Andrea Semplicini

Downregulation of the Longevity-Associated Protein Sirtuin 1 in Insulin Resistance and Metabolic Syndrome: Potential Biochemical Mechanisms

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.

Reduced expression of regulator of G-protein signaling 2 (RGS2) in hypertensive patients increases calcium mobilization and ERK1/2 phosphorylation induced by angiotensin II.

Context RGS2 (regulators of G-protein signaling) is a negative regulator of Gαq protein signaling, which mediates the action of several vasoconstrictors. RGS2-deficient mouse line exhibits a hypertensive phenotype and a prolonged response to vasoconstrictors. Objective To compare RGS2 expression in peripheral blood mononuclear cells (PBMs) and cultured fibroblasts from normotensive subjects and hypertensive patients. Methods PBMs were isolated from 100 controls and 150 essential hypertensives. Additionally, fibroblasts were isolated from skin biopsy of 11 normotensives and 12 hypertensives and cultured up to the third passage. Quantitative mRNA and protein RGS2 expression were performed by real-time quantitative reverse transcriptase-polymerase chain reaction and by immunoblotting, respectively. Free Ca2+ measurement was performed in monolayers of 24-h serum-deprived cells, using FURA-2 AM. Phosphorylation of the extracellular signal-regulated kinases ERK1/2 was measured by immunoblotting. Polymorphism (C1114G) in the 3′ untranslated region of the RGS2 gene was investigated by direct sequencing and real-time polymerase chain reaction (PCR). Results RGS2 mRNA expression was significantly lower in PBM and in fibroblasts from hypertensives, in comparison to normotensives. C1114G polymorphism was associated with RGS2 expression, with the lowest values in GG hypertensives. The 1114G allele frequency was increased in hypertensives compared with normotensives. Angiotensin II-stimulated intracellular Ca2+ increase and ERK1/2 phosphorylation were higher in fibroblasts from hypertensive patients compared with control subjects, and in those with the G allele, independently of the blood pressure status. The angiotensin II-stimulated Ca2+ mobilization and ERK1/2 phosphorylation were negatively correlated with RGS2 mRNA expression. Conclusion Low expression of RGS2 contributes to increased G-protein-coupled signaling in hypertensive patients. The allele G is associated with low RGS2 expression and blood pressure increase in humans.

G-Protein β3-Subunit Gene 825T Allele and Hypertension: A Longitudinal Study in Young Grade I Hypertensives

Introduction Essential hypertension affects approximately 25% of individuals in Western societies, with an increased prevalence in older subjects. It has long been recognized that a significant part of the susceptibility for hypertension is inherited. However, unlike monogenic disorders, hypertension develops on the genetic background of multiple gene alterations in concert with environmental factors, eg, nutrition and physical activity. The hunt for hypertension susceptibility genes is nourished from different aspects. One goal is easily understood: if a causative mutation or polymorphism is found, there exists, at least theoretically, the possibility to modify the activity of the gene product through existing or novel drugs. Moreover, since hypertension is not a disorder per se but a major risk factor for stroke, left ventricular hypertrophy, myocardial, and renal insufficiency, genetic testing could identify individuals at highest risk in order to provide them with optimized medical care to prevent the aforementioned sequels. Finally, one could envisage a scenario in which certain genotypes may be used to guide antihypertensive therapy in terms of drug class and dosage.Abstract—The 825T allele of the GNB3 gene has been associated with essential hypertension and obesity in cross-sectional studies. We have therefore planned a longitudinal cohort study to assess whether the GNB3 825T allele is predictive of blood pressure increase in young subjects with grade I hypertension. We genotyped at the GNB3 825 locus 461 participants of the Hypertension and Ambulatory Recording Venetia Study (HARVEST) study (age, 18 to 45 years) at low cardiovascular risk, according to 1999 ISH/WHO criteria. The study end point was eligibility for antihypertensive medication, that is, progression to grade II hypertension during the first year of observation or office systolic blood pressure ≥150 mm Hg and/or office diastolic blood pressure ≥95 mm Hg in two later consecutive visits during follow-up. At baseline, there was no statistically significant difference among genotypes with respect to body mass index, blood pressure, and heart rate. During follow-up (mean, 4.7 years), 113 (51.1%) patients with CC genotype and 145 (60.4%) patients with TT/TC genotype reached the end point. According to survival analysis, the patients carrying the 825T allele had an increased risk of reaching the blood pressure end point (CI, 1.108 to 1.843; P =0.006). In young patients with grade I hypertension, the 825T allele is associated with increased risk of progression to more severe hypertension requiring antihypertensive therapy. The GNB3 825T allele may be considered a genetic marker of predisposition for hypertension.

Angiotensin II-stimulated collagen production in cardiac fibroblasts is mediated by reactive oxygen species

Objective The aim of the present study was to determine whether inhibition of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] oxidase and of various superoxide generating systems could affect the collagen production, the mRNA and protein expression of collagen types I and III in control and angiotensin II-treated cardiac fibroblasts. Methods Cardiac fibroblasts from passage 2 from normal male adult rats were cultured to confluency and incubated in serum-free Dulbeccos modified Eagles medium for 24 h. The cells were then preincubated with(out) the tested inhibitors for 1 h and then further incubated with(out) angiotensin II (1 μmol/l) for 24 h. Collagen production was measured spectrophotometrically with picrosirius red as dye and with [3H]proline incorporation; collagen type I and III content by enzyme-linked immunosorbent assay and collagen type I and III mRNA expression by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). NAD(P)H-dependent superoxide anion production was assayed as superoxide dismutase-inhibitable cytochrome c reduction. Intracellular formation of reactive oxygen species was assessed with 2’,7’-dichlorofluorescein diacetate as fluorescent probe. Results Angiotensin II stimulated the collagen production, the collagen I and III content and mRNA expression in cardiac fibroblasts, and apocynin, a membrane NAD(P)H oxidase inhibitor, abolished this induction. Rotenone, allopurinol, indomethacin, nordihydroguiaretic acid, ketoconazole and nitro-L-arginine (inhibitors of mitochondrial NAD(P)H oxidase, xanthine oxidase, cyclooxygenase, lipoxygenase, cytochrome P450 oxygenase and nitric oxide synthase, respectively) did not affect the angiotensin II-induced collagen production. Angiotensin II increased the NAD(P)H-dependent superoxide anion production and the intracellular generation of reactive oxygen species in cardiac fibroblasts, and apocynin abrogated this rise. Conclusions Our data show that in adult rat cardiac fibroblasts the membrane-associated NAD(P)H oxidase complex is the predominant source of superoxide anion and reactive oxygen species generation in angiotensin II-stimulated adult cardiac fibroblasts. Inhibition of this NAD(P)H oxidase complex with apocynin completely blocked the angiotensin II-stimulated collagen production, and collagen I and III protein and mRNA expression.

Controlled study of the effect of angiotensin converting enzyme inhibition versus calcium-entry blockade on insulin sensitivity in overweight hypertensive patients : Trandolapril Italian Study (TRIS)

OBJECTIVE The aim of this study was to evaluate the effect of trandolapril, an angiotensin converting enzyme inhibitor, on blood pressure, forearm blood flow and insulin sensitivity in comparison with nifedipine gastrointestinal therapeutic system. PATIENTS AND METHODS This is a multicentre, two-way parallel-group, open-label comparative study in 90 overweight hypertensive patients, who were randomly assigned to treatment for 8 weeks with either trandolapril or nifedipine. At baseline and after treatment, all patients underwent an oral glucose tolerance test, an evaluation of their metabolic profiles and a euglycaemic hyperinsulinaemic clamp test. In a subgroup of 18 patients, a forearm study was carried out. RESULTS Blood pressure fell by the second week of treatment and remained significantly reduced compared with baseline in both treatment groups. Plasma triglyceride levels were also significantly reduced after trandolapril therapy, but no significant changes occurred in the other metabolic parameters during treatment with either drug. During the euglycaemic hyperinsulinaemic clamp, whole-body glucose use was similar in the two treatment groups at baseline, and a moderate but statistically significant increase in insulin sensitivity was observed after trandolapril treatment (trandolapril: 5.0 +/- 0.2 versus 4.5 +/- 0.2 mg/kg per min; nifedipine: 4.1 +/- 0.3 versus 4.2 +/- 0.3 mg/kg per min; P < 0.05, versus baseline and trandolapril versus nifedipine treatment). Skeletal muscle glucose uptake was significantly higher after trandolapril than after nifedipine therapy (5.0 +/- 0.7 and 3.0 +/- 0.4 mg/min, respectively; P < 0.01). As forearm blood flow was similar in the two treatment groups at baseline and was unchanged after 8 weeks of therapy, skeletal muscle glucose extraction was significantly greater in the ACE inhibitor treated-group than in the nifedipine comparative group (trandolapril: baseline 21 +/- 2, treatment 24 +/- 3 mg/dl; nifedipine: baseline 18 +/- 3, treatment 16 +/- 2 mg/dl; P < 0.05, trandolapril versus nifedipine treatment). CONCLUSIONS During short-term treatment, ACE inhibition with trandolapril was able to moderately improve insulin sensitivity, in comparison with calcium blockade, and this effect appeared to be independent of the haemodynamic action of the drug.

Silencing regulator of G protein signaling-2 (RGS-2) increases angiotensin II signaling: insights into hypertension from findings in Bartter's/Gitelman's syndromes.

Objective Regulator of G-protein signaling (RGS)-2 is a regulator of angiotensin II (Ang II) signaling. In Bartters syndrome/Gitelmans syndrome (BS/GS), we have demonstrated increased RGS-2 levels and blunted Ang II signaling which contribute to their reduced vasomotor tone and remodeling. The present study investigates the effect of silencing RGS-2 in fibroblasts from six BS/GS patients on intracellular Ca2+ ( ) mobilization and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, established Ang II-mediated responses. Methods Fibroblasts were RGS-2 silenced by transfecting chemically synthesized small interfering RNA. Silencing efficiency and Ang II-induced ERK 1/2 phosphorylation were evaluated by western blot and Ang II-induced using Fura-2 AM. Results RGS-2 expression in not silenced BS/GS fibroblasts from patients is increased compared with healthy controls [0.34 ± 0.02 vs. 0.19 ± 0.01 densitometric units (d.u.), P = 0.0005]. Silencing RGS-2 in BS/GS patients was achieved to the level of controls. Ang II-induced release and ERK 1/2 phosphorylation were reduced in not silenced cells from BG/GS patients compared with controls (112.16 ± 13.2 vs. 130.33 ± 13.64 mmol/l, P = 0.011 and 0.64 ± 0.08 vs. 0.91 ± 0.03 mmol/l, P < 0.006, respectively). Silencing RGS-2 in BS/GS patients increased Ang II-induced release and ERK 1/2 phosphorylation in silenced cells compared with not silenced cells [59.3 ± 10.8 (peak-basal) vs. 40.5 ± 14.1 nmol/l, P = 0.017 and 0.84 ± 0.06 vs. 0.64 ± 0.08 nmol/l, P < 0.03, respectively], whereas they were not different compared with controls (60.1 ± 4.3 and 0.91 ± 0.03 nmol/l). Integrating the response over time showed increased area under the curve (AUC) of BS/GS silenced cells compared with that of not silenced cells (P = 0.013). Conclusion This is the first report of silencing RGS-2 effect on Ang II signaling in a human clinical condition of altered vascular tone regulation and remodeling and establishes RGS-2 as a key regulatory element of Ang II signaling in humans.

Kinetics and stoichiometry of the human red cell Na+/H+ exchanger

SummaryWe have investigated the kinetic properties of the human red blood cell Na+/H+ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H+ (Hi) and external Na+ (Nao) in red blood cells of normal subjects.Red blood cells with different cell Na+ (Nai) and pH (pHi) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na+ influx were measured by varying pHi (from 5.7 to 7.4), external pH (pHo), Nai and Nao and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na+ influx (Nai<2.0 mmol/liter cell, Nao= 150mm) increased sigmoidally (Hill coefficient 2.5) when pHi fell below 7.0 and the external pHo was 8.0, but increased linearly at pHo 6.0. The net Na+ influx driven by an outward H+ gradient was estimated from the difference of Na+ influx at the two pHo levels (pHo 8 and pHo 6). The H+-driven Na+ influx reached saturation between pHi 5.9 and 6.1. TheVmax had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheKm for Hi to activate H+-driven Na+ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 μm) partially (59±10%) inhibited red cell Na+/H+ exchange. The stoichiometric ratio between H+-driven Na+ influx and Na+-driven H+ efflux was 1∶1. The dependence of Na+ influx from Nao was studied at pHi 6.0, and Nai lower than 2 mmol/liter cell at pHo 6.0 and 8.0. The meanKm for Nao of the H+-gradient-driven Na+ influx was 55±7mm.An increase in Nai from 2 to 20 mmol/liter cell did not change significantly H+-driven net Na+ influx as estimated from the difference between unidirectional22Na influx and efflux. Na+/Na+ exchange was negligible in acid-loaded, DIDS-treated cells. Na+ and H+ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na+ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na+/H+ exchanger.It is concluded that human red cell Na+/H+ exchange performs 1∶1 exchange of external Na+ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H+ and external Na+ is similar to other cells, but it displays a larger variability in theVmax between individuals.

Sodium-Lithium Countertransport Has Low Affinity for Sodium in Hyperinsulinemic Hypertensive Subjects

We recently reported that incubation of red blood cells with insulin markedly decreases the affinity for external Na+ and increases the maximal transport rate (Vmax) of Na(+)-Li+ countertransport. The association of hypertension with insulin resistance and its compensatory hyperinsulinemia led us to investigate the relationship between insulin levels in vivo and the Na+ activation kinetics of this antiporter. We studied normotensive (n = 28) and hypertensive (n = 25) subjects after they had fasted overnight and determined their plasma glucose and insulin concentrations. Insulin levels were higher in the hypertensive subjects (11.7 +/- 1.5 microU/mL, mean +/- SEM) than in the normotensive subjects (8.2 +/- 1.2 microU/mL), but glucose levels were similar and within normal limits. Antiporter activity was measured as sodium-stimulated Li+ efflux by a new procedure that uses isosmotic conditions to raise external Na+ to 280 mmol/L. In normotensive subjects, Vmax was reached between 50 and 100 mmol/L Na+, whereas in most hypertensive subjects, Na+ concentrations higher than 150 mmol/L were needed. This different kinetic behavior was because the Na+ concentration for half-maximal activation (Km) was twofold higher in hypertensive subjects (58.9 +/- 5.3 mmol/L) than in normotensive subjects (29.8 +/- 2.6 mmol/L, P < .001). Hypertensive subjects with fasting insulin levels greater than 10 microU/mL (n = 12) had a higher Km for Na+ than subjects with insulin levels less than 10 microU/mL (n = 13) (73.4 +/- 8.7 versus 45.6 +/- 3.9 mmol/L, respectively, P < .01) and similar Vmax (0.57 +/- 0.05 versus 0.41 +/- 0.05 mmol.L-1.h-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Renal vein renin measurements accurately identify renovascular hypertension caused by total occlusion of the renal artery.

Objective To assess the usefulness of indexes derived from renal vein renin measurements. Design A 12-year prospective study. Setting A tertiary institutional referral centre. Patients and methods Between 1988 and 2000, we studied 152 consecutive hypertensive patients with a high pre-test probability of renovascular hypertension (RVH). RVH was diagnosed retrospectively on the basis of reduction in blood pressure after correction of ischaemia at follow-up. Renal vein renin measurements were used to calculate the ratios: Visch/Vctl (renal vein renin ratio; RVRR); Vctl/Viivc; (Visch − Viivc)/Viivc; (Vctl − Viivc)/Viivc, where Visch and Vctl indicate plasma renin activity (PRA) in the ischaemic and contralateral renal veins, respectively, and Viivc denotes PRA in the infrarenal inferior vena cava. A receiver operator characteristics (ROC) curve analysis was used to determine the cut-off value of renal vein renin measurement indexes that provided the best discrimination between patients with and without RVH and to identify patients with RVH caused by total occlusion of the renal artery. Results Sixty-seven patients were diagnosed as having RVH: 51 had significant renal artery stenoses (RVH non-occluded) and 16 had total renal artery occlusion (RVH occluded). Of the remaining 85 patients in whom RVH was excluded (non-RVH group), 27 had reno-parenchymal hypertension and 58 had essential hypertension. Of the renal vein renin measurement indexes, only RVRR and (Visch − Viivc)/Viivc in RVH-occluded patients differed significantly (P < 0.005) from those in the non-RVH group and showed the best performance by ROC curve analysis. This analysis also showed that, at any cut-off value, RVRR was far more accurate for identification of RVH-occluded patients than for identification of RVH non-occluded patients, both in the subgroup with unilateral and, even more so, in those with bilateral renal artery lesions. The best trade-off between sensitivity and false-positive rate was provided by cut-off values of 1.55 and 1.70 of the RVRR for identification of non-occluded and occluded RVH, respectively. Conclusions RVRR is more useful for establishing an indication for nephrectomy in patients with renal artery occlusion than for identifying those patients with renal artery stenosis who will benefit from revascularization. In patients with RVH with bilateral renal artery lesions, lateralization of renin secretion occurs only in the presence of total renal artery occlusion. Different cut-off values are necessary for identification of non-occluded and occluded RVH.

Rho kinase and PAI-1 in Bartter's/Gitelman's syndromes: relationship to angiotensin II signaling.

Objective Angiotensin II (Ang II)-mediated activation of Rho kinase (ROK) is involved in the pathophysiology of hypertension and cardiovascular remodeling. ROK also controls plasminogen activator inhibitor-1 (PAI-1) which promotes vascular fibrosis contributing to atherogenesis. Bartters and Gitelmans syndromes (BS/GS) are useful models to investigate abnormalities of vascular tone regulation, due to their reduced short- and long-term signaling pathways of Ang II. This study evaluated, using BS/GS as a model, ROK and PAI-1 gene and protein expression and the effect of Ang II co-incubation on ROK and PAI-1 gene and protein expression. Design, methods and results We measured ROK and PAI-1 gene and protein expression [reverse transcription-polymerase chain reaction (RT-PCR) and Western blot] in mononuclear cells (PBM) from one BS and eight GS patients. The effect of Ang II on ROK and PAI-1 gene and protein expression was also evaluated and compared with 10 controls. ROK gene and protein expression was reduced in BS/GS [0.47 ± 0.11 densitometric units (d.u.) versus 0.70 ± 0.04 d.u., P = 0.0038 and 0.39 ± 0.07 d.u. versus 0.55 ± 0.07 d.u., P = 0.0026, respectively]. The basal level of PAI-1 gene and protein expression did not differ (0.40 ± 0.03 d.u. versus 0.39 ± 0.02 d.u. and 0.81 ± 0.02 d.u. versus 0.83 ± 0.02 d.u., respectively). Ang II increased ROK and PAI-1 gene and protein expression only in controls: from 0.70 ± 0.04 to 0.90 ± 0.06 d.u., P = 0.007 (ROK mRNA); from 0.55 ± 0.07 to 0.86 ± 0.07 d.u., P = 0.0005 (ROK protein); from 0.40 ± 0.02 to 0.63 ± 0.03 d.u., P = 0.001 (PAI-1 mRNA); and from 0.83 ± 0.02 to 1.34 ± 0.16 d.u., P = 0.0023 (PAI-1 protein). Conclusions This study confirms BS/GS as a human model to investigate interrelated systems involved in the pathophysiology of hypertension and throws more light on the cellular mechanisms of BS/GS reduced Ang II short- and long-term signaling pathways.