20% lower insulinogenic index (P = 0.03) than homozygous carriers of the Ala allele. These results suggest that variation in the coding region of SLC2A10 does not contribute substantially to the pathogenesis of type 2 diabetes in the examined study population. However, the codon 206 polymorphism may be related to the interindividual variation in fasting and oral glucose-induced serum insulin levels.
Portal Serotonin Infusion and Glucose Disposal in Conscious Dogs
Authors: Moore Mary Courtney , Geho W. Blair , Lautz Margaret , Farmer Ben , Neal Doss W. , Cherrington Alan D.
Published: March 2007

Whether serotonin (5-hydroxytryptamine [5-HT]) enhances net hepatic glucose uptake (NHGU) during glucose infusion was examined in conscious 42-h-fasted dogs, using arteriovenous difference and tracer ([3-3H]glucose) techniques. Experiments consisted of equilibration (−120 to −30 min), basal (−30 to 0 min), and experimental (0–390 min) periods. During the experimental period, somatostatin, fourfold basal intraportal insulin, basal intraportal glucagon, and peripheral glucose (to double the hepatic glucose load) were infused. In one group of dogs (SER; n = 8), saline was infused intraportally from 0 to 90 min (P1), and 5-HT was infused intraportally at 10, 20, and 40 μg · kg−1 · min−1 from 90 to 150 (P2), 150 to 210 (P3), and 210 to 270 (P4) min, respectively. In the other group (SAL; n = 8), saline was infused intraportally from 0 to 270 min. NHGU in SAL was 12.4 ± 2.3, 14.9 ± 2.7, 13.4 ± 2.1, and 15.1 ± 1.8 μmol · kg−1 · min−1 in P1 to P4, respectively, whereas NHGU in SER averaged 13.2 ± 3.0, 16.4 ± 2.4, 19.0 ± 2.4 (P < 0.05 vs. SAL), and 22.0 ± 2.9 μmol · kg−1 · min−1 (P < 0.05 vs. SAL). Nonhepatic glucose uptake (μmol · kg−1 · min−1) in SAL was 31.7 ± 4.9, 43.9 ± 5.1, 55.1 ± 5.6, and 66.2 ± 8.6 during P1 to P4, respectively, whereas in SER, the corresponding values were 26.1 ± 5.7, 31.6 ± 9.4, 35.1 ± 7.6 (P < 0.05 vs. SAL), and 34.7 ± 7.7 (P < 0.05 vs. SAL). Intraportal 5-HT enhances NHGU but blunts nonhepatic glucose uptake, raising the possibility that hepatic-targeted 5-HT or 5-HT receptor agonists might reduce postprandial hyperglycemia.
Role of β-Cell Prohormone Convertase (PC)1/3 in Processing of Pro-Islet Amyloid Polypeptide
Authors: Marzban Lucy , Trigo-Gonzalez Genny , Zhu Xiaorong , Rhodes Christopher J. , Halban Philippe A. , Steiner Donald F. , Verchere C. Bruce
Published: March 2007

Islet amyloid polypeptide (IAPP) (amylin), the major component of islet amyloid, is produced by cleavage at the COOH- and NH2-termini of its precursor, proIAPP, likely by the β-cell prohormone convertases (PC) 1/3 and PC2. Mice lacking PC2 can process proIAPP at its COOH- but not its NH2-terminal cleavage site, suggesting that PC1/3 is capable of initiating proIAPP cleavage at its COOH-terminus. To determine the precise role of PC1/3 in proIAPP processing, Western blot analysis was performed on islets isolated from mice lacking PC1/3 (PC1/3−/−). These islets contained not only fully processed IAPP as in PC1/3+/+ islets, but also elevated levels of a COOH-terminally unprocessed intermediate form, suggesting impaired processing at the COOH-terminus. Next, GH3 cells that do not normally express proIAPP or detectable levels of PC1/3 or PC2 were cotransduced with adenoviruses expressing rat proIAPP and either PC2 or PC1/3. As expected, in GH3 cells transduced to express only proIAPP, no processing was observed. Coexpression of proIAPP and PC2 resulted in production of mature IAPP, whereas in cells that coexpressed proIAPP and PC1/3 only a 6-kDa intermediate was produced. We conclude that PC1/3 is important for processing of proIAPP at the COOH-terminus, but in its absence, PC2 can initiate complete processing of proIAPP to IAPP by cleaving the precursor at either its NH2- or COOH-terminal cleavage sites.
Ceramide Content Is Increased in Skeletal Muscle From Obese Insulin-Resistant Humans
Authors: Adams John M. , Pratipanawatr Thongchai , Berria Rachele , Wang Elaine , DeFronzo Ralph A. , Sullards M. Cameron , Mandarino Lawrence J.
Published: March 2007

Increased intramyocellular lipid concentrations are thought to play a role in insulin resistance, but the precise nature of the lipid species that produce insulin resistance in human muscle are unknown. Ceramides, either generated via activation of sphingomyelinase or produced by de novo synthesis, induce insulin resistance in cultured cells by inhibitory effects on insulin signaling. The present study was undertaken to determine whether ceramides or other sphingolipids are increased in muscle from obese insulin-resistant subjects and to assess whether ceramide plays a role in the insulin resistance of Akt in human muscle. Lean insulin-sensitive and obese insulin-resistant subjects (n = 10 each) received euglycemic-hyperinsulinemic clamps with muscle biopsies basally and after 30, 45, or 60 min of insulin infusion. The rate of glucose infusion required to maintain euglycemia (reflecting glucose uptake) was reduced by >50%, as expected, in the obese subjects at each time point (P < 0.01). Under basal conditions, total muscle ceramide content was increased nearly twofold in the obese subjects (46 ± 9 vs. 25 ± 2 pmol/2 mg muscle, P < 0.05). All species of ceramides were increased similarly in the obese subjects; in contrast, no other sphingolipid was increased. Stimulation of Akt phosphorylation by insulin in the obese subjects was significantly reduced after 30 min (0.96 ± 0.11 vs. 1.84 ± 0.38 arbitrary units) or 45–60 min (0.68 ± 0.17 vs. 1.52 ± 0.26) of insulin infusion (P < 0.05 for both). Muscle ceramide content was significantly correlated with the plasma free fatty acid concentration (r = 0.51, P < 0.05). We conclude that obesity is associated with increased intramyocellular ceramide content. This twofold increase in ceramide may be involved in the decrease in Akt phosphorylation observed after insulin infusion and could theoretically play a role in the reduced ability of insulin to stimulate glucose uptake in skeletal muscle from obese subjects.
Islet-Sparing Effects of Protein Tyrosine Phosphatase-1b Deficiency Delays Onset of Diabetes in IRS2 Knockout Mice
Authors: Kushner Jake A. , Haj Fawaz G. , Klaman Lori D. , Dow Matthew A. , Kahn Barbara B. , Neel Benjamin G. , White Morris F.
Published: March 2007

Protein tyrosine phosphatase-1b (Ptp1b) inhibits insulin and leptin signaling by dephosphorylating specific tyrosine residues in their activated receptor complexes. Insulin signals are mediated by tyrosine phosphorylation of the insulin receptor and its downstream targets, such as Irs1 and Irs2. Irs2 plays an especially important role in glucose homeostasis because it mediates some peripheral actions of insulin and promotes pancreatic β-cell function. To determine whether the deletion of Ptp1b compensates for the absence of Irs2, we analyzed mice deficient in both Ptp1b and Irs2. Pancreatic β-cell area decreased in Ptp1b−/− mice, consistent with decreased insulin requirements owing to increased peripheral insulin sensitivity. By contrast, peripheral insulin sensitivity and β-cell area increased in Irs2−/−::Ptp1b−/− mice, which improved glucose tolerance in Irs2−/−::Ptp1b−/− mice and delayed diabetes until 3 months of age. However, β-cell function eventually failed to compensate for absence of Irs2. Our studies demonstrate a novel role for Ptp1b in regulating β-cell homeostasis and indicate that Ptp1b deficiency can partially compensate for lack of Irs2.
Intracerebroventricular Neuropeptide Y Infusion Precludes Inhibition of Glucose and VLDL Production by Insulin
Authors: van den Hoek Anita M. , Voshol Peter J. , Karnekamp Barbara N. , Buijs Ruud M. , Romijn Johannes A. , Havekes Louis M. , Pijl Hanno
Published: March 2007

Recent evidence demonstrates that hypothalamic insulin signaling is required for inhibition of endogenous glucose production. The downstream mechanisms that are responsible for the effects of hypothalamic insulin receptor activation on hepatic fuel flux remain to be determined. To establish whether downregulation of neuropeptide Y (NPY) release by insulin is mandatory for its capacity to suppress glucose production, we examined the effects of a continuous intracerebroventricular (ICV) infusion of NPY (10 μg/h for 3–5 h) on glucose flux during a hyperinsulinemic-euglycemic clamp in mice. We also evaluated the effects of ICV NPY administration on free fatty acid and glycerol flux and VLDL production in this experimental context. In basal conditions, none of the metabolic parameters was affected by NPY infusion. In hyperinsulinemic conditions, peripheral glucose disposal was not different between vehicle- and NPY-infused animals. In contrast, hyperinsulinemia suppressed endogenous glucose production by ∼8% vs. 30% in NPY- vs. vehicle-infused mice, respectively (P < 0.05). Also, VLDL production was significantly higher during hyperinsulinemia in NPY- compared with vehicle-infused mice (97.5 ± 18.0 vs. 54.7 ± 14.9 μmol · kg−1 · h−1; P < 0.01). These data suggest that the neurophysiological action of insulin to downregulate hypothalamic NPY release is a prerequisite for its ability to suppress hepatic fuel production, whereas it is not mandatory for its capacity to modulate glucose disposal or lipolysis.
Evidence Against the Involvement of Oxidative Stress in Fatty Acid Inhibition of Insulin Secretion
Authors: Moore Patrick C. , Ugas Marco A. , Hagman Derek K. , Parazzoli Susan D. , Poitout Vincent
Published: March 2007

Prolonged exposure to elevated levels of fatty acids adversely affects pancreatic β-cell function. Here we investigated 1) whether ceramide synthesis, which we reported to mediate fatty acid inhibition of insulin gene expression, also inhibits insulin secretion and 2) whether fatty acid inhibition of insulin secretion involves the generation of reactive oxygen species (ROS), nitric oxide (NO), or prostaglandin E2 (PGE2). A 72-h culture of islets in the presence of palmitate or oleate resulted in a marked decrease in glucose-induced insulin release assessed in 1-h static incubations. This effect was reproduced by exogenous diacylglycerol, but not by a cell-permeable analog of ceramide. Culture in the presence of fatty acids was not associated with an increase in intracellular peroxide or NO levels, neither was insulin secretion restored by antioxidants or an inhibitor of NO production. Exposure to fatty acids led to an increase in PGE2 release, but an inhibitor of cyclooxygenase 2 was unable to prevent fatty acid inhibition of insulin secretion. These results indicate that fatty acid inhibition of insulin secretion 1) is not mediated by de novo ceramide synthesis, ROS, NO, or PGE2, and 2) is likely to be caused by the generation of signals or metabolites downstream of diacylglycerol.
Alterations in Plasma Vitamin E Distribution in Type 2 Diabetic Patients With Elevated Plasma Phospholipid Transfer Protein Activity
Authors: Schneider Martina , Vergès Bruno , Klein Alexis , Miller Elizabeth R. , Deckert Valérie , Desrumaux Catherine , Masson David , Gambert Philippe , Brun Jean-Marcel , Fruchart-Najib Jamila , Blache Denis , Witztum Joseph L. , Lagrost Laurent
Published: March 2007

Mouse studies indicated that plasma phospholipid transfer protein (PLTP) determines the plasma distribution of vitamin E, a potent lipophilic antioxidant. Vitamin E distribution, antioxidant status, and titer of anti–oxidized LDLs (oxLDL) autoantibodies were evaluated in plasma from control subjects (n = 31) and type 2 diabetic patients (n = 31) with elevated plasma PLTP concentration. Unlike diabetic and control HDLs, which displayed similar vitamin E contents, diabetic VLDLs and diabetic LDLs contained fewer vitamin E molecules than normal counterparts. Plasma PLTP concentration in diabetic plasmas correlated negatively with vitamin E in VLDL+LDL, but positively with vitamin E in HDL, with an even stronger correlation with the VLDL+LDL–to–HDL vitamin E ratio. Circulating levels of oxLDL were significantly higher in diabetic plasmas than in control plasmas. Whereas the titer of IgG autoantibodies to modified LDL did not differ significantly between diabetic patients and control subjects, diabetic plasmas showed significantly lower levels of potentially protective IgM autoantibodies. The present observations support a pathophysiological role of PLTP in decreasing the vitamin E content of apolipoprotein B–containing lipoproteins, but not of HDL in plasma of type 2 diabetic patients, contributing to a greater potential for LDL oxidation.
Genome-wide Scan for Metabolic Syndrome and Related Quantitative Traits in Hong Kong Chinese and Confirmation of a Susceptibility Locus on Chromosome 1q21-q25
Authors: Ng Maggie C.Y. , So Wing-Yee , Lam Vincent K.L. , Cockram Clive S. , Bell Graeme I. , Cox Nancy J. , Chan Juliana C.N.
Published: March 2007

We conducted autosomal genome scans to map loci for metabolic syndrome (MES) and related traits in the Hong Kong Family Diabetes Study. We selected 55 families with 137 affected members (121 affected relative pairs) for nonparametric linkage analysis on MES. We also selected 179 families with 897 members (2,127 relative pairs) for variance component-based linkage analyses on seven MES-related traits: waist circumference, systolic and diastolic blood pressure (BP), triglyceride, HDL cholesterol, fasting plasma glucose, and insulin resistance index (insulin resistance index by homeostasis model assessment [HOMA%IR]). Analyses revealed three regions that showed suggestive linkage for MES and also showed overlapping signals for metabolic traits: chromosome 1 at 169.5–181.5 cM (logarithm of odds [LOD] = 4.50 for MES, 3.71 for waist circumference, and 1.24 for diastolic BP), chromosome 2 at 44.1–57.3 cM (LOD = 2.22 for MES, 2.07 for fasting plasma glucose, and 1.29 for diastolic BP), and chromosome 16 at 45.2–65.4 cM (LOD = 1.75 for MES, 1.61 for HOMA%IR, and 1.25 for HDL cholesterol). Other regions that showed suggestive linkages included chromosome 5q for diastolic BP; 2q, 3q, 6q, 9q, 10q, and 17q for triglyceride; 12p, 12q, and 22q for HDL-C; and 6q for HOMA%IR. Simulation studies demonstrated genome-wide significant linkage of the chromosome 1 region to both MES and waist circumference (Pgenome-wide = 0.002 and 0.019, respectively). In summary, we have found a susceptibility locus on chromosome 1q21-q25 involved in the pathogenesis of multiple metabolic abnormalities, in particular obesity. Our results confirm the findings of previous studies on diabetes and related phenotypes. We also suggest the locations of other loci that may contribute to the development of MES in Hong Kong Chinese.
Permanent Neonatal Diabetes due to Mutations in KCNJ11 Encoding Kir6.2
Authors: Sagen Jørn V. , Ræder Helge , Hathout Eba , Shehadeh Naim , Gudmundsson Kolbeinn , Bævre Halvor , Abuelo Dianne , Phornphutkul Chanika , Molnes Janne , Bell Graeme I. , Gloyn Anna L. , Hattersley Andrew T. , Molven Anders , Søvik Oddmund , Njølstad Pål R.
Published: March 2007

Permanent neonatal diabetes (PND) can be caused by mutations in the transcription factors insulin promoter factor (IPF)-1, eukaryotic translation initiation factor-2α kinase 3 (EIF2AK3), and forkhead box-P3 and in key components of insulin secretion: glucokinase (GCK) and the ATP-sensitive K+ channel subunit Kir6.2. We sequenced the gene encoding Kir6.2 (KCNJ11) in 11 probands with GCK-negative PND. Heterozygous mutations were identified in seven probands, causing three novel (F35V, Y330C, and F333I) and two known (V59M and R201H) Kir6.2 amino acid substitutions. Only two probands had a family history of diabetes. Subjects with the V59M mutation had neurological features including motor delay. Three mutation carriers tested had an insulin secretory response to tolbutamide, but not to glucose or glucagon. Glibenclamide was introduced in increasing doses to investigate whether sulfonylurea could replace insulin. At a glibenclamide dose of 0.3–0.4 mg · kg−1 · day−1, insulin was discontinued. Blood glucose did not deteriorate, and HbA1c was stable or fell during 2–6 months of follow-up. An oral glucose tolerance test performed in one subject revealed that glucose-stimulated insulin release was restored. Mutations in Kir6.2 were the most frequent cause of PND in our cohort. Apparently insulin-dependent patients with mutations in Kir6.2 may be managed on an oral sulfonylurea with sustained metabolic control rather than insulin injections, illustrating the principle of pharmacogenetics applied in diabetes treatment.
Molecular Mechanisms of Insulin Resistance That Impact Cardiovascular Biology
Authors: Wang Cecilia C. Low , Goalstone Marc L. , Draznin Boris
Published: March 2007

Insulin resistance is concomitant with type 2 diabetes, obesity, hypertension, and other features of the metabolic syndrome. Because insulin resistance is associated with cardiovascular disease, both scientists and physicians have taken great interest in this disorder. Insulin resistance is associated with compensatory hyperinsulinemia, but individual contributions of either of these two conditions remain incompletely understood and a subject of intense investigation. One possibility is that in an attempt to overcome the inhibition within the metabolic insulin-signaling pathway, hyperinsulinemia may continue to stimulate the mitogenic insulin-signaling pathway, thus exerting its detrimental influence. Here we discuss some of the effects of insulin resistance and mechanisms of potentially detrimental influence of hyperinsulinemia in the presence of metabolic insulin resistance.
Ciliary Neurotrophic FactorAx15 Alters Energy Homeostasis, Decreases Body Weight, and Improves Metabolic Control in Diet-Induced Obese and UCP1-DTA Mice
Authors: Blüher Susann , Moschos Stergios , Bullen John , Kokkotou Efi , Maratos-Flier Eleftheria , Wiegand Stanley J. , Sleeman Mark W. , Mantzoros Christos S.
Published: March 2007

Ciliary neurotrophic factor (CNTF) potently reduces appetite and body weight in rodents and humans. We studied the short- and long-term effects of CNTFAx15, a second-generation CNTF analog, in diet-induced obese C57BL/6J mice and brown adipose tissue (BAT)-deficient obese UCP1-DTA (uncoupling protein 1–diphtheria toxin A) mice. CNTFAx15 administration (0.1, 0.3, or 1.0 μg · g−1 · day−1 s.c.) for 3 or 7 days reduced food intake and body weight (mainly body fat mass). The effect of CNTFAx15 on food intake and body weight was more pronounced in CNTFAx15-treated diet-induced obese C57BL/6J mice compared with pair-fed controls and was associated with suppressed expression of hypothalamic neuropeptide Y and agouti gene–related protein. Moreover, CNTFAx15 increased uncoupling protein 1 mRNA expression in BAT and energy expenditure in diet-induced obese C57BL/6J mice. Longitudinal observations revealed a sustained reduction in body weight for several days post-CNTFAx15 treatment of CNTFAx15-treated but not pair-fed mice, followed by a gradual regain in body weight over 28 days. Finally, CNTFAx15 administration improved the metabolic profile in both diet-induced obese C57BL/6J and UCP1-DTA mice and resulted in a significantly improved glycemic response to oral glucose tolerance tests in CNTFAx15-treated UCP1-DTA compared with pair-fed mice of similar body weight. These data suggest that CNTFAx15 may act through a pathway downstream of the putative point responsible for leptin resistance in diet-induced obese C57BL/6J and UCP1-DTA mice to alter food intake, body weight, body composition, and metabolism. CNTFAx15 has delayed and persistent effects in diet-induced obese C57BL/6J mice, which account for a reduction in body weight over and above what would be expected based on decreased foot intake alone.
Elevated Alanine Aminotransferase Predicts New-Onset Type 2 Diabetes Independently of Classical Risk Factors, Metabolic Syndrome, and C-Reactive Protein in the West of Scotland Coronary Prevention Study
Authors: Sattar Naveed , Scherbakova Olga , Ford Ian , O’Reilly Denis St. J. , Stanley Adrian , Forrest Ewan , MacFarlane Peter W. , Packard Chris J. , Cobbe Stuart M. , Shepherd James
Published: March 2007

We examined the association of serum alanine aminotransferase (ALT) with features of the metabolic syndrome and whether it predicted incident diabetes independently of routinely measured factors in 5,974 men in the West of Scotland Coronary Prevention Study. A total of 139 men developed new diabetes over 4.9 years of follow-up. ALT, but not aspartate aminotransferase, levels increased progressively with the increasing number of metabolic syndrome abnormalities from (means ± SD) 20.9 ± 7.6 units/l in those with none to 28.1 ± 10.1 units/l in those with four or more (P < 0.001). In a univariate analysis, men with ALT in the top quartile (ALT ≥29 units/l) had an elevated risk for diabetes (hazard ratio 3.38 [95% CI 1.99–5.73]) versus those in the bottom quartile (<17 units/l). ALT remained a predictor with adjustment for age, BMI, triglycerides, HDL cholesterol, systolic blood pressure, glucose, and alcohol intake (2.04 [1.16–3.58] for the fourth versus first quartile). In stepwise regression, incorporating ALT and C-reactive protein (CRP) together with metabolic syndrome criteria, elevated ALT (≥29 units/l), and CRP (≥3 mg/l) predicted incident diabetes, but low HDL cholesterol and hypertension did not. Thus, elevated ALT levels within the “normal” range predict incident diabetes. The simplicity of ALT measurement and its availability in routine clinical practice suggest that this enzyme activity could be included in future diabetes prediction algorithms.
Activation of Nulcear Factor-κB by Hyperglycemia in Vascular Smooth Muscle Cells Is Regulated by Aldose Reductase
Authors: Ramana Kota V. , Friedrich Brian , Srivastava Sanjay , Bhatnagar Aruni , Srivastava Satish K.
Published: March 2007

Activation of the polyol pathway has been linked to the development of secondary diabetic complications. However, the underlying molecular mechanisms remain unclear. To probe the contribution of this pathway, we examined whether inhibition of aldose reductase, which catalyzes the first step of the pathway, affects hyperglycemia-induced activation of the inflammatory transcription factor nuclear factor (NF)-κB. Treatment of vascular smooth muscle cells with the aldose reductase inhibitors tolrestat and sorbinil prevented high-glucose–induced protein kinase C (PKC) activation, nuclear translocation of NF-κB, phosphorylation of IKK, and the increase in the expression of intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and aldose reductase. High-glucose–induced NF-κB activation was also prevented by the PKC inhibitors chelerythrine and calphostin C. Ablation of aldose reductase by small interference RNA (siRNA) prevented high-glucose–induced NF-κB and AP-1 activation but did not affect the activity of SP-1 or OCT-1. Stimulation with iso-osmotic mannitol activated NF-κB and increased the expression of aldose reductase but not ICAM-1 and VCAM-1. Treatment with aldose reductase inhibitors or aldose reductase siRNA did not affect mannitol-induced NF-κB or AP-1 activation. Administration of tolrestat (15 mg · kg−1 · day−1) decreased the abundance of activated NF-κB in balloon-injured carotid arteries of diabetic rats. Collectively, these results suggest that inhibition of aldose reductase, which prevents PKC-dependent nonosmotic NF-κB activation, may be a useful approach for treating vascular inflammation caused by diabetes.
Attenuation of Extracellular Matrix Accumulation in Diabetic Nephropathy by the Advanced Glycation End Product Cross-Link Breaker ALT-711 via a Protein Kinase C-α−Dependent Pathway
Authors: Thallas-Bonke Vicki , Lindschau Carsten , Rizkalla Bishoy , Bach Leon A. , Boner Geoffrey , Meier Matthias , Haller Hermann , Cooper Mark E. , Forbes Josephine M.
Published: March 2007

This study investigated the role of advanced glycation end products (AGEs) in mediating protein kinase C (PKC) isoform expression in diabetic nephropathy. In vitro, vascular smooth muscle cells incubated in a high-glucose (25-mmol/l) medium demonstrated translocation and increased expression of PKC-α as compared with those from a low-glucose (5-mmol/l) environment. Coincubation with the cross-link breaker ALT-711 and, to a lesser extent, with aminoguanidine, an inhibitor of AGE formation, attenuated the increased expression and translocation of PKC-α. Streptozotocin-induced diabetic rats were randomized to no treatment, treatment with ALT-711, or treatment with aminoguanidine. Diabetes induced increases in PKC-α as well as in the -βI, -βII, and -ε isoforms. Treatment with ALT-711 and aminoguanidine, which both attenuate renal AGE accumulation, abrogated these increases in PKC expression. However, translocation of phosphorylated PKC-α from the cytoplasm to the membrane was reduced only by ALT-711. ALT-711 treatment attenuated expression of vascular endothelial growth factor and the extracellular matrix proteins, fibronectin and laminin, in association with reduced albuminuria. Aminoguanidine had no effect on VEGF expression, although some reduction of fibronectin and laminin was observed. These findings implicate AGEs as important stimuli for the activation of PKC, particularly PKC-α, in the diabetic kidney, which can be directly inhibited by ALT-711.
Poly(ADP-Ribose) Polymerase Is Involved in the Development of Diabetic Retinopathy via Regulation of Nuclear Factor-κB
Authors: Zheng Ling , Szabó Csaba , Kern Timothy S.
Published: March 2007

The current study investigated the role of poly(ADP-ribose) polymerase (PARP) in the development of diabetic retinopathy. Activity of PARP was increased in whole retina and in endothelial cells and pericytes of diabetic rats. Administration of PJ-34 (a potent PARP inhibitor) for 9 months to diabetic rats significantly inhibited the diabetes-induced death of retinal microvascular cells and the development of early lesions of diabetic retinopathy, including acellular capillaries and pericyte ghosts. To further investigate how PARP activation leads to cell death in diabetes, we investigated the possibility that PARP acts as a coactivator of nuclear factor-κB (NF-κB) in the retinal cells. In bovine retinal endothelial cells (BRECs), PARP interacted directly with both subunits of NF-κB (p50 and p65). More PARP was complexed to the p50 subunit in elevated glucose concentration (25 mmol/l) than at 5 mmol/l glucose. PJ-34 blocked the hyperglycemia-induced increase in NF-κB activation in BRECs. PJ-34 also inhibited diabetes-induced increase expression of intercellular adhesion molecule-1, a product of NF-κB–dependent transcription in retina, and subsequent leukostasis. Inhibition of PARP or NF-κB inhibited the hyperglycemia (25 mmol/l glucose)-induced cell death in retinal endothelial cells. Thus, PARP activation plays an important role in the diabetes-induced death of retinal capillary cells, at least in part via its regulation of NF-κB.
Association of Glomerulopathy With the 5′-End Polymorphism of the Aldose Reductase Gene and Renal Insufficiency in Type 2 Diabetic Patients
Authors: Zhao Hai-Lu , Tong Peter C.Y. , Lai Fernand M.M. , Tomlinson Brian , Chan Juliana C.N.
Published: March 2007

The expression of nephropathy in type 2 diabetes has several levels of abnormalities. To define the primary abnormalities of diabetic nephropathy, we conducted an autopsy study of 186 consecutive patients with type 2 diabetes to determine correlations among the aldose reductase gene, renal histopathologies, extracellular matrix, glomerular function, and clinical characteristics. Compared with cases of near-normal renal structure (n = 51) and atypical diabetic glomerulopathy (n = 75), patients with classic diabetic glomerulopathy (n = 60) had advanced glomerular disease, as reflected by elevated plasma creatinine levels (133.2 ± 59.8 vs. 166.0 ± 65.7 vs. 243.8 ± 82.6 μmol/l; P < 0.001), glomerular matrix fractions (20.8 ± 6.7 vs. 33.5 ± 16.8 vs. 39.2 ± 14.3%; P < 0.001), and risk of renal failure (odds ratio [OR] 1 vs. 3.5 vs. 21.4; P < 0.001). Compared with noncarriers of the aldose reductase z-2 allele (n = 92) and z-2 heterozygotes (n = 77), z-2 homozygotes (n = 17) had elevated plasma creatinine (164.1 ± 73.7 vs. 190.6 ± 60.9 vs. 241.1 ± 86.2 μmol/l; P < 0.001) and an increased risk of classic diabetic glomerulopathy (OR 1 vs. 0.9 vs. 3.3; P = 0.026). Overexpression of transforming growth factor-β1, mesangial cell transdifferentiation by expression of α-smooth muscle actin, and aberrant deposition of collagen type IV, fibronectin, and laminin were found in classic diabetic glomerulopathy. These data suggest genetic, biochemical, pathophysiological, and clinical correlations among the aldose reductase gene, extracellular matrix, classic diabetic glomerulopathy, and renal insufficiency. Gene mutation, cellular transdifferentiation, growth factor upregulation, extracellular matrix expansion, and glomerular filtration impairment are the primary abnormalities in type 2 diabetic patients with nephropathy.
Activating Mutations in the KCNJ11 Gene Encoding the ATP-Sensitive K+ Channel Subunit Kir6.2 Are Rare in Clinically Defined Type 1 Diabetes Diagnosed Before 2 Years
Authors: Edghill Emma L. , Gloyn Anna L. , Gillespie Kathleen M. , Lambert A. Paul , Raymond Neil T. , Swift Peter G. , Ellard Sian , Gale Edwin A.M. , Hattersley Andrew T.
Published: March 2007

We have recently shown that permanent neonatal diabetes can be caused by activating mutations in KCNJ11 that encode the Kir6.2 subunit of the β-cell ATP-sensitive K+ channel. Some of these patients were diagnosed after 3 months of age and presented with ketoacidosis and marked hyperglycemia, which could have been diagnosed as type 1 diabetes. We hypothesized that KCNJ11 mutations could present clinically as type 1 diabetes. We screened the KCNJ11 gene for mutations in 77 U.K. type 1 diabetic subjects diagnosed under the age of 2 years. One patient was found to be heterozygous for the missense mutation R201C. She had low birth weight, was diagnosed at 5 weeks, and did not have a high risk predisposing HLA genotype. A novel variant, R176C, was identified in one diabetic subject but did not cosegregate with diabetes within the family. In conclusion, we have shown that heterozygous activating mutations in the KCNJ11 gene are a rare cause of clinically defined type 1 diabetes diagnosed before 2 years. Although activating KCNJ11 mutations are rare in patients diagnosed with type 1 diabetes, the identification of a KCNJ11 mutation may have important treatment implications.
G-Protein Signaling Participates in the Development of Diabetic Cardiomyopathy
Authors: Harris Ian S. , Treskov Ilya , Rowley Michael W. , Heximer Scott , Kaltenbronn Kevin , Finck Brian N. , Gross Richard W. , Kelly Daniel P. , Blumer Kendall J. , Muslin Anthony J.
Published: March 2007

Diabetic patients develop a cardiomyopathy that consists of ventricular hypertrophy and diastolic dysfunction. Although the pathogenesis of this condition is poorly understood, previous studies implicated abnormal G-protein activation. In this work, mice with cardiac overexpression of the transcription factor peroxisome proliferator–activated receptor-α (PPAR-α) were examined as a model of diabetic cardiomyopathy. PPAR-α transgenic mice develop spontaneous cardiac hypertrophy, contractile dysfunction, and “fetal” gene induction. We examined the role of abnormal G-protein activation in the pathogenesis of cardiac dysfunction by crossing PPAR-α mice with transgenic mice with cardiac-specific overexpression of regulator of G-protein signaling subtype 4 (RGS4), a GTPase activating protein for Gq and Gi. Generation of compound transgenic mice demonstrated that cardiac RGS4 overexpression ameliorated the cardiomyopathic phenotype that occurred as a result of PPAR-α overexpression without affecting the metabolic abnormalities seen in these hearts. Next, transgenic mice with increased or decreased cardiac Gq signaling were made diabetic by injection with streptozotocin (STZ). RGS4 transgenic mice were resistant to STZ-induced cardiac fetal gene induction. Transgenic mice with cardiac-specific expression of mutant Gαq, Gαq-G188S, that is resistant to RGS protein action were sensitized to the development of STZ-induced cardiac fetal gene induction and bradycardia. These results establish that Gq-mediated signaling plays a critical role in the pathogenesis of diabetic cardiomyopathy.
Pancreatic Polypeptide Is Involved in the Regulation of Body Weight in Pima Indian Male Subjects
Authors: Koska Juraj , DelParigi Angelo , de Courten Barbora , Weyer Christian , Tataranni P. Antonio
Published: March 2007

Pancreatic polypeptide (PP) is released from the pancreas in response to a meal. In humans, low-circulating PP levels have been observed in obesity, and administration of pharmacological doses of PP has been shown to decrease food intake. The aim of the present study was to investigate whether low circulating PP is associated with weight gain in Pima Indians. Plasma PP concentrations were measured after an overnight fast and 30 min after a standardized mixed meal in 33 nondiabetic male subjects who had a follow-up visit 4.9 ± 2.5 years later. Cross-sectionally, fasting and postprandial PP levels were negatively associated with body size and adiposity. Prospectively, the change in PP response to the meal was negatively associated with the change in body weight (r = −0.53, P = 0.002). In contrast, a high fasting PP level was positively associated with change in body weight (r = 0.45, P = 0.009). In conclusion, our results provide evidence that, even within the physiological range, PP contributes to the regulation of energy balance in humans. However this contribution appears to be more complex than anticipated because of the opposite effect of fasting and postprandial PP on the risk of future weight gain.
Islet Graft Assessment in the Edmonton Protocol
Authors: Street Cale N. , Lakey Jonathan R.T. , Shapiro A.M. James , Imes Sharleen , Rajotte Ray V. , Ryan Edmond A. , Lyon James G. , Kin Tatsuya , Avila Jose , Tsujimura Toshiaki , Korbutt Gregory S.
Published: March 2007

The success of the Edmonton Protocol for islet transplantation has provided new hope in the treatment of type 1 diabetes. This study reports on the assessment of 83 human islet grafts transplanted using the Edmonton Protocol since 1999. Cellular composition, as assessed by immunohistochemistry, showed a lower islet purity (∼40%) than has been reported in previous studies using dithizone staining to quantitate islet equivalents. Furthermore, grafts were found to contain substantial populations of exocrine and ductal tissue. Total cellular insulin transplanted was 8,097.6 ± 3,164.4 μg/patient, and was significantly lower in bottom gradient layer grafts than top gradient layer or whole/combined grafts (P < 0.0005). A static incubation test for islet function gave a stimulation index of 3–4, although this measure did not correlate with posttransplant metabolic outcome. Furthermore, we confirmed a previously reported trend in which donor age affects islet yield and purity. It is important to note that a significant positive correlation was observed between the number of islet progenitor (ductal-epithelial) cells transplanted and long-term metabolic success as assessed an by intravenous glucose tolerance test at ∼2 years posttransplant. In summary, careful assessment of islet graft composition is needed in a clinical transplantation program to accurately estimate islet purity and assess the contribution of other cell types present, such as islet progenitor cells.
Palmitate-Induced Interleukin-6 Expression in Human Coronary Artery Endothelial Cells
Authors: Staiger Harald , Staiger Katrin , Stefan Norbert , Wahl Hans Günther , Machicao Fausto , Kellerer Monika , Häring Hans-Ulrich
Published: March 2007

Obesity-linked insulin resistance is associated with chronic inflammation and cardiovascular complications. Free fatty acids (FFAs) are prominent candidates for the molecular link between these disorders. In this study, we determined whether FFAs contribute to vascular inflammation via induction of interleukin (IL)-6 in coronary artery endothelial cells (CAECs) and coronary artery smooth muscle cells (CASMCs) and whether this is reflected in vivo. In contrast to our findings regarding IL-6 and gp130 (the glycoprotein of 130 kDa) expression, IL-6 receptor mRNA expression was very low in these cells. Palmitate, but not linoleate, induced a significant increase in IL-6 mRNA expression in CAECs (P < 0.001) and, to a less relevant extent, in CASMCs (P < 0.01). gp130 remained unaffected. As to potency, palmitate was comparable with the IL-6−inducer IL-1β. To substantiate our in vitro data, we examined the plasma FFA pattern in 54 healthy human subjects and studied the relation of individual FFAs with plasma IL-6. IL-6 levels correlated with palmitate, but not with other abundant FFAs, even after adjusting for body fat (r = 0.33, P < 0.05) and total FFAs (r = 0.29, P < 0.05). We show here that the common plasma FFA palmitate induces high levels of IL-6 in CAECs. Furthermore, palmitate correlates with IL-6 in vivo. This points to a potential contribution of palmitate to vascular inflammation.
Hyperglycemia Regulates Hypoxia-Inducible Factor-1α Protein Stability and Function
Authors: Catrina Sergiu-Bogdan , Okamoto Kensaku , Pereira Teresa , Brismar Kerstin , Poellinger Lorenz
Published: March 2007

Hyperglycemia and hypoxia are suggested to play essential pathophysiological roles in the complications of diabetes, which may result from a defective response of the tissues to low oxygen tension. In this study, we show that in primary dermal fibroblasts and endothelial cells, hyperglycemia interferes with the function of hypoxia-inducible factor-1 (HIF-1), a transcription factor that is essential for adaptive responses of the cell to hypoxia. Experiments using proteasomal and prolyl hydroxylases inhibitors indicate that hyperglycemia inhibits hypoxia-induced stabilization of HIF-1α protein levels against degradation and suggest that mechanisms in addition to proline hydroxylation may be involved. This effect of hyperglycemia was dose dependent and correlates with a lower transcription activation potency of HIF-1α, as assessed by transient hypoxia-inducible reporter gene assay. Regulation of HIF-1α function by hyperglycemia could be mimicked by mannitol, suggesting hyperosmolarity as one critical parameter. The interference of hyperglycemia with hypoxia-dependent stabilization of HIF-1α protein levels was confirmed in vivo, where only very low levels of HIF-1α protein could be detected in diabetic wounds, as compared with chronic venous ulcers. In conclusion, our data demonstrate that hyperglycemia impairs hypoxia-dependent protection of HIF-1α against proteasomal degradation and suggest a mechanism by which diabetes interferes with cellular responses to hypoxia.
Development of Late-Stage Diabetic Nephropathy in OVE26 Diabetic Mice
Authors: Zheng Shirong , Noonan William T. , Metreveli Naira S. , Coventry Susan , Kralik Patricia M. , Carlson Edward C. , Epstein Paul N.
Published: March 2007

OVE26 mice are a transgenic model of severe early-onset type 1 diabetes. These mice develop diabetes within the first weeks of life and can survive well over a year with no insulin treatment, and they maintain near normal body weight. To determine whether OVE26 mice provide a valuable model of chronic diabetic nephropathy (DN), OVE26 diabetic mice were compared with their nondiabetic littermates for functional and structural characteristics of DN. OVE26 mice exhibited pronounced polyuria and significant albuminuria by 2 months of age (305 μg/24 h in OVE26 vs. 20 μg/24 h in controls). Albumin excretion rate increased progressively with age and exceeded 15,000 μg/24 h at 9 months of age. The profound loss of albumin led to hypoalbuminemia in some diabetic animals. Albuminuria coincided with an elevation in blood pressure as measured by tail cuff. The glomerular filtration rate (GFR) in OVE26 mice measured using fluorescein isothiocynate inulin clearance demonstrated that GFR increased significantly from 2 to 3 months of age and then decreased significantly from 5 to 9 months. GFR in 9-month-old diabetic mice was significantly lower than that of 9-month-old control mice. The decline in GFR coincided with a significant increase in renal vascular resistance. Structural studies showed an almost twofold increase in kidney weight between 2 and 5 months. Diabetic mice also showed progressively enlarged glomeruli and expanded mesangium with diffuse and nodular expansion of mesangial matrix. Tubulointerstitial fibrosis was also observed in these mice. Glomerular basement membrane was thickened in OVE26 mice. In summary, OVE26 mice demonstrate that most of the characteristics of human DN can be produced by chronic hyperglycemia in a murine model. This model will be useful for improved understanding and treatment of DN.
Heterogeneity in the Magnitude of the Insulin Gene Effect on HLA Risk in Type 1 Diabetes
Authors: Motzo Costantino , Contu Daniela , Cordell Heather J. , Lampis Rosanna , Congia Mauro , Marrosu Maria Giovanna , Todd John A. , Devoto Marcella , Cucca Francesco
Published: March 2007

There is still uncertainty concerning the joint action of the two established type 1 diabetes susceptibility loci, the HLA class II DQB1 and DRB1 genes (IDDM1) and the insulin gene (INS) promoter (IDDM2). Some previous studies reported independence, whereas others suggested heterogeneity in the relative effects of the genotypes at these disease loci. In this study, we have assessed the combined effects of the HLA-DQB1/DRB1 and INS genotypes in 944 type 1 diabetic patients and 1,023 control subjects, all from Sardinia. Genotype variation at INS significantly influenced disease susceptibility in all HLA genotype risk categories. However, there was a significant heterogeneity (P = 2.4 × 10−4) in the distribution of the INS genotypes in patients with different HLA genotypes. The INS predisposing genotype was less frequent (74.9%) in high-risk HLA genotype–positive patients than in those with HLA intermediate-risk (86.1%) and low-risk (84.8%) categories. Gene-gene interaction modeling led to rejection of the additive model, whereas a multiplicative model showed a better, albeit still partial, fit to the observed data. These genetic results are consistent with an interaction between the protein products of the HLA and INS alleles, in which both the affinity of the various HLA class II molecules for a preproinsulin-derived peptide and the levels of this peptide in the thymus act jointly as key regulators of type 1 diabetes autoimmunity.
Polymorphism in the Calsequestrin 1 (CASQ1) Gene on Chromosome 1q21 Is Associated With Type 2 Diabetes in the Old Order Amish
Authors: Fu Mao , Damcott Coleen M. , Sabra Mona , Pollin Toni I. , Ott Sandra H. , Wang Jian , Garant Michael J. , O’Connell Jeffrey R. , Mitchell Braxton D. , Shuldiner Alan R.
Published: March 2007

Calsequestrin (CASQ)1 is involved in intracellular storage and release of calcium, a process that has been shown to mediate glucose transport in muscle. Its gene, CASQ1, is encoded on chromosome 1q21, a region that has been linked to type 2 diabetes in the Amish and several other populations. We screened all 11 exons, exon-intron junctions, and the proximal regulatory region of CASQ1 for mutations. We detected four novel single nucleotide polymorphisms (SNPs) (−1470C→T, −1456delG, −1366insG, and 593C→T). Ten informative SNPs within CASQ1 were genotyped in Amish subjects with type 2 diabetes (n = 145), impaired glucose tolerance (n = 148), and normal glucose tolerance (n = 358). Rs2275703 and rs617698 in introns 4 and 2 were significantly associated with type 2 diabetes (P = 0.008 and 0.04, respectively); three other SNPs showed borderline evidence for association to type 2 diabetes (P = 0.076–0.093). Furthermore, in nondiabetic subjects (n = 754), both rs2275703 and rs617698 were significantly associated with glucose area under the curve during an oral glucose tolerance test (P = 0.035 and 0.013, respectively). Haplotype analysis suggested that no haplotype could explain these associations better than rs2275703. These findings, coupled with similar findings in Utah Caucasians, suggest that sequence variation in CASQ1 may influence risk of type 2 diabetes.