Giulio Ceolotto

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.

CYP1A2 genotype modifies the association between coffee intake and the risk of hypertension.

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.

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.

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)

Adrenomedullin stimulates DNA synthesis of rat adrenal zona glomerulosa cells through activation of the mitogen-activated protein kinase-dependent cascade.

Background Adrenal zona glomerulosa cells are provided with adrenomedullin receptors. Adrenomedullin has recently been found to enhance proliferation of cultured rat vascular smooth muscle cells and zona glomerulosa cells. Objective To investigate whether adrenomedullin affects rat zona glomerulosa proliferative activity through the tyrosine kinase and extracellular signal regulated kinases (ERKs) pathways. Methods Dispersed rat zona glomerulosa cells were cultured in vitro for 24 h and then exposed to adrenomedullin (10−7 mol/l), alone or in the presence of tyrphostin-23 (10−5 mol/l) or PD-98059 (10−4 mol/l), for 24 or 48 h. To assess the rate of DNA synthesis, 5-bromo-2 ′-deoxyuridine (BrdU, 20 mg/ml) was also added to the medium and BrdU-positive cells were detected by immunocytochemistry. The expression of ERKs and the effect of adrenomedullin on ERKs phosphorylation and activity were assayed in dispersed zona glomerulosa cells. Results Adrenomedullin significantly increased the percentage of BrdU-positive (phase-S) zona glomerulosa cells; this effect was blocked by either the tyrosine kinase inhibitor, tyrphostin-23, or the mitogen-activated protein kinase kinase (MEK-1) inhibitor, PD-98059. Both zona glomerulosa and zona fasciculata/reticularis express ERK-1 (44 kDa) and ERK-2 (42 kDa) isoforms. However, adrenomedullin phosphorylated ERK-1 and ERK-2 only in the zona glomerulosa; this effect was blunted by the MEK-1 inhibitor, PD98059, and by the calcitonin gene-related peptide type 1 (CGRP-1) receptor antagonist, CGRP8−−37, but not by the adrenomedullin C-terminal fragment, ADM22−−52. Conclusion Adrenomedullin stimulates the growth of rat zona glomerulosa cells through activation of CGRP-1 receptor, linked to the tyrosine kinase–MEK-1–ERKs signalling pathway. These results confirm the complex role played by this peptide in the regulation of zona glomerulosa cell physiology.

Abnormal regulation of G protein alpha(i2) subunit in skin fibroblasts from insulin-resistant hypertensive individuals.

Background Studies in experimental animals and human cells have demonstrated increased intracellular calcium (Cai2+) signalling and Gαi signal transduction associated with hypertension. We have recently shown that angiotensin II-induced mobilization of Cai2+ is enhanced in fibroblasts from hypertensive individuals in comparison with that in normotensive individuals and that it is blunted by insulin and pertussis toxin in insulin-sensitive, but not in insulin-resistant, patients. This suggests that Gi-mediated signal transduction is reduced in insulin-resistant hypertension. Objective To investigate the expression and regulation of Gαi2 subunit in insulin-sensitive and insulin-resistant hypertensive individuals. Methods G protein αi2 subunit mRNA was measured in cultured skin fibroblasts from patients with insulin-sensitive and insulin-resistant hypertension, by real-time reverse transcriptase polymerase chain reaction. We also investigated the effects of short-term exposure to fetal calf serum, angiotensin II and insulin, alone and in combination, on the expression of Gαi2 in vitro. Spectrofluorophotometric measurement of free Cai2+ was performed in monolayers of 24 h serum-deprived cells in basal conditions and after exposure to angiotensin II, with and without pre-incubation with insulin. Results Expression of Gαi2 was significantly greater in fibroblasts from hypertensive individuals than in normotensive individuals and the increase was unrelated to age and body mass. The difference was largely accounted for by greater values in insulin-sensitive than in insulin-resistant hypertensive individuals. In fibroblasts from those with insulin-sensitive hypertension, angiotensin II and insulin were additive to fetal calf serum in increasing the expression of Gαi2. In these patients, insulin blunted the angiotensin-II induced Cai2+ transient. In contrast, in those with insulin-resistant hypertension, Gαi2 was lower and unresponsive to angiotensin II and insulin. Finally, in fibroblasts from insulin-resistant patients, insulin was unable to reduce the angiotensin II-induced Cai2+ peak. Conclusions A subnormal Gαi2-mediated signal transduction may be involved in the pathogenesis of cellular insulin resistance in hypertension. This novel Gαi2-mediated signal transduction associated with insulin sensitivity in fibroblasts may help to control excessive angiotensin II signalling.

Angiotensin II-induced over-activation of p47phox in fibroblasts from hypertensives: which role in the enhanced ERK1/2 responsiveness to angiotensin II?

Background Fibroblasts are involved in the remodeling of the heart and of the vasculature associated to arterial hypertension, and an abnormal extracellular signal-regulated kinase 1/2 (ERK1/2) activation by angiotensin II (Ang II) plays a pivotal role in this process. However, the intracellular pathways leading to cell hypertrophy and hyperplasia, as well as to collagen production, are still incompletely known. Objective To investigate the role of superoxide anion (O2−) and of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase in Ang II-stimulated ERK1/2 over-activation in fibroblasts from hypertensive patients. Methods O2− production was measured in skin fibroblasts from hypertensives (HT, n = 11) and from normotensive controls (NT, n = 10) by electron spin resonance technique. ERK1/2 phosphorylation and p47phox NAD(P)H oxidase subunit translocation were measured by western blot. Results Ang II (1 μmol/l) induced a larger p47phox subunit translocation and increased intracellular O2− production to a larger extent in HT in comparison to NT and this effect was blocked by apocynin, an inhibitor of the NAD(P)H oxidase. Ang II increased ERK1/2 phosphorylation more in HT than in NT. The Ang II-induced ERK1/2 phosphorylation was inhibited by apocynin in a dose-dependent manner in NT, but not in HT. Conclusions The chain of cellular events leading to increased ERK1/2 responsiveness to Ang II in hypertension include an exaggerated response of p47phox, NAD(P)H oxidase and O2−, but it is partially resistant to apocynin. Therefore, NAD(P)H-dependent reactive oxygen species (ROS) production is not the only determinant of the exaggerated ERK1/2 responsiveness in fibroblasts of hypertensives (HT).

RGS2 expression and aldosterone: renin ratio modulate response to drug therapy in hypertensive patients.

Objective RGS2 (regulators of G-protein signalling) is a negative regulator of Gαq protein signalling, which mediates the action of several vasoconstrictors. Low RGS2 expression increases G-protein-coupled signalling in hypertensive patients. The aim of the present study was to correlate RGS2 expression in peripheral blood mononuclear cells (PBMs) with response to antihypertensive therapy in never-treated patients with essential hypertension. Methods and design RGS2 expression was measured by real-time quantitative RT-PCR in peripheral blood mononuclear cells (PBMs) from 102 essential hypertensives. The diagnosis of essential hypertension was based on all clinically required tests, including the captopril suppression test. Antihypertensive treatment was given in accordance to international guidelines. End-point of the study was systolic blood pressure (BP) less than 140 mmHg and diastolic BP less than 90 mmHg with three or less different antihypertensive agents, which identified responders to treatment. Resistant hypertension was defined as the failure to control systolic and/or diastolic BP despite at least three different classes of antihypertensive agents, including a diuretic. Results During follow-up, 85 (83%) patients reached the end point (responders). Resistant hypertensives (n = 17, 17%) were older, had higher baseline BP, plasma aldosterone and aldosterone: renin ratio (ARR) and lower plasma renin activity than patients who reached the end point. RGS2 was negatively correlated to systolic BP at enrolment and significantly lower in PBMs from resistant hypertensives in comparison with patients that reached BP goal. According to logistic regression analysis, high RGS2 expression was predictor of reaching BP goal, whereas high ARR after captopril, age and systolic pressure at enrolment were predictor of resistant hypertension. Conclusion RGS2 expression affects the response to antihypertensive treatment. Reduced RGS2 expression contributes to resistance to antihypertensive agents through poor negative feedback on the effects of aldosterone and of other vasoactive agents.

Modulatory effect of insulin on release of calcium from human fibroblasts by angiotensin II

Background Angiotensin II stimulates synthesis and deposition of collagen and might contribute to the vascular and cardiac dysfunction associated with arterial hypertension. Insulin attenuates angiotensin II-induced responses of intracellular Ca2+ concentration ([Ca2+]) in many cell types but this effect is less in insulin-resistant states. The mechanisms of the interaction between insulin and angiotensin II are still not known. Objective To characterize the effects of angiotensin II on intracellular [Ca2+] and the effects of insulin on the angiotensin II-induced response of intracellular [Ca2+] in human skin fibroblasts. Methods Spectrofluorophotometric measurements of intracellular [Ca2+] in monolayers of cultured human skin fibroblasts from 15 normotensive patients were performed using Fura-2 at 510 nm emission with excitation wavelengths of 340 and 380 nm. Results Basal intracellular [Ca2+] in quiescent (24 h serum-deprived) human fibroblasts was 75 ± 3 nmol/l (n = 20). Administration of angiotensin II elevated intracellular [Ca2+] dose-dependently with a concentration for half-maximal effect of 20 nmol/l. Administration of 100 nmol/l angiotensin II stimulated a rapid and transient increase in intracellular [Ca2+] (from 75 ± 3 to130 ± 2 nmol/l, n = 20). Removal of extracellular calcium did not change peak intracellular [Ca2+], but it did reduce the time to recovery of [Ca2+] (from 64 ± 4 to 48 ± 2s, n = 10, P < 0.01), suggesting that an angiotensin II-induced transmembrane calcium influx had occurred. This hypothesis was confirmed by quenching studies with manganese. The angiotensin II-induced changes in intracellular [Ca2+] were completely blocked by administration of 100 nmol/l of the angiotensin II type 1 receptor inhibitor losartan but not by administration of 100 nmol/l of the angiotensin II type 2 receptor blocker CGP42112A. Acute (20 min) exposure to 100 nmol/l insulin did not alter basal intracellular [Ca2+] in quiescent fibroblasts, but significantly blunted angiotensin II-stimulated peak of [Ca2+] (to 101 ± 3 nmol/l, P < 0.01, n = 18) and delayed recovery of [Ca2+] (to 99 ± 5 s, P < 0.01). The inhibitory effect of insulin was observed both with and without extracellular Ca2+. Conclusions Our results demonstrate that administration of angiotensin II increases intracellular [Ca2+] in human skin fibroblasts by release of Ca2+ from intracellular Ca2+ stores and by influx of Ca2+ and that administration of insulin attenuates the response of [Ca2+] to angiotensin II but prolongs the time to recovery of [Ca2+].