Interaction of glucose and long chain fatty acids (C18) on antioxidant defences and free radical damage in porcine vascular smooth muscle cells in vitro.

Aims/hypothesis: Abnormalities of glucose and fatty acid metabolism in diabetes are believed to contribute to the development of oxidative stress and the long term vascular complications of the disease therefore the interactions of glucose and long chain fatty acids on free radical damage and endogenous antioxidant defences were investigated in vascular smooth muscle cells. Methods: Porcine vascular smooth muscle cells were cultured in 5 mmol/l or 25 mmol/l glucose for ten days. Fatty acids, stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and gamma-linolenic acid (18:3) were added with defatted bovine serum albumin as a carrier for the final three days. Results. Glucose (25 mmol/l) alone caused oxidative stress in the cells as evidenced by free radical-mediated damage to DNA, lipids, and proteins. The addition of fatty acids (0.2 mmol/l) altered the profile of free radical damage; the response was J-shaped with respect to the degree of unsaturation of each acid, and oleic acid was associated with least damage. The more physiological concentration (0.01 mmol/l) of gamma-linolenic acids was markedly different in that, when added to 25 mmol/l glucose it resulted in a decrease in free radical damage to DNA, lipids and proteins. This was due to a marked increase in levels of the antioxidant, glutathione, and increased gene expression of the rate-limiting enzyme in glutathione synthesis, gamma-glutamylcysteine synthetase. Conclusion/Interpretation: The results clearly show that glucose and fatty acids interact in the production of oxidative stress in vascular smooth muscle cells.

Glucose-induced phosphatidylinositol 3-kinase and mitogen-activated protein kinase-dependent upregulation of the platelet-derived growth factor-B receptor potentiates vascular smooth muscle cell chemotaxis

The aim of this study was to investigate the effects of elevated D-glucose concentrations on vascular smooth muscle cell (VSMC) expression of the platelet-derived growth factor (PDGF) beta receptor and VSMC migratory behavior. Immunoprecipitation, immunofluorescent staining, and RT-PCR of human VSMCs showed that elevated D-glucose induced an increase in the PDGF beta receptor that was inhibited by phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway inhibitors. Exposure to 25 mmol/l D-glucose (HG) induced increased phosphorylation of protein kinase B (PKB) and extracellular-regulated kinase (ERK). All HG chemotaxis assays (with either 10 days' preincubation in HG or no preincubation) in a FCS or PDGF-BB gradient showed positive chemotaxis, whereas those in 5 mmol/l D-glucose did not. Assays were also run with concentrations ranging from 5 to 25 mmol/l D-glucose. Chemotaxis was induced at concentrations >9 mmol/l D-glucose. An anti-PDGF beta receptor antibody inhibited glucose-potentiated VSMC chemotaxis, as did the inhibitors for the PI3K and MAPK pathways. This study has shown that small increases in D-glucose concentration, for a short period, increase VSMC expression of the PDGF beta receptor and VSMC sensitivity to chemotactic factors in serum, leading to altered migratory behavior in vitro. It is probable that similar processes occur in vivo with glucose-enhanced chemotaxis of VSMCs, operating through PDGF beta receptor-operated pathways, contributing to the accelerated formation of atheroma in diabetes.

Assocation Between Variation in the Actin-Binding Gene Caldesmon and Diabetic Nephropathy in Type 1 Diabetes

Dysfunction of the actin cytoskeleton is a key event in the pathogenesis of diabetic nephropathy. We previously reported that certain cytoskeletal genes are upregulated in mesangial cells exposed to a high extracellular glucose concentration. One such gene, caldesmon, lies on chromosome 7q35, a region linked to nephropathy in family studies, making it a candidate susceptibility gene for diabetic nephropathy. We screened all exons, untranslated regions, and a 5-kb region upstream of the gene for variation using denaturing high-performance liquid chromatography technology. An A>G single nucleotide polymorphism (SNP) at position -579 in the promoter region was associated with nephropathy in a case-control study using 393 type 1 diabetic patients from Northern Ireland (odds ratio [OR] 1.38, 95% CI 1.02–1.86, P = 0.03). A similar trend was found in an independent sample from a second center. When the sample groups were combined (n = 606), the association between the -579G allele and nephropathy remained significant (OR 1.35, 1.07–1.70, P = 0.01). The haplotype structure in the surrounding 7-kb region was determined. No single haplotype was more strongly associated with nephropathy than the -579A>G SNP. These results suggest a role for the caldesmon gene in susceptibility to diabetic nephropathy in type 1 diabetes.

Effect of eucaloric high- and low-sucrose diets with identical macronutrient profile on insulin resistance and vascular risk - A randomized controlled trial

The long-term impact of dietary carbohydrate type, in particular sucrose, on insulin resistance and the development of diabetes and atherosclerosis is not established. Current guidelines for the healthy population advise restriction of sucrose intake. We investigated the effect of high- versus low-sucrose diet (25 vs. 10%, respectively, of total energy intake) in 13 healthy subjects aged 33 +/- 3 years (mean +/- SE), BMI 26.6 +/- 0.9 kg/m(2), in a randomized crossover design with sequential 6-week dietary interventions separated by a 4-week washout. Weight maintenance, eucaloric diets with identical macronutrient profiles and fiber content were designed. All food was weighed and distributed. Insulin action was assessed using a two-step euglycemic clamp; glycemic profiles were assessed by the continuous glucose monitoring system and vascular compliance by pulse-wave analysis. There was no change in weight across the study. Peripheral glucose uptake and suppression of endogenous glucose production were similar after each diet. Glycemic profiles and measures of vascular compliance did not change. A rise in total and LDL cholesterol was observed. In this study, a high-sucrose intake as part of an eucaloric, weight-maintaining diet had no detrimental effect on insulin sensitivity, glycemic profiles, or measures of vascular compliance in healthy nondiabetic subjects.

The regulation and role of osteopontin in malignant transformation and cancer.

Osteopontin (OPN) is a predominantly secreted extracellular matrix glycophosphoprotein which binds to alpha v-containing integrins and has an important role in malignant cell attachment and invasion. High OPN expression in the primary tumor is associated with early metastasis and poor outcome in human breast and other cancers. Forced OPN overexpression in benign cells may induce neoplastic-like cell behaviour including increased attachment and invasion in vitro as well as the ability to metastasize in vivo. Conversely, OPN inhibition by antisense cDNA impedes cell growth and tumor forming capacity. OPN is not mutationally activated in cancer but its expression is regulated by Wnt/Tcf signaling, steroid receptors, growth factors, ras, Ets and AP-1 transcription factors. Presumably these factors are implicated in induction of OPN overexpression in cancer. Greater understanding of the role of OPN in neoplastic change and its transcriptional regulation may enable development of novel cancer treatment strategies

Chemical ablation of gastric inhibitory polypeptide receptor action by daily (Pro3) GIP administration improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure in obesity-diabetes.

Glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide [GIP]) is an important incretin hormone secreted by endocrine K-cells in response to nutrient ingestion. In this study, we investigated the effects of chemical ablation of GIP receptor (GIP-R) action on aspects of obesity-related diabetes using a stable and specific GIP-R antagonist, (Pro3)GIP. Young adult ob/ob mice received once-daily intraperitoneal injections of saline vehicle or (Pro3)GIP over an 11-day period. Nonfasting plasma glucose levels and the overall glycemic excursion (area under the curve) to a glucose load were significantly reduced (1.6-fold; P <0.05) in (Pro3)GIP-treated mice compared with controls. GIP-R ablation also significantly lowered overall plasma glucose (1.4-fold; P <0.05) and insulin (1.5-fold; P <0.05) responses to feeding. These changes were associated with significantly enhanced (1.6-fold; P <0.05) insulin sensitivity in the (Pro3)GIP-treated group. Daily injection of (Pro3)GIP reduced pancreatic insulin content (1.3-fold; P <0.05) and partially corrected the obesity-related islet hypertrophy and ß-cell hyperplasia of ob/ob mice. These comprehensive beneficial effects of (Pro3)GIP were reversed 9 days after cessation of treatment and were independent of food intake and body weight, which were unchanged. These studies highlight a role for GIP in obesity-related glucose intolerance and emphasize the potential of specific GIP-R antagonists as a new class of drugs for the alleviation of insulin resistance and treatment of type 2 diabetes.

Cox-2 Promotes Chromogranin A Expression and Bioactivity: Evidence for a PGE2-Dependent Mechanism and the Involvement of a Proximal cAMP-Responsive Element.

Abstract The prostanoid biosynthetic enzyme cyclooxygenase-2 (Cox-2) is upregulated in several neuroendocrine tumors. The aim of the current study was to employ a neuroendocrine cell (PC12) model of Cox-2 over-expression to identify gene products that might be implicated in the oncogenic and/or inflammatory actions of this enzyme in the setting of neuroendocrine neoplasia. Expression array and real-time PCR analysis demonstrated that levels of the neuroendocrine marker chromogranin A (CGA) were 2-fold and 3.2-fold higher, respectively, in Cox-2 over-expressing cells (PCXII) vs their control (PCMT) counterparts. Immunocytochemical and immunoblotting analyses confirmed that both intracellular and secreted levels of CGA were elevated in response to Cox-2 induction. Moreover, exogenous addition of prostaglandin E2 (1u�­M), mimicked this effect in PCMT cells, while treatment of PCXII cells with the Cox-2 selective inhibitor NS-398 (100 nM) reduced CGA expression levels, thereby confirming the biospecificity of this finding. Levels of neurone specific enolase (NSE) were similar in the two cell lines, suggesting that the effect of Cox-2 on CGA expression was specific and not due to a global enhancement of neuroendocrine marker expression/differentiation. Cox-2-dependent CGA upregulation was associated with significantly increased chromaffin granule number and intracellular and secreted levels of dopamine. CGA promoter-driven reporter gene expression studies provided evidence that prostaglandin E2-dependent upregulation required a proximal cAMP-responsive element (CRE; -71 - -64 bp). This study is the first to demonstrate that Cox-2 upregulates both CGA expression and bioactivity in a neuroendocrine cell line and has major implications for the role of this polypeptide in the pathogenesis of neuroendocrine cancers in which Cox-2 is upregulated.

A longitudinal study of infant feeding and obesity throughout the life course

Background: The Centers for Disease Control and Prevention and the US Department of Health and Human Services promote breastfeeding as a strategy for reducing childhood overweight. We evaluated the relation between infant feeding and the development of overweight and obesity throughout life course. Methods: We investigated the association between infant feeding and obesity among 35 526 participants in the Nurses' Health Study II who were followed prospectively from 1989 to 2001. Mothers of participants provided information by mailed questionnaires on the duration of breast- and bottle-feeding, as well as the type of milk or milk substitute in the bottle. Information on body shape at ages 5 and 10, weight at age 18, current weight between 1989 and 2001, and height was reported by the participants. Results: The duration of breastfeeding, including exclusive breastfeeding, was not related to being overweight (25 body mass index (BMI)