OBJECTIVE: Sirtuins (SIRTs) are NAD+-dependent deacetylases that regulate metabolism and life span. We used peripheral blood mononuclear cells (PBMCs) to determine ex vivo whether insulin resistance/metabolic syndrome influences SIRTs. We also assessed the potential mechanisms linking metabolic alterations to SIRTs in human monocytes (THP-1) in vitro. RESEARCH DESIGN AND METHODS: SIRT1-SIRT7 gene and protein expression was determined in PBMCs of 54 subjects (41 with normal glucose tolerance and 13 with metabolic syndrome). Insulin sensitivity was assessed by the minimal model analysis. Subclinical atherosclerosis was assessed by carotid intima-media thickness (IMT). In THP-1 cells exposed to high glucose or fatty acids in vitro, we explored SIRT1 expression, p53 acetylation, Jun NH2-terminal kinase (JNK) activation, NAD+ levels, and nicotinamide phosphoribosyltransferase (NAMPT) expression. The effects of SIRT1 induction by resveratrol and of SIRT1 gene silencing were also assessed. RESULTS: In vivo, insulin resistance and metabolic syndrome were associated with low PBMC SIRT1 gene and protein expression. SIRT1 gene expression was negatively correlated with carotid IMT. In THP-1 cells, high glucose and palmitate reduced SIRT1 and NAMPT expression and reduced the levels of intracellular NAD+ through oxidative stress. No effect was observed in cells exposed to linoleate or insulin. High glucose and palmitate increased p53 acetylation and JNK phosphorylation; these effects were abolished in siRNA SIRT1–treated cells. Glucose- and palmitate-mediated effects on NAMPT and SIRT1 were prevented by resveratrol in vitro. CONCLUSIONS: Insulin resistance and subclinical atherosclerosis are associated with SIRT1 downregulation in monocytes. Glucotoxicity and lypotoxicity play a relevant role in quenching SIRT1 expression.

Primary aldosteronism is the most common form of secondary hypertension. Mutations in the KCNJ5 gene have been described recently in aldosterone-producing adenomas (APAs). The aim of this study was to investigate the prevalence of KCNJ5 mutations in unselected patients with primary aldosteronism and their clinical, biological and molecular correlates. KCNJ5 sequencing was performed on somatic (APA, n=380) and peripheral (APA, n=344; bilateral adrenal hyperplasia, n=174) DNA of patients with primary aldosteronism, collected through the European Network for the Study of Adrenal Tumors. Transcriptome analysis was performed in 102 tumors. Somatic KCNJ5 mutations (p.Gly151Arg or p.Leu168Arg) were found in 34% (129 of 380) of APA. They were significantly more prevalent in females (49%) than males (19%; P <10 ⁻³ ) and in younger patients (42.1±1.0 versus 47.6±0.7 years; P <10 ⁻³ ) and were associated with higher preoperative aldosterone levels (455±26 versus 376±17 ng/L; P =0.012) but not with therapeutic outcome after surgery. Germline KCNJ5 mutations were found neither in patients with APA nor those with bilateral adrenal hyperplasia. Somatic KCNJ5 mutations were specific for APA, because they were not identified in 25 peritumoral adrenal tissues or 16 cortisol-producing adenomas. Hierarchical clustering of transcriptome profiles showed that APAs with p.Gly151Arg or p.Leu168Arg mutations were indistinguishable from tumors without KCNJ5 mutations. In conclusion, although a large proportion of sporadic APAs harbors somatic KCNJ5 mutations, germline mutations are not similarly causative for bilateral adrenal hyperplasia. KCNJ5 mutation carriers are more likely to be females; younger age and higher aldosterone levels at diagnosis suggest that KCNJ5 mutations may be associated with a more florid phenotype of primary aldosteronism.

Objective— Hyperglycemia is the main determinant of long-term diabetic complications, mainly through induction of oxidative stress. NAD(P)H oxidase is a major source of glucose-induced oxidative stress. In this study, we tested the hypothesis that rosiglitazone (RSG) is able to quench oxidative stress initiated by high glucose through prevention of NAD(P)H oxidase activation. Methods and Results— Intracellular ROS were measured using the fluoroprobe TEMPO-9-AC in HUVECs exposed to control (5 mmol/L) and moderately high (10 mmol/L) glucose concentrations. NAD(P)H oxidase and AMPK activities were determined by Western blot. We found that 10 mmol/L glucose increased significantly ROS production in comparison with 5 mmol/L glucose, and that this effect was completely abolished by RSG. Interestingly, inhibition of AMPK, but not PPARγ, prevented this effect of RSG. AMPK phosphorylation by RSG was necessary for its ability to hamper NAD(P)H oxidase activation, which was indispensable for glucose-induced oxidative stress. Downstream of AMPK activation, RSG exerts antioxidative effects by inhibiting PKC. Conclusions— This study demonstrates that RSG activates AMPK which, in turn, prevents hyperactivity of NAD(P)H oxidase induced by high glucose, possibly through PKC inhibition. Therefore, RSG protects endothelial cells against glucose-induced oxidative stress with an AMPK-dependent and a PPARγ-independent mechanism.

Objectives The longitudinal relationship between coffee use and hypertension is still controversial. Cytochrome P450 1A2 (CYP1A2) is the main responsible enzyme for the metabolism of caffeine. The aim of the present study was to investigate the effect of coffee intake on the risk of developing hypertension needing antihypertensive treatment in individuals stratified by CYP1A2 genotype. Design We assessed prospectively 553 young White individuals screened for stage 1 hypertension. Coffee intake was ascertained from regularly administered questionnaires. Incident physician-diagnosed hypertension was the outcome measure. Genotyping of CYP1A2 SNP was performed by real time PCR. Results During a median follow-up of 8.2 years, 323 individuals developed hypertension. For carriers of the slow *1F allele (59%), hazard ratios of hypertension from multivariable Cox analysis were 1.00 in abstainers (reference), 1.72 (95%CI, 1.21–2.44) in moderate coffee drinkers (P = 0.03), and 3.00 (1.53–5.90) in heavy drinkers (P = 0.001). In contrast, hazard ratios for coffee drinkers with the rapid *1A/*1A genotype were 0.80 (0.52–1.23, P = 0.29) for moderate drinkers and 0.36 (0.14–0.89, P = 0.026) for heavy drinkers. In a two-way ANCOVA, a gene × coffee interactive effect was found on follow-up changes in systolic (P = 0.000) and diastolic (P = 0.007) blood pressure. Urinary epinephrine was higher in coffee drinkers than abstainers but only among individuals with slow *1F allele (P = 0.001). Conclusion These data show that the risk of hypertension associated with coffee intake varies according to CYP1A2 genotype. Carriers of slow *1F allele are at increased risk and should thus abstain from coffee, whereas individuals with *1A/*1A genotype can safely drink coffee.

Background. A silent polymorphism (+1166 A/C single-nucleotide polymorphism) localized in the 3′-UTR (untranslated region) of the human angiotensin II type-1 r

Diabetes compromises the bone marrow (BM) microenvironment and reduces the number of circulating CD34+ cells. Diabetic autonomic neuropathy (DAN) may impact the BM, because the sympathetic nervous system is prominently involved in BM stem cell trafficking. We hypothesize that neuropathy of the BM affects stem cell mobilization and vascular recovery after ischemia in patients with diabetes. We report that, in patients, cardiovascular DAN was associated with fewer circulating CD34+ cells. Experimental diabetes (streptozotocin-induced and ob/ob mice) or chemical sympathectomy in mice resulted in BM autonomic neuropathy, impaired Lin−cKit+Sca1+ (LKS) cell and endothelial progenitor cell (EPC; CD34+Flk1+) mobilization, and vascular recovery after ischemia. DAN increased the expression of the 66-kDa protein from the src homology and collagen homology domain (p66Shc) and reduced the expression of sirtuin 1 (Sirt1) in mice and humans. p66Shc knockout (KO) in diabetic mice prevented DAN in the BM, and rescued defective LKS cell and EPC mobilization. Hematopoietic Sirt1 KO mimicked the diabetic mobilization defect, whereas hematopoietic Sirt1 overexpression in diabetes rescued defective mobilization and vascular repair. Through p66Shc and Sirt1, diabetes and sympathectomy elevated the expression of various adhesion molecules, including CD62L. CD62L KO partially rescued the defective stem/progenitor cell mobilization. In conclusion, autonomic neuropathy in the BM impairs stem cell mobilization in diabetes with dysregulation of the life-span regulators p66Shc and Sirt1.