n-3 Fatty acids preserve muscle mass and insulin sensitivity in a rat model of energy restriction.

In obese subjects, the loss of fat mass during energy restriction is often accompanied by a loss of muscle mass. The hypothesis that n-3 PUFA, which modulate protein homoeostasis via effects on insulin sensitivity, could contribute to maintain muscle mass during energy restriction was tested in rats fed a high-fat diet (4 weeks) rich in 18 : 1 n-9 (oleic acid, OLE-R), 18 : 3 n-3 (α-linolenic acid, ALA-R) or n-3 long-chain (LC-R) fatty acid and then energy restricted (8 weeks). A control group (OLE-ad libitum (AL)) was maintained with AL diet throughout the study. Rats were killed 10 min after an i.v. insulin injection. All energy-restricted rats lost weight and fat mass, but only the OLE-R group showed a significant muscle loss. The Gastrocnemius muscle was enriched with ALA in the ALA-R group and with LC-PUFA in the ALA-R and LC-R groups. The proteolytic ubiquitin-proteasome system was differentially affected by energy restriction, with MAFbx and muscle ring finger-1 mRNA levels being decreased in the LC-R group (-30 and -20 %, respectively). RAC-α serine/threonine-protein kinase and insulin receptor substrate 1 phosphorylation levels increased in the LC-R group (+70 %), together with insulin receptor mRNA (+50 %). The ALA-R group showed the same overall activation pattern as the LC-R group, although to a lesser extent. In conclusion, dietary n-3 PUFA prevent the loss of muscle mass associated with energy restriction, probably by an improvement in the insulin-signalling pathway activation, in relation to enrichment of plasma membranes in n-3 LC-PUFA.

Role of smooth muscle cell mineralocorticoid receptor in vascular tone.

Identification of the mineralocorticoid receptor (MR) in the vasculature (i.e., endothelial and smooth muscle cells) raised the question of its role in vascular function and blood pressure control. Using a mouse model with conditional inactivation of MR in vascular smooth muscle cell (VSMC) (MR(SMKO)), we have recently shown that the VSMC MR is crucial for aldosterone-salt-induced carotid stiffening. In the present study, we have investigated the specific contribution of the VSMC MR in the regulation of vascular tone in large vessels. In MR(SMKO) mice, contractions induced by potassium chloride and calcium (Ca(2+)) are decreased in the aorta, whereas contraction is normal in response to phenylephrine and caffeine. The difference in response to Ca(2+) suggests that the VSMC-specific deficiency of the MR modifies VSM Ca(2+) signaling but without altering the intracellular Ca(2+) store handling. The relaxation induced by acetylcholine is not affected by the absence of MR. However, the relaxation induced by Ach in the presence of indomethacin and the relaxation induced by sodium nitroprussiate are significantly reduced in MR(SMKO) mice compared to controls. Since endothelial nitric oxide synthase (eNOS) activity is increased in mutant mice, their altered relaxation reflects impairment of the nitric oxide (NO) signaling pathway. In addition to altered NO and Ca(2+) signaling, the activity of myosin light chain and its regulators, myosin light chain kinase (MLCK) and myosin phosphatase (MLCP), is reduced. In conclusion, MR expressed in VSMC is required for NO and Ca(2+) signaling pathways and contractile protein activity leading to an altered contraction/relaxation coupling.

Inactivation of serum response factor contributes to decrease vascular muscular tone and arterial stiffness in mice.

RATIONALE: Vascular smooth muscle (SM) cell phenotypic modulation plays an important role in arterial stiffening associated with aging. Serum response factor (SRF) is a major transcription factor regulating SM genes involved in maintenance of the contractile state of vascular SM cells. OBJECTIVE: We investigated whether SRF and its target genes regulate intrinsic SM tone and thereby arterial stiffness. METHODS AND RESULTS: The SRF gene was inactivated SM-specific knockout of SRF (SRF(SMKO)) specifically in vascular SM cells by injection of tamoxifen into adult transgenic mice. Fifteen days later, arterial pressure and carotid thickness were lower in SRF(SMKO) than in control mice. The carotid distensibility/pressure and elastic modulus/wall stress curves showed a greater arterial elasticity in SRF(SMKO) without modification in collagen/elastin ratio. In SRF(SMKO), vasodilation was decreased in aorta and carotid arteries, whereas a decrease in contractile response was found in mesenteric arteries. By contrast, in mice with inducible SRF overexpression, the in vitro contractile response was significantly increased in all arteries. Without endothelium, the contraction was reduced in SRF(SMKO) compared with control aortic rings owing to impairment of the NO pathway. Contractile components (SM-actin and myosin light chain), regulators of the contractile response (myosin light chain kinase, myosin phosphatase target subunit 1, and protein kinase C-potentiated myosin phosphatase inhibitor) and integrins were reduced in SRF(SMKO). CONCLUSIONS: SRF controls vasoconstriction in mesenteric arteries via vascular SM cell phenotypic modulation linked to changes in contractile protein gene expression. SRF-related decreases in vasomotor tone and cell-matrix attachment increase arterial elasticity in large arteries.

Selective involvement of serum response factor in pressure-induced myogenic tone in resistance arteries.

OBJECTIVE: In resistance arteries, diameter adjustment in response to pressure changes depends on the vascular cytoskeleton integrity. Serum response factor (SRF) is a dispensable transcription factor for cellular growth, but its role remains unknown in resistance arteries. We hypothesized that SRF is required for appropriate microvascular contraction. METHODS AND RESULTS: We used mice in which SRF was specifically deleted in smooth muscle or endothelial cells, and their control. Myogenic tone and pharmacological contraction was determined in resistance arteries. mRNA and protein expression were assessed by quantitative real-time PCR (qRT-PCR) and Western blot. Actin polymerization was determined by confocal microscopy. Stress-activated channel activity was measured by patch clamp. Myogenic tone developing in response to pressure was dramatically decreased by SRF deletion (5.9±2.3%) compared with control (16.3±3.2%). This defect was accompanied by decreases in actin polymerization, filamin A, myosin light chain kinase and myosin light chain expression level, and stress-activated channel activity and sensitivity in response to pressure. Contractions induced by phenylephrine or U46619 were not modified, despite a higher sensitivity to p38 blockade; this highlights a compensatory pathway, allowing normal receptor-dependent contraction. CONCLUSIONS: This study shows for the first time that SRF has a major part to play in the control of local blood flow via its central role in pressure-induced myogenic tone in resistance arteries.

Natural Isotope Abundances of Carbon and Nitrogen in Tissue Proteins and Amino Acids as Biomarkers of the Decreased Carbohydrate Oxidation and Increased Amino Acid Oxidation Induced by Caloric Restriction under a Maintained Protein Intake in Obese Rats.

A growing body of evidence supports a role for tissue-to-diet 15N and 13C discrimination factors (Δ15N and Δ13C), as biomarkers of metabolic adaptations to nutritional stress, but the underlying mechanisms remain poorly understood. In obese rats fed ad libitum or subjected to gradual caloric restriction (CR), under a maintained protein intake, we measured Δ15N and Δ13C levels in tissue proteins and their constitutive amino acids (AA) and the expression of enzymes involved in the AA metabolism. CR was found to lower protein mass in the intestine, liver, heart and, to a lesser extent, some skeletal muscles. This was accompanied by Δ15N increases in urine and the protein of the liver and plasma, but Δ15N decreases in the proteins of the heart and the skeletal muscles, alongside Δ13C decreases in all tissue proteins. In Lys, Δ15N levels rose in the plasma, intestine, and some muscles, but fell in the heart, while in Ala, and to a lesser extent Glx and Asx, Δ13C levels fell in all these tissues. In the liver, CR was associated with an increase in the expression of genes involved in AA oxidation. During CR, the parallel rises of Δ15N in urine, liver, and plasma proteins reflected an increased AA catabolism occurring at the level of the liver metabolic branch point, while Δ15N decreases in cardiac and skeletal muscle proteins indicated increased protein and AA catabolism in these tissues. Thus, an increased protein and AA catabolism results in opposite Δ15N effects in splanchnic and muscular tissues. In addition, the Δ13C decrease in all tissue proteins, reflects a reduction in carbohydrate (CHO) oxidation and routing towards non-indispensable AA, to achieve fuel economy.

The expression of aromatase in gonadotropes is regulated by estradiol and gonadotropin-releasing hormone in a manner that differs from the regulation of luteinizing hormone.

The role of estrogens is dual: they suppress basal expression of gonadotropins and enhance GnRH responsiveness at the time of the LH surge. Estrogens are synthesized by cytochrome P450 aromatase (P450arom), encoded by the cyp19 gene. We focused on the cyp19 gene in rat and showed that it is expressed in gonadotropes through promoters PII and PI.f, using RT-PCR and dual fluorescence labeling with anti-P450arom and -LH antibodies. Real-time PCR quantification revealed that aromatase mRNA levels varied during the estrous cycle and were significantly increased after ovariectomy. This effect is prevented by estradiol (E2) as well as GnRH antagonist administration, suggesting that GnRH may mediate the steroid effect. Interestingly, the long-acting GnRH agonist that induces LH desensitization does not modify aromatase expression in ovariectomized rats. Administration of E2 in ovariectomized rats receiving either GnRH agonist or GnRH antagonist clearly demonstrated that E2 also reduces cyp19 expression at the pituitary level. The selective estrogen receptor-alpha ligand propyl pyrazole triol and the selective estrogen receptor-beta ligand diarylpropionitrile both mimic the E2 effects. By contrast, propyl pyrazole triol reduces LH beta expression whereas diarylpropionitrile does not. In addition, using transient transfection assays in an L beta T2 gonadotrope cell line, we provided evidence that GnRH agonist stimulated, in a dose-dependant manner, cyp19 promoters PII and PI.f and that E2 decreased the GnRH stimulation. In conclusion, our data demonstrate that GnRH is an important signal in the regulation of cyp19 in gonadotrope cells. Both common and specific intracellular factors were responsible for dissociated variations of LH beta and cyp19 expression.

Aromatase gene expression and regulation in the female rat pituitary.

Aromatase cytochrome P450, the key enzyme of estrogen biosynthesis from androgens, is encoded by CYP19. Its structure shows some peculiarities: exons II to X encode the protein, while multiple alternative exons I encode unique 5'-untranslated regions of the aromatase mRNA transcripts. Immunohistochemistry studies in the rat have shown that pituitary aromatase expression is sex-dependent and varies across the estrous cycle, suggesting that estrogens might be involved in the regulation of aromatase activity and might act locally as a paracrine or autocrine factor in the pituitary. In the present study, we used RT-PCR to characterize aromatase transcripts and real-time PCR to quantify the expression of the total aromatase mRNA at the different stages of the estrous cycle and from an ovariectomy and estradiol replacement model. We identified the two previously described aromatase transcripts with a specific 5'untranslated region of the brain 1f and the gonadal PII transcripts. Total aromatase mRNA expression in the pituitary varied significantly during the estrous cycle, with the highest level occurring on the day of metestrus. After ovariectomy, we observed an increase of aromatase mRNA levels, and this effect was completely prevented by estradiol administration. These results suggest that pituitary aromatase mRNA expression is downregulated by estrogens.

Vascular Smooth Muscle Mineralocorticoid Receptor Contributes to Coronary and Left Ventricular Dysfunction After Myocardial Infarction.

Mineralocorticoid receptor (MR) antagonists slow down the progression of heart failure after myocardial infarction (MI), but the cell-specific role of MR in these benefits is unclear. In this study, the role of MR expressed in vascular smooth muscle cells (VSMCs) was investigated. Two months after coronary artery ligation causing MI, mice with VSMC-specific MR deletion (MI-MR(SMKO)) and mice treated with the MR antagonist finerenone (MI-fine) had improved left ventricular compliance and elastance when compared with infarcted control mice (MI-CTL), as well as reduced interstitial fibrosis. Importantly, the coronary reserve assessed by magnetic resonance imaging was preserved (difference in myocardial perfusion before and after induction of vasodilatation, mL mg(-1) min(-1): MI-CTL: 1.1 ± 0.5, nonsignificant; MI-MR(SMKO): 4.6 ± 1.6 [P<0.05]; MI-fine: 3.6 ± 0.7 [P<0.01]). The endothelial function, tested on isolated septal coronary arteries by analyzing the acetylcholine-induced nitric oxide-dependent relaxation, was also improved by MR deletion in VSMCs or by finerenone treatment (relaxation %: MI-CTL: 36 ± 5, MI-MR(SMKO): 54 ± 3, and MI-fine: 76 ± 4; P<0.05). Such impairment of the coronary endothelial function on MI involved an oxidative stress that was reduced when MR was deleted in VSMCs or by finerenone treatment. Moreover, short-term incubation of coronary arteries isolated from noninfarcted animals with low-dose angiotensin-II (10(-9) mol/L) induced oxidative stress and impaired acetylcholine-induced relaxation in CTL but neither in MR(SMKO) nor in mice pretreated with finerenone. In conclusion, deletion of MR in VSMCs improved left ventricular dysfunction after MI, likely through maintenance of the coronary reserve and improvement of coronary endothelial function. MR blockage by finerenone had similar effects.

Smooth muscle cell mineralocorticoid receptors are mandatory for aldosterone-salt to induce vascular stiffness.

Arterial stiffness is recognized as a risk factor for many cardiovascular diseases. Aldosterone via its binding to and activation of the mineralocorticoid receptors (MRs) is a main regulator of blood pressure by controlling renal sodium reabsorption. Although both clinical and experimental data indicate that MR activation by aldosterone is involved in arterial stiffening, the molecular mechanism is not known. In addition to the kidney, MR is expressed in both endothelial and vascular smooth muscle cells (VSMCs), but the specific contribution of the VSMC MR to aldosterone-induced vascular stiffness remains to be explored. To address this question, we generated a mouse model with conditional inactivation of the MR in VSMC (MR(SMKO)). MR(SMKO) mice show no alteration in renal sodium handling or vascular structure, but they have decreased blood pressure when compared with control littermate mice. In vivo at baseline, large vessels of mutant mice presented with normal elastic properties, whereas carotids displayed a smaller diameter when compared with those of the control group. As expected after aldosterone/salt challenge, the arterial stiffness increased in control mice; however, it remained unchanged in MR(SMKO) mice, without significant modification in vascular collagen/elastin ratio. Instead, we found that the fibronectin/α5-subunit integrin ratio is profoundly altered in MR(SMKO) mice because the induction of α5 expression by aldosterone/salt challenge is prevented in mice lacking VSMC MR. Altogether, our data reveal in the aldosterone/salt hypertension model that MR activation specifically in VSMC leads to the arterial stiffening by modulation of cell-matrix attachment proteins independent of major vascular structural changes.