bluuebunny/crossref_metadata_2025 huggingface dataset:
Early Decrease of Skin Blood Flow in Response to Locally Applied Pressure in Diabetic Subjects
Authors: Fromy Berengere , Abraham Pierre , Bouvet Celine , Bouhanick Beatrice , Fressinaud Philippe , Saumet Jean Louis
Published: March 2007

Pressure ulcers are common debilitating complications of diabetes that are caused by tissue ischemia. Skin blood flow in response to locally applied pressure might be impaired in diabetic patients because of the combined effects of a typically low skin temperature and alterations in microcirculatory function, and could be worsened by neuropathy. We measured skin blood flow by laser Doppler flowmetry over the internal anklebone in response to local pressure applied at 5.0 mmHg/min in three groups of diabetic patients (with clinical and subclinical neuropathy and without neuropathy) and in healthy matched control subjects at usual room temperature. Compared with in matched control subjects with comparable skin temperatures (29.3 ± 0.4 vs. 28.7 ± 0.4°C), in diabetic patients the skin blood flow response to locally applied pressure was further impeded, even in those without neuropathy. Indeed, skin blood flow decreased significantly from baseline at much lower applied pressure (7.5 mmHg) in diabetic subjects, again even in those without neuropathy, than in control subjects (48.8 mmHg). The large difference between these pressures could partially explain diabetic patients’ high risk of developing decubitus and plantar ulcers.
Type 2 Diabetes, APOE Gene, and the Risk for Dementia and Related Pathologies
Authors: Peila Rita , Rodriguez Beatriz L. , Launer Lenore J.
Published: March 2007

Type 2 diabetes may be a risk factor for dementia, but the associated pathological mechanisms remains unclear. We evaluated the association of diabetes alone or combined with the apolipoprotein E (APOE) gene with incident dementia and neuropathological outcomes in a population-based cohort of 2,574 Japanese-American men enrolled in the Honolulu-Asia Aging Study, including 216 subjects who underwent autopsy. Type 2 diabetes was ascertained by interview and direct glucose testing. Dementia was assessed in 1991 and 1994 by clinical examination and magnetic resonance imaging and was diagnosed according to international guidelines. Logistic regression was used to assess the RR of developing dementia, and log-linear regression was used to estimate the incident rate ratio (IRR) of neuropathological outcomes. Diabetes was associated with total dementia (RR 1.5 [95% CI 1.01–2.2]), Alzheimer’s disease (AD; 1.8 [1.1–2.9]), and vascular dementia (VsD; 2.3 [1.1–5.0]). Individuals with both type 2 diabetes and the APOE ε4 allele had an RR of 5.5 (CI 2.2–13.7) for AD compared with those with neither risk factor. Participants with type 2 diabetes and the ε4 allele had a higher number of hippocampal neuritic plaques (IRR 3.0 [CI 1.2–7.3]) and neurofibrillary tangles in the cortex (IRR 3.5 [1.6–7.5]) and hippocampus (IRR 2.5 [1.5–3.7]), and they had a higher risk of cerebral amyloid angiopathy (RR 6.6, 1.5–29.6). Type 2 diabetes is a risk factor for AD and VsD. The association between diabetes and AD is particularly strong among carriers of the APOE ε4 allele. The neuropathological data are consistent with the clinical results.
Increased Proinsulin Levels and Decreased Acute Insulin Response Independently Predict the Incidence of Type 2 Diabetes in the Insulin Resistance Atherosclerosis Study
Authors: Hanley Anthony J.G. , D’Agostino Ralph , Wagenknecht Lynne E. , Saad Mohammed F. , Savage Peter J. , Bergman Richard , Haffner Steven M.
Published: March 2007

Previous studies have indicated that β-cell dysfunction predicts the development of diabetes, although it is unknown whether the use of combinations of insulin secretory measures further improves prediction. The Insulin Resistance Atherosclerosis Study is a prospective, multicenter, epidemiological study of the relationship between insulin sensitivity and the risk of diabetes and cardiovascular disease. At baseline, fasting concentrations of insulin, intact proinsulin (PI), and split PI were measured, and acute insulin response (AIR) was determined during a frequently sampled intravenous glucose tolerance test (FSIGTT). Subjects who were nondiabetic at baseline (n = 903) were reexamined after 5 years of follow-up; 148 had developed diabetes. In separate logistic regression models adjusted for age, sex, clinic, and ethnicity, 1 SD differences in measures of β-cell dysfunction were associated with diabetes incidence (AIR: odds ratio [OR] 0.37, 95% CI 0.27–0.52; intact PI: OR 1.90, 95% CI 1.57–2.30; split PI: OR 1.94, 95% CI 1.63–2.31). After additional adjustment for BMI, impaired glucose tolerance, and insulin sensitivity, these measures continued to be significantly associated with risk of diabetes (all P < 0.0001). Furthermore, in models that included both PI and AIR, each was an independent predictor, and individuals who had combined low AIR and high PI experienced the highest diabetes risk. In conclusion, both low AIR and high PI independently predicted diabetes in a well-characterized multiethnic population. Although fasting PI is simpler to assess, determining AIR from an FSIGTT may further improve prediction. If pharmacological agents to prevent diabetes are proved to be efficacious in ongoing clinical trials, then it may be beneficial to perform FSIGTTs to identify better (for intensive intervention) prediabetic subjects who would ultimately require lifelong pharmacological therapy.
Peroxisome Proliferator-Activated Receptor-α Regulates Fatty Acid Utilization in Primary Human Skeletal Muscle Cells
Authors: Muoio Deborah M. , Way James M. , Tanner Charles J. , Winegar Deborah A. , Kliewer Steven A. , Houmard Joseph A. , Kraus William E. , Dohm G. Lynis
Published: March 2007

In humans, skeletal muscle is a major site of peroxisome proliferator–activated receptor-α (PPAR-α) expression, but its function in this tissue is unclear. We investigated the role of hPPAR-α in regulating muscle lipid utilization by studying the effects of a highly selective PPAR-α agonist, GW7647, on [14C]oleate metabolism and gene expression in primary human skeletal muscle cells. Robust induction of PPAR-α protein expression occurred during muscle cell differentiation and corresponded with differentiation-dependent increases in oleate oxidation. In mature myotubes, 48-h treatment with 10–1,000 nmol/l GW7647 increased oleate oxidation dose-dependently, up to threefold. Additionally, GW7647 decreased oleate esterification into myotube triacylglycerol (TAG), up to 45%. This effect was not abolished by etomoxir, a potent inhibitor of β-oxidation, indicating that PPAR-α–mediated TAG depletion does not depend on reciprocal changes in fatty acid catabolism. Consistent with its metabolic actions, GW7647 induced mRNA expression of mitochondrial enzymes that promote fatty acid catabolism; carnitine palmityltransferase 1 and malonyl-CoA decarboxylase increased ∼2-fold, whereas pyruvate dehydrogenase kinase 4 increased 45-fold. Expression of several genes that regulate glycerolipid synthesis was not changed by GW7647 treatment, implicating involvement of other targets to explain the TAG-depleting effect of the compound. These results demonstrate a role for hPPAR-α in regulating muscle lipid homeostasis.
Impaired β-Cell Function, Incretin Effect, and Glucagon Suppression in Patients With Type 1 Diabetes Who Have Normal Fasting Glucose
Authors: Greenbaum Carla J. , Prigeon Ronald L. , D’Alessio David A.
Published: March 2007

We have recently described a novel phenotype in a group of subjects with type 1 diabetes that is manifested by glucose >11.1 mmol/l 120 min after an oral glucose load, but with normal fasting glucose levels. We now describe the metabolic characteristics of these subjects by comparing parameters of islet hormone secretion and glucose disposal in these subjects to age-matched nondiabetic control subjects. The patients with type 1 diabetes had fasting glucose, insulin, and glucagon values similar to those of control subjects. Additionally, the insulin secretory response to intravenous arginine at euglycemia was similar in the control and diabetic groups (264 ± 33.5 and 193 ± 61.3 pmol/l; P = 0.3). However, marked differences in β-cell function were found in response to hyperglycemia. Specifically, the first-phase insulin response was lower in diabetic subjects (329.1 ± 39.6 vs. 91.3 ± 34.1 pmol/l; P < 0.001), as was the slope of glucose potentiation of the insulin response to arginine (102 ± 18.7 vs. 30.2 ± 6.1 pmol/l per mmol/l; P = 0.005) and the maximum insulin response to arginine (2,524 ± 413 vs. 629 ± 159 pmol/l; P = 0.001). Although plasma levels of glucagon-like peptide (GLP)-1 and gastric inhibitory peptide (GIP) did not differ between control and diabetic subjects, the incretin effect was lower in the diabetic patients (70.3 ± 5.4 vs. 52.1 ± 5.9%; P = 0.03). Finally, there was a lack of suppression of glucagon in the patients after both oral and intravenous glucose administration, which may have contributed to their postprandial hyperglycemia. Glucose effectiveness did not differ between patients and control subjects, nor did insulin sensitivity, although there was a tendency for the patients to be insulin resistant (9.18 ± 1.59 vs. 5.22 ± 1.17 pmol · l−1 · min−1; P = 0.08). These data characterize a novel group of subjects with type 1 diabetes manifested solely by hyperglycemia following an oral glucose load in whom islet function is normal at euglycemia, but who have marked defects in both α- and β-cell secretion at hyperglycemia. This pattern of abnormalities may be characteristic of islet dysfunction early in the development of type 1 diabetes.
Expression Profiling of Palmitate- and Oleate-Regulated Genes Provides Novel Insights Into the Effects of Chronic Lipid Exposure on Pancreatic β-Cell Function
Authors: Busch Anna K. , Cordery Damien , Denyer Gareth S. , Biden Trevor J.
Published: March 2007

Chronic lipid exposure is implicated in β-cell dysfunction in type 2 diabetes. We therefore used oligonucleotide arrays to define global alterations in gene expression in MIN6 cells after 48-h pretreatment with oleate or palmitate. Altogether, 126 genes were altered ≥1.9-fold by palmitate, 62 by oleate, and 46 by both lipids. Importantly, nine of the palmitate-regulated genes are known to be correspondingly changed in models of type 2 diabetes. A tendency toward β-cell de-differentiation was also apparent with palmitate: pyruvate carboxylase and mitochondrial glycerol 3-phosphate dehydrogenase were downregulated, whereas lactate dehydrogenase and fructose 1,6-bisphosphatases were induced. Increases in the latter (also seen with oleate), along with glucosamine-phosphate n-acetyl transferase, imply upregulation of the hexosamine biosynthesis pathway in palmitate-treated cells. However, palmitate also increased expression of calcyclin and 25-kDa synaptosomal-associated protein (SNAP25), which control distal secretory processes. Consistent with these findings, secretory responses to noncarbohydrate stimuli, especially palmitate itself, were upregulated in palmitate-treated cells (much less so with oleate). Indeed, glucose-stimulated secretion was slightly sensitized by chronic palmitate exposure but inhibited by oleate treatment, whereas both lipids enhanced basal secretion. Oleate and palmitate also induced expression of chemokines (MCP-1 and GRO1 oncogene) and genes of the acute phase response (serum amyloid A3). Increases in transcriptional modulators such as ATF3, CCAAT/enhancer binding protein-β (C/EBPβ), C/EBPδ, and c-fos were also seen. The results highlight links between regulated gene expression and phenotypic alterations in palmitate versus oleate-pretreated β-cells.
Genetic Effects on Age-Dependent Onset and Islet Cell Autoantibody Markers in Type 1 Diabetes
Authors: Graham Jinko , Hagopian William A. , Kockum Ingrid , Li Lou Sheng , Sanjeevi Carani B. , Lowe Robert M. , Schaefer Jonathan B. , Zarghami Marjan , Day Heather L. , Landin-Olsson Mona , Palmer Jerry P. , Janer-Villanueva Marta , Hood Leroy , Sundkvist Göran , Lernmark Åke , Breslow Norman , Dahlquist Gisela ,   for the Swedish Childhood Diabetes Study Group, Blohmé Göran ,   for the Diabetes Incidence in Sweden Study Group
Published: March 2007

Age-dependent associations between type 1 diabetes risk genes HLA, INS VNTR, and CTLA-4 and autoantibodies to GAD65 (GADAs), ICA512/IA-2, insulin, and islet cells were determined by logistic regression analysis in 971 incident patients with type 1 diabetes and 702 control subjects aged 0–34 years. GADAs were associated with HLA-DQ2 in young but not in older patients (P = 0.009). Autoantibodies to insulin were negatively associated with age (P < 0.0001) but positively associated with DQ8 (P = 0.03) and with INS VNTR (P = 0.04), supporting possible immune tolerance induction. ICA512/IA-2 were negatively associated with age (P < 0.0001) and with DQ2 (P < 0.0001) but positively associated with DQ8 (P = 0.04). Males were more likely than females to be negative for GADA (P < 0.0001), autoantibodies to islet cells (P = 0.04), and all four autoantibody markers (P = 0.004). The CTLA-4 3′ end microsatellite marker was not associated with any of the autoantibodies. We conclude that age and genetic factors such as HLA-DQ and INS VNTR need to be combined with islet autoantibody markers when evaluating the risk for type 1 diabetes development.
Detection of GAD65-Specific T-Cells by Major Histocompatibility Complex Class II Tetramers in Type 1 Diabetic Patients and At-Risk Subjects
Authors: Reijonen Helena , Novak Erik J. , Kochik Sharon , Heninger Anne , Liu Andrew W. , Kwok William W. , Nepom Gerald T.
Published: March 2007

Soluble HLA-DR401 or -DR404 tetramers containing a peptide corresponding to an immunodominant epitope from human GAD65 were used to analyze peripheral blood T-cells of newly diagnosed type 1 diabetic patients and at-risk subjects. Peripheral blood mononuclear cells were expanded on antigen-presenting cells presenting GAD65 peptide and subsequently activated with specific plate-bound class II-peptide monomers. T-cell activation defined in flow cytometry by CD4high and/or CD25 markers were observed in all type 1 diabetic patients and some at-risk subjects, but not in normal control subjects. The activated T-cells stained positive with tetramers containing the GAD65 epitope 555-567. Tetramer-positive cells were CD4high T-cells with high avidity for an immunodominant GAD65 T-cell epitope. Phenotyping of T-cells utilizing HLA class II tetramers provides a new tool to characterize the autoimmune response in type 1 diabetes.
Fas/Fas Ligand Interactions Play an Essential Role in the Initiation of Murine Autoimmune Diabetes
Authors: Nakayama Maki , Nagata Masao , Yasuda Hisafumi , Arisawa Kenji , Kotani Reiko , Yamada Katsumi , Chowdhury Shahead Ali , Chakrabarty Sagarika , Jin Zhen Zi , Yagita Hideo , Yokono Koichi , Kasuga Masato
Published: March 2007

Apoptosis via Fas/Fas ligand (FasL) interactions has been proposed to be a major T-cell-mediated effector mechanism in autoimmune diabetes. To elucidate the role of Fas/FasL interactions in NOD diabetes, the effects of neutralizing anti-FasL antibody on autoimmune responses were evaluated. Islet-specific CD8+ and CD4+ T-cells expressed FasL upon activation and mediated FasL-dependent cytotoxicity against Fas-expressing target cells in vitro, although their cytotoxicity against islet cells was not blocked by anti-FasL antibody. Moreover, administration of anti-FasL antibody failed to inhibit diabetes in vivo in the CD8+ T-cell adoptive transfer model. On the other hand, blockade of Fas/FasL interactions significantly inhibited CD4+ T-cell-dependent diabetes in adoptive transfer models. These results suggest a substantial contribution of Fas/FasL interactions to CD4+, but not CD8+, T-cell-mediated destruction of pancreatic β-cells. When anti-FasL antibody was administered to NOD mice between 5 and 15 weeks of age, the onset of diabetes was slightly delayed but the incidence was not decreased. However, administration of anti-FasL antibody at 2–4 weeks of age completely prevented insulitis and diabetes. These results suggest that Fas/FasL interactions contribute to CD4+ T-cell-mediated β-cell destruction and play an essential role in the initiation of autoimmune NOD diabetes.
Hepatocyte Nuclear Factor-1α Recruits the Transcriptional Co-Activator p300 on the GLUT2 Gene Promoter
Authors: Ban Nobuhiro , Yamada Yuichiro , Someya Yoshimichi , Miyawaki Kazumasa , Ihara Yu , Hosokawa Masaya , Toyokuni Shinya , Tsuda Kinsuke , Seino Yutaka
Published: March 2007

Mutations in the hepatocyte nuclear factor (HNF)-1α gene have been linked to subtype 3 of maturity-onset diabetes of the young (MODY), a disease characterized by a primary defect in insulin secretion. Here we show that the human GLUT2 gene is closely regulated by HNF-1α via sequences downstream of the transcriptional start site by interaction with transcriptional co-activator p300. The promoter region of the human GLUT2 gene was subcloned into luciferase expression plasmids that were transfected together with HNF-1α expression plasmid into a pancreatic β-cell line, HIT-T15, to evaluate transcriptional activities. HNF-1α enhanced human GLUT2 promoter activity sixfold. Site-direct mutagenesis and footprint analyses showed that the HNF-1α binding site (+200 to +218) is critical in human GLUT2 gene expression. Furthermore, mammalian two-hybrid and immunoprecipitation studies revealed the transactivation domain of HNF-1α (amino acids 391–540) to interact with both the NH2-terminal region (amino acids 180–662) and the COOH-terminal region (amino acids 1,818–2,079) of p300. These findings demonstrated that HNF-1α binds to the 5′-untranslated region of GLUT2 and that p300 acts as a transcriptional co-activator for HNF-1α. In addition, these results provided new insight into the regulatory function of HNF-1α by suggesting a molecular basis for human GLUT2 gene expression.
Unexpected Sensitivity of Nonobese Diabetic Mice With a Disrupted Poly(ADP-Ribose) Polymerase-1 Gene to Streptozotocin-Induced and Spontaneous Diabetes
Authors: Gonzalez Cristina , Ménissier de Murcia Josiane , Janiak Philip , Bidouard Jean-Pierre , Beauvais Catherine , Karray Saoussen , Garchon Henri-Jean , Lévi-Strauss Matthieu
Published: March 2007

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that consumes NAD in response to DNA strand breaks. Its excessive activation seems particularly deleterious to pancreatic β-cells, as exemplified by the complete resistance of PARP-1-deficient mice to the toxic diabetes induced by streptozotocin. Because of the possible implication of this enzyme in type 1 diabetes, many human trials using nicotinamide, an inhibitor of PARP-1, have been conducted either in patients recently diagnosed or in subjects highly predisposed to this disease. To analyze the role of this enzyme in murine type 1 diabetes, we introgressed a disrupted PARP-1 allele onto the autoimmune diabetes-prone nonobese diabetic (NOD) mouse strain. We showed that these mice were protected neither from spontaneous nor from cyclophosphamide-accelerated diabetes. Surprisingly they were also highly sensitive to the diabetes induced by a single high dose of streptozotocin, standing in sharp contrast with C57BL/6 mice that bear the same inactivated PARP-1 allele. Our results suggest that NOD mice are characterized not only by their immune dysfunction but also by a peculiarity of their islets leading to a PARP-1-independent mechanism of streptozotocin-induced β-cell death.
Gestational Diabetes Leads to the Development of Diabetes in Adulthood in the Rat
Authors: Boloker Judd , Gertz Shira J. , Simmons Rebecca A.
Published: March 2007

We have developed a model of gestational diabetes in the rat to determine whether an altered metabolic intrauterine milieu is directly linked to the development of diabetes later in life. Uteroplacental insufficiency is induced in the pregnant rat on day 19 of gestation. Sham-operated animals serve as controls. Offspring are growth retarded at birth; however, they catch up by 5–7 weeks of age. At ∼8 weeks of age, they are bred to normal males. During pregnancy, these animals develop progressive hyperglycemia and hyperinsulinemia accompanied by impaired glucose tolerance and insulin resistance. Offspring, designated as infants of a diabetic mother (IDMs), are heavier at birth and remain heavy throughout life. IDMs are insulin resistant very early in life, and glucose homeostasis is progressively impaired. Defects in insulin secretion are detectable as early as 5 weeks of age. By 26 weeks of age, IDMs are overtly diabetic. These data demonstrate that the altered metabolic milieu of the diabetic pregnancy causes permanent defects in glucose homeostasis in the offspring that lead to the development of diabetes later in life.
MEDICA 16 Inhibits Hepatic Acetyl-CoA Carboxylase and Reduces Plasma Triacylglycerol Levels in Insulin-Resistant JCR
Authors: Atkinson Laura L. , Kelly Sandra E. , Russell James C. , Bar-Tana Jacob , Lopaschuk Gary D.
Published: March 2007

Intracellular triacylglycerol (TG) content of liver and skeletal muscle contributes to insulin resistance, and a significant correlation exists between TG content and the development of insulin resistance. Because acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme for liver fatty acid biosynthesis and a key regulator of muscle fatty acid oxidation, we examined whether ACC plays a role in the accumulation of intracellular TG. We also determined the potential role of 5′-AMP-activated protein kinase (AMPK) in this process, since it can phosphorylate and inhibit ACC activity in both liver and muscle. TG content, ACC, and AMPK were examined in the liver and skeletal muscle of insulin-resistant JCR:LA-cp rats during the time frame when insulin resistance develops. At 12 weeks of age, there was a threefold elevation in liver TG content and a sevenfold elevation in skeletal muscle TG content. Hepatic ACC activity was significantly elevated in 12-week-old JCR:LA-cp rats compared with lean age-matched controls (8.75 ± 0.53 vs. 3.30 ± 0.18 nmol · min−1 · mg−1, respectively), even though AMPK activity was also increased. The observed increase in hepatic ACC activity was accompanied by a 300% increase in ACC protein expression. There were no significant differences in ACC activity, ACC protein expression, or AMPK activity in the skeletal muscle of the 12-week JCR:LA-cp rats. Treatment of 12-week JCR:LA-cp rats with MEDICA 16 (an ATP-citrate lyase inhibitor) resulted in a decrease in hepatic ACC and AMPK activities, but had no effect on skeletal muscle ACC and AMPK. Our data suggest that alterations in ACC or AMPK activity in muscle do not contribute to the development of insulin resistance. However, increased liver ACC activity in the JCR:LA-cp rat appears to contribute to the development of lipid abnormalities, although this increase does not appear to occur secondary to a decrease in AMPK activity.
Variation in Three Single Nucleotide Polymorphisms in the Calpain-10 Gene Not Associated With Type 2 Diabetes in a Large Finnish Cohort
Authors: Fingerlin Tasha E. , Erdos Michael R. , Watanabe Richard M. , Wiles Kerry R , Stringham Heather M. , Mohlke Karen L. , Silander Kaisa , Valle Timo T. , Buchanan Thomas A. , Tuomilehto Jaakko , Bergman Richard N. , Boehnke Michael , Collins Francis S.
Published: March 2007

Variations in the calpain-10 gene have recently been reported to be associated with type 2 diabetes in a Mexican-American population. We typed three single nucleotide polymorphisms (SNPs) in the calpain-10 gene (SNPs 43, 56, and 63) to test for association between variation at these loci and type 2 diabetes and diabetes-related traits in 1,603 Finnish subjects: two samples of 526 (Finland-U.S. Investigation of NIDDM Genetics [FUSION] 1) and 255 (FUSION 2) index case subjects with type 2 diabetes, 185 and 414 unaffected spouses and offspring of FUSION 1 index case subjects or their affected siblings, and 223 elderly normal glucose-tolerant control subjects. We found no significant differences in allele, genotype, haplotype, or haplogenotype frequencies between index case subjects with diabetes and the elderly and spouse control populations (all P > 0.087). Although variation in these three SNPs was associated with variation in some type 2 diabetes-related traits within each of the case and control groups, no consistent pattern of the implicated variant or combination of variants was discerned. We conclude that variation in these three SNPs in the calpain-10 gene is unlikely to confer susceptibility to type 2 diabetes in this Finnish cohort.
Amino Acid Kinetics During the Anhepatic Phase of Liver Transplantation
Authors: Battezzati Alberto , Caumo Andrea , Fattorini Annalisa , Sereni Lucia Piceni , Coppa Jorgelina , Romito Raffaele , Ammatuna Mario , Regalia Enrico , Mazzaferro Vincenzo , Luzi Livio
Published: March 2007

Alanine and glutamine are interorgan nitrogen/carbon carriers for ureagenesis and gluconeogenesis, which are mainly but not necessarily only hepatic. The liver is central to alanine and glutamine metabolism, but most organs can produce and use them. We studied amino acid kinetics after liver removal to depict initial events of liver failure and to provide a model to study extrahepatic gluconeogenesis and nitrogen disposal in humans. We measured amino acid kinetics with [5,5,5-2H3]leucine and [3-13C]alanine or [1,2-13C2]glutamine tracers in 21 subjects during and after the anhepatic phase of liver transplantation: 12 were at 7 months posttransplantation, and 7 were healthy control subjects. Anhepatic leucine kinetics, including proteolysis, was unchanged. Alanine plasma and whole-body contents increased 3× and 2×, with a halved metabolic clearance and a doubled production, 2% greater than disposal. Free whole-body glutamine decreased 25% but increased 50% in plasma. Glutamine clearance was halved, and the production decreased by 25%, still 2% greater than disposal. Liver replacement decreased alanine and glutamine concentrations, leaving leucine unchanged. Liver removal caused doubled alanine fluxes, minor changes in glutamine, and no changes in leucine. The initial events after liver removal are an accumulation of three-carbon compounds, an acceleration of alanine turnover, and limited nitrogen storage in alanine and glutamine.
Central Leptin Gene Therapy Blocks High-Fat Diet-Induced Weight Gain, Hyperleptinemia, and Hyperinsulinemia
Authors: Dube Michael G. , Beretta Elena , Dhillon Harveen , Ueno Naohiko , Kalra Pushpa S. , Kalra Satya P.
Published: March 2007

Recombinant adeno-associated virus (rAAV), encoding either rat leptin (rAAV-lep) or green fluorescent protein (rAAV-GFP, control), was injected intracerebroventricularly in rats consuming a high-fat diet (HFD; 45 kcal%). Caloric consumption and body weight were monitored weekly until the rats were killed at 9 weeks. Untreated control rats consuming regular rat diet (RCD; 11 kcal%) were monitored in parallel. Body weight gain was accelerated in rAAV-GFP + HFD control rats relative to those consuming RCD, despite equivalent kcal consumption. At 9 weeks, serum leptin, free fatty acids, triglycerides, and insulin were elevated in HFD control rats. In contrast, rAAV-lep treatment reduced intake and blocked the HFD-induced increase in weight, adiposity, and metabolic variables. Blood glucose was slightly reduced but within the normal range, and serum ghrelin levels were significantly elevated in rAAV-lep + HFD rats. Uncoupling protein-1 (UCP1) mRNA in brown adipose tissue (BAT), an index of energy expenditure through nonshivering thermogenesis, was decreased in rats consuming HFD. Treatment with rAAV-lep significantly augmented BAT UCP1 mRNA expression, indicating increased thermogenic energy expenditure. These findings demonstrate that central leptin gene therapy efficiently prevents weight gain, increased adiposity, and hyperinsulinemia in rats consuming an HFD by decreasing energy intake and increasing thermogenic energy expenditure.
Antidiabetic Effects of Panax ginseng Berry Extract and the Identification of an Effective Component
Authors: Attele Anoja S. , Zhou Yun-Ping , Xie Jing-Tian , Wu Ji An , Zhang Liu , Dey Lucy , Pugh William , Rue Paul A. , Polonsky Kenneth S. , Yuan Chun-Su
Published: March 2007

We evaluated antihyperglycemic and anti-obese effects of Panax ginseng berry extract and its major constituent, ginsenoside Re, in obese diabetic C57BL/6J ob/ ob mice and their lean littermates. Animals received daily intraperitoneal injections of Panax ginseng berry extract for 12 days. On day 12, 150 mg/kg extract–treated ob/ob mice became normoglycemic (137 ± 6.7 mg/dl) and had significantly improved glucose tolerance. The overall glucose excursion during the 2-h intraperitoneal glucose tolerance test decreased by 46% (P < 0.01) compared with vehicle-treated ob/ob mice. The improvement in blood glucose levels in the extract-treated ob/ ob mice was associated with a significant reduction in serum insulin levels in fed and fasting mice. A hyperinsulinemic-euglycemic clamp study revealed a more than twofold increase in the rate of insulin-stimulated glucose disposal in treated ob/ ob mice (112 ± 19.1 vs. 52 ± 11.8 μmol · kg−1 · min−1 for the vehicle group, P < 0.01). In addition, the extract-treated ob/ob mice lost a significant amount of weight (from 51.7 ± 1.9 g on day 0 to 45.7 ± 1.2 on day 12, P < 0.01 vs. vehicle-treated ob/ob mice), associated with a significant reduction in food intake (P < 0.05) and a very significant increase in energy expenditure (P < 0.01) and body temperature (P < 0.01). Treatment with the extract also significantly reduced plasma cholesterol levels in ob/ob mice. Additional studies demonstrated that ginsenoside Re plays a significant role in antihyperglycemic action. This antidiabetic effect of ginsenoside Re was not associated with body weight changes, suggesting that other constituents in the extract have distinct pharmacological mechanisms on energy metabolism.
Sensitivity to Acute Insulin-Mediated Suppression of Plasma Free Fatty Acids Is Not a Determinant of Fasting VLDL Triglyceride Secretion in Healthy Humans
Authors: Carpentier André , Patterson Bruce W. , Leung Nathalie , Lewis Gary F.
Published: March 2007

One important mechanism whereby obesity-associated insulin resistance leads to VLDL overproduction is thought to be by the increased flux of free fatty acids (FFAs) from extrahepatic tissues to liver, which arises as a direct consequence of impaired insulin action in adipose tissue and skeletal muscle. The aim of the present study was to address whether direct measures of peripheral tissue insulin sensitivity with regard to FFAs and glucose in the fasting state are good predictors of postabsorptive VLDL triglyceride secretion rate (VLDL-TG ASR) in humans, independent of obesity. Eighteen healthy control subjects, after an overnight fast, underwent three studies 3 weeks apart, in random order. Study 1: VLDL-TG levels, fractional clearance rate (per h), and VLDL-TG ASR were determined after an intravenous bolus of [1,1,2,3,3-2H5] glycerol. Study 2: Insulin sensitivity (SI), acute insulin response (AIR), and acute C-peptide response to glucose were assessed by frequently sampled intravenous glucose tolerance test using the minimal model approach. Study 3: Insulin-mediated suppression of plasma FFAs (k) and insulin clearance were assessed in response to a low-dose stepwise intravenous insulin infusion. BMI (R2 = 0.54), AIR, and fasting insulin levels were positively and SI negatively correlated with VLDL-TG ASR, but there was no significant association with plasma FFAs or k. Only BMI remained significantly associated with VLDL-TG ASR in multivariate analysis. The best multivariate model for VLDL-TG ASR (R2 = 0.61, P = 0.0008) included BMI (P = 0.0008) and SI (P = 0.12, inversely correlated). VLDL-TG secretion is predicted by BMI, independently of direct measures of insulin sensitivity. The sensitivity to insulin’s acute suppressive effect on plasma FFA levels during fasting is not an important determinant of postabsorptive VLDL-TG secretion in humans.
Female Rats Do Not Exhibit Free Fatty Acid–Induced Insulin Resistance
Authors: Hevener Andrea , Reichart Donna , Janez Andrej , Olefsky Jerrold
Published: March 2007

It is well described that excessive lipid metabolism can cause insulin resistance in both animals and humans, and this has been implicated as a causative factor in the development of insulin resistance and type 2 diabetes in humans. Recently, we have shown that intravenous lipid emulsion (liposyn) infusion during a 120-min euglycemic-hyperinsulinemic clamp led to significant reductions in insulin action and fatty acid translocase (FAT/CD36) skeletal muscle protein expression. After reviewing the literature, it became evident that essentially all past studies, including our own, were conducted in male animals. Therefore, to determine whether there were sex determinants of fat-induced insulin resistance, we assessed the impact of free fatty acid (FFA) elevation on insulin action in female rats. Here, we report that a fourfold elevation in plasma FFA concentration induced a 40% reduction in the insulin-stimulated glucose disposal rate, a 30% decline in insulin-stimulated skeletal muscle insulin substrate receptor-1 (IRS-1) phosphorylation, a 48% decrease in IRS-1–associated phosphatidylinositol (PI) 3-kinase activity, and a 50% reduction in muscle FAT/CD36 protein expression in male rats. In striking contrast, we found no effect of FFA elevation to cause insulin resistance, changes in IRS-1/PI 3-kinase, or FAT/CD36 protein levels in female animals. Our findings indicate that female animals are protected from lipid-induced reductions in insulin action.
Hyperglycemia-Induced Apoptosis in Mouse Myocardium
Authors: Cai Lu , Li Wei , Wang Guangwu , Guo Luping , Jiang Youchun , Kang Y. James
Published: March 2007

Diabetic cardiomyopathy is related directly to hyperglycemia. Cell death such as apoptosis plays a critical role in cardiac pathogenesis. Whether hyperglycemia induces myocardial apoptosis, leading to diabetic cardiomyopathy, remains unclear. We tested the hypothesis that apoptotic cell death occurs in the diabetic myocardium through mitochondrial cytochrome c–mediated caspase-3 activation pathway. Diabetic mice produced by streptozotocin and H9c2 cardiac myoblast cells exposed to high levels of glucose were used. In the hearts of diabetic mice, apoptotic cell death occurred as detected by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) assay. Correspondingly, caspase-3 activation as determined by enzymatic assay and mitochondrial cytochrome c release detected by Western blotting analysis were observed. Supplementation of insulin inhibited diabetes-induced myocardial apoptosis as well as suppressed hyperglycemia. To explore whether apoptosis in diabetic hearts is related directly to hyperglycemia, we exposed cardiac myoblast H9c2 cells to high levels of glucose (22 and 33 mmol/l) in cultures. Apoptotic cell death was detected by TUNEL assay and DAPI nuclear staining. Caspase-3 activation with a concomitant mitochondrial cytochrome c release was also observed. Apoptosis or activation of caspase-3 was not observed in the cultures exposed to the same concentrations of mannitol. Inhibition of caspase-3 with a specific inhibitor, Ac-DEVD-cmk, suppressed apoptosis induced by high levels of glucose. In addition, reactive oxygen species (ROS) generation was detected in the cells exposed to high levels of glucose. These results suggest that hyperglycemia directly induces apoptotic cell death in the myocardium in vivo. Hyperglycemia-induced myocardial apoptosis is mediated, at least in part, by activation of the cytochrome c–activated caspase-3 pathway, which may be triggered by ROS derived from high levels of glucose.
The Diabetes-Prone BB Rat Carries a Frameshift Mutation inIan4, a Positional Candidate ofIddm1
Authors: Hornum Lars , Rømer John , Markholst Helle
Published: March 2007

Diabetes-prone (DP) BB rats spontaneously develop insulin-dependent diabetes resembling human type 1 diabetes. They also exhibit lifelong T-cell lymphopenia. Functional and genetic data support the hypothesis that the gene responsible for the lymphopenia, Lyp, is also a diabetes susceptibility gene, named Iddm1. We constructed a 550-kb P1-derived artificial chromosome contig of the region. Here, we present a corrected genetic map reducing the genetic interval to 0.2 cM and the physical interval to 150–290 kb. A total of 13 genes and six GenomeScan models are assigned to the homologous human DNA segment on HSA7q36.1, 8 of which belong to the family of immune-associated nucleotides (Ian genes). Two of these are orthologous to mouse Ian1 and -4, both excellent candidates for Iddm1. In normal rats, they are expressed in the thymus and T-cell regions of the spleen. In the thymus of lymphopenic rats, Ian1 exhibits wild-type expression patterns, whereas Ian4 expression is reduced. Mutational screening of their coding sequences revealed a frameshift mutation in Ian4 among lymphopenic rats. The mutation results in a truncated protein in which the COOH-terminal 215 amino acids—including the anchor localizing the protein to the outer mitochondrial membrane—are replaced by 19 other amino acids. We propose that Ian4 is identical to Iddm1.
Critical Role for Cataplerosis via Citrate in Glucose-Regulated Insulin Release
Authors: Flamez Daisy , Berger Veerle , Kruhøffer Mogens , Orntoft Torben , Pipeleers Daniel , Schuit Frans C.
Published: March 2007

The molecular mechanisms mediating acute regulation of insulin release by glucose are partially known. The process involves at least two pathways that can be discriminated on basis of their (in)dependence of closure of ATP-sensitive potassium (K+ATP) channels. The mechanism of the K+ATP channel–independent pathway was proposed to involve cataplerosis, the export of mitochondrial intermediates into the cytosol and in the induction of fatty acid–derived signaling molecules. In the present article, we have explored in fluorescence-activated cell sorter (FACS)-purified rat β-cells the molecular steps involved in chronic glucose regulation of the insulin secretory response. When compared with culture in 10 mmol/l glucose, 24 h culture in 3 mmol/l glucose shifts the phenotype of the cells into a state with low further secretory responsiveness to glucose, lower rates of glucose oxidation, and lower rates of cataplerosis. Microarray mRNA analysis indicates that this shift can be attributed to differences in expression of genes involved in the K+ATP channel–dependent pathway, in cataplerosis and in fatty acid/cholesterol biosynthesis. This response was paralleled by glucose upregulation of the transcription factor sterol regulatory element binding protein 1c (SREBP1c) (ADD1) and downregulation of peroxisome proliferator—activated receptor (PPAR)-α and PPAR-β (PPARδ). The functional importance of cataplerosis via citrate for glucose-induced insulin release was further supported by the observation that two ATP-citrate lyase inhibitors, radicicol and (−)-hydroxycitrate, block part of glucose-stimulated release in β-cells. In conclusion, chronic glucose regulation of the glucose-responsive secretory phenotype is associated with coordinated changes in gene expression involved in the K+ATP channel–dependent pathway, in cataplerosis via citrate and in acyl CoA/cholesterol biosynthesis.
Glucokinase Is the Likely Mediator of Glucosensing in Both Glucose-Excited and Glucose-Inhibited Central Neurons
Authors: Dunn-Meynell Ambrose A. , Routh Vanessa H. , Kang Ling , Gaspers Larry , Levin Barry E.
Published: March 2007

Specialized neurons utilize glucose as a signaling molecule to alter their firing rate. Glucose-excited (GE) neurons increase and glucose-inhibited (GI) neurons reduce activity as ambient glucose levels rise. Glucose-induced changes in the ATP-to-ADP ratio in GE neurons modulate the activity of the ATP-sensitive K+ channel, which determines the rate of cell firing. The GI glucosensing mechanism is unknown. We postulated that glucokinase (GK), a high–Michaelis constant (Km) hexokinase expressed in brain areas containing populations of GE and GI neurons, is the controlling step in glucosensing. Double-label in situ hybridization demonstrated neuron-specific GK mRNA expression in locus ceruleus norepinephrine and in hypothalamic neuropeptide Y, pro-opiomelanocortin, and γ-aminobutyric acid neurons, but it did not demonstrate this expression in orexin neurons. GK mRNA was also found in the area postrema/nucleus tractus solitarius region by RT-PCR. Intracarotid glucose infusions stimulated c-fos expression in the same areas that expressed GK. At 2.5 mmol/l glucose, fura-2 Ca2+ imaging of dissociated ventromedial hypothalamic nucleus neurons demonstrated GE neurons whose intracellular Ca2+ oscillations were inhibited and GI neurons whose Ca2+ oscillations were stimulated by four selective GK inhibitors. Finally, GK expression was increased in rats with impaired central glucosensing (posthypoglycemia and diet-induced obesity) but was unaffected by a 48-h fast. These data suggest a critical role for GK as a regulator of glucosensing in both GE and GI neurons in the brain.
Sustained Exposure of L6 Myotubes to High Glucose and Insulin Decreases Insulin-Stimulated GLUT4 Translocation but Upregulates GLUT4 Activity
Authors: Huang Carol , Somwar Romel , Patel Nish , Niu Wenyan , Török Dóra , Klip Amira
Published: March 2007

Hyperglycemia and hyperinsulinemia are cardinal features of acquired insulin resistance. In adipose cell cultures, high glucose and insulin cause insulin resistance of glucose uptake, but because of altered GLUT4 expression and contribution of GLUT1 to glucose uptake, the basis of insulin resistance could not be ascertained. Here we show that GLUT4 determines glucose uptake in L6 myotubes stably overexpressing myc-tagged GLUT4. Preincubation for 24 h with high glucose and insulin (high Glc/Ins) reduced insulin-stimulated GLUT4 translocation by 50%, without affecting GLUT4 expression. Insulin receptor and insulin receptor substrate-1 tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and Akt phosphorylation also diminished, as did insulin-mediated glucose uptake. However, basal glucose uptake rose by 40% without any gain in surface GLUT4. High Glc/Ins elevated basal p38 mitogen-activated protein kinase (MAPK) phosphorylation and activity, and a short inhibition of p38 MAPK with SB202190 corrected the rise in basal glucose uptake, suggesting that p38 MAPK activity contributes to this rise. We propose that in a cellular model of skeletal muscle, chronic exposure to high Glc/Ins reduced the acute, insulin-elicited GLUT4 translocation. In addition, basal state GLUT4 activity was augmented to partially compensate for the translocation defect, resulting in a more robust glucose uptake than what would be predicted from the amount of cell surface GLUT4 alone.
Redirection of Human Autoreactive T-Cells Upon Interaction With Dendritic Cells Modulated by TX527, an Analog of 1,25 Dihydroxyvitamin D3
Authors: van Halteren Astrid G.S. , van Etten Evelyne , de Jong Esther C. , Bouillon Roger , Roep Bart O. , Mathieu Chantal
Published: March 2007

The active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), is a potent immunomodulator known to affect T-cells through targeting antigen-presenting cells such as dendritic cells (DCs). We studied the effects of a novel nonhypercalcemic 1,25(OH)2D3 analog, TX527, on DC differentiation, maturation, and function with respect to stimulation of a committed human GAD65-specific autoreactive T-cell clone. Continuous addition of TX527 impaired interleukin (IL)-4 and granulocyte/macrophage colony-stimulating factor (GM-CSF)-driven DC differentiation as well as lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-induced maturation into Th1-promoting DC (DC1), as characterized by marked changes in DC morphology and abrogation of IL-12p70 release upon CD40 ligation. Addition of TX527 during maturation did not affect DC morphology but significantly changed DC cytokine profiles. The potential of treated DCs to alter the response pattern of committed autoreactive T-cells was found to depend on the timing of TX527 exposure. Continuously TX527-treated DCs significantly inhibited T-cell proliferation and blocked IFN-γ, IL-10, but not IL-13 production, whereas DCs treated during maturation failed to inhibit T-cell proliferation but affected IL-10 and IFN-γ production. Collectively, we provide evidence that nonhypercalcemic TX527 is a potent in vitro DC modulator, yielding DCs with the potential to change cytokine responses of committed autoreactive T-cells.
Influence of Experimental Diabetes on the Microcirculation of Injured Peripheral Nerve
Authors: Kennedy James M. , Zochodne Douglas W.
Published: March 2007

Regeneration of diabetic axons has delays in onset, rate, and maturation. It is possible that microangiopathy of vasa nervorum, the vascular supply of the peripheral nerve, may render an unfavorable local environment for nerve regeneration. We examined local nerve blood flow proximal and distal to sciatic nerve transection in rats with long-term (8 month) experimental streptozotocin diabetes using laser Doppler flowmetry and microelectrode hydrogen clearance polarography. We then correlated these findings, using in vivo perfusion of an India ink preparation, by outlining the lumens of microvessels from unfixed nerve sections. There were no differences in baseline nerve blood flow between diabetic and nondiabetic uninjured nerves, and vessel number, density, and area were unaltered. After transection, there were greater rises in blood flow in proximal stumps of nondiabetic nerves than in diabetic animals associated with a higher number, density, and caliber of epineurial vessels. Hyperemia also developed in distal stumps of nondiabetic nerves but did not develop in diabetic nerves. In these stumps, diabetic rats had reduced vessel numbers and smaller mean endoneurial vessel areas. Failed or delayed upregulation of nerve blood flow after peripheral nerve injury in diabetes may create a relatively ischemic regenerative microenvironment.
Activation of Nuclear Factor-κB Induced by Diabetes and High Glucose Regulates a Proapoptotic Program in Retinal Pericytes
Authors: Romeo Giulio , Liu Wei-Hua , Asnaghi Veronica , Kern Timothy S. , Lorenzi Mara
Published: March 2007

To reconstruct the events that may contribute to the accelerated death of retinal vascular cells in diabetes, we investigated in situ and in vitro the activation of nuclear factor-κB (NF-κB), which is triggered by cellular stress and controls several programs of gene expression. The retinal capillaries of diabetic eye donors showed an increased number of pericyte nuclei positive for NF-κB, when compared with nondiabetic donors, whereas endothelial cells were negative. Microvascular cell apoptosis and acellular capillaries were increased only in the diabetic donors with numerous NF-κB-positive pericytes. Likewise, high glucose in vitro activated NF-κB in retinal pericytes but not in endothelial cells, and increased apoptosis only in pericytes. Studies with NF-κB inhibitors suggested that in pericytes, basal NF-κB has prosurvival functions, whereas NF-κB activation induced by high glucose is proapoptotic. Pericytes exposed to high glucose showed increased expression of Bax and of tumor necrosis factor-α, which were prevented by the NF-κB inhibitors and mimicked by transfection with the p65 subunit of NF-κB, and failed to increase the levels of the NF-κB-dependent inhibitors of apoptosis. Colocalization of activated NF-κB and Bax overexpression was observed in the retinal pericytes of diabetic donors. A proapoptotic program triggered by NF-κB selectively in retinal pericytes in response to hyperglycemia is a possible mechanism for the early demise of pericytes in diabetic retinopathy.