Very-low-calorie ketogenic diet vs hypocaloric balanced diet in the prevention of high-frequency episodic migraine: the EMIKETO randomized, controlled trial.

BACKGROUND: Migraine is the second world's cause of disability. Among non-pharmacological treatments, nutritional intervention, particularly ketogenic diet, represents one of the most promising approaches. METHODS: This a prospective, single center, randomized, controlled study aimed at evaluating the efficacy of a very low-calorie ketogenic diet (VLCKD) compared to a hypocaloric balanced diet (HBD) in migraine prophylaxis in patients affected by high-frequency episodic migraine (HFEM) with a Body Mass Index (BMI) > 27 kg/m2. Fifty-seven patients were randomly assigned to a VLCKD (group 1) or HBD (group 2). Group 1 patients followed a VLCKD for 8 weeks, followed by a low calorie diet (LCD, weeks 9-12), and a HBD (weeks 13-24), whereas group 2 patients followed a HBD from week 0 to 24. Anthropometric indexes, urine and blood chemistry were assessed at enrollment, baseline, weeks 4, 8, 12, and 24. Migraine characteristics were evaluated at baseline, weeks 8, 12 and 24. Change in monthly migraine days (MMDs) at weeks 5-8 compared to baseline was the primary endpoint. Secondary endpoints encompassed changes in visual analogue scale (VAS), Headache Impact Test-6 (HIT-6) and Short Form Health Survey-36 (SF-36) scores. We also studied effects on circulating lymphocytes and markers of inflammation, changes in plasma aldosterone and renin levels before and after VLCKD or HBD treatment. RESULTS: Reduction from baseline in MMDs was greater in VLCKD compared to HBD group at week 8 (p = 0.008), at week 12 (p = 0.007), when ketosis had been interrupted by carbohydrates reintroduction, and at week 24 (p = 0.042), when all patients were following the same dietary regimen. Quality of life scores (SF-36) were improved in VLCKD group at week 8 and 12, and were also improved in HBD group, but only at week 12. Weight-loss was significantly higher in VLCKD group at week 8 (p = 0.002) and week 12 (p = 0.020). At the end of the study weight loss was maintained in VLCKD group whereas a slight weight regain was observed in HBD group. Inflammatory indexes, namely C reactive protein (CRP), neutrophil to lymphocyte ratio (NLR) and total white blood cell count (WBC) were significantly reduced (p < 0.05) in VLCKD group at week 12. Aldosterone plasma level were significantly increased in both groups at week 8, particularly in VLCKD group. However, electrolytes and renin plasma levels were never altered throughout the study in both groups. CONCLUSIONS: VLCKD is more effective than HBD in reducing MMD in patients with HFEM and represents an effective prophylaxis in patients with overweight/obesity. Trial registration ClinicalTrials.gov identifier: NCT04360148.

The novel non-steroidal MR antagonist finerenone improves metabolic parameters in high-fat diet-fed mice and activates brown adipose tissue via AMPK-ATGL pathway.

Mineralocorticoid receptor antagonists (MRAs) are recommended for the treatment of heart failure and hypertension, mainly due to their natriuretic and anti-fibrotic mode of action. Rodent studies have shown that MRAs can prevent adverse metabolic consequences of obesity but an elucidation of underlying molecular mechanisms is missing. Here, we investigated metabolic effects of the novel non-steroidal MRA finerenone (FIN) in a mouse model of high-fat diet (HFD)-induced obesity and the signaling pathways activated by MR antagonism at level of interscapular brown adipose tissue (iBAT). C57BL/6J male mice were fed a normal diet or a HFD (with60% kcal from fat) containing or not FIN for 3 months. Metabolic parameters, adipose tissue morphology, gene and protein expression analysis were assessed. We also used brown adipocyte cultures (T37i cells) to investigate the effects of FIN-mediated MR antagonism upon lipid and mitochondrial metabolism. HFD + FIN-treated mice showed improved glucose tolerance together with increased multilocularity and higher expression of thermogenic markers at the level of iBAT, without differences in white adipose depots, suggesting an iBAT-specific effect of FIN. Mechanistically, FIN increased activation of AMP-activated protein kinase which, in turn, stimulated adipose triglyceride lipase activation, with subsequent increased expression of uncoupling protein-1 in brown adipocytes.

Macrophage NCOR1 protects from atherosclerosis by repressing a pro-atherogenic PPARγ signature.

AIMS: Nuclear receptors and their cofactors regulate key pathophysiological processes in atherosclerosis development. The transcriptional activity of these nuclear receptors is controlled by the nuclear receptor corepressors (NCOR), scaffolding proteins that form the basis of large corepressor complexes. Studies with primary macrophages demonstrated that the deletion of Ncor1 increases the expression of atherosclerotic molecules. However, the role of nuclear receptor corepressors in atherogenesis is unknown. METHODS AND RESULTS: We generated myeloid cell-specific Ncor1 knockout mice and crossbred them with low-density lipoprotein receptor (Ldlr) knockouts to study the role of macrophage NCOR1 in atherosclerosis. We demonstrate that myeloid cell-specific deletion of nuclear receptor corepressor 1 (NCOR1) aggravates atherosclerosis development in mice. Macrophage Ncor1-deficiency leads to increased foam cell formation, enhanced expression of pro-inflammatory cytokines, and atherosclerotic lesions characterized by larger necrotic cores and thinner fibrous caps. The immunometabolic effects of NCOR1 are mediated via suppression of peroxisome proliferator-activated receptor gamma (PPARγ) target genes in mouse and human macrophages, which lead to an enhanced expression of the CD36 scavenger receptor and subsequent increase in oxidized low-density lipoprotein uptake in the absence of NCOR1. Interestingly, in human atherosclerotic plaques, the expression of NCOR1 is reduced whereas the PPARγ signature is increased, and this signature is more pronounced in ruptured compared with non-ruptured carotid plaques. CONCLUSIONS: Our findings show that macrophage NCOR1 blocks the pro-atherogenic functions of PPARγ in atherosclerosis and suggest that stabilizing the NCOR1-PPARγ binding could be a promising strategy to block the pro-atherogenic functions of plaque macrophages and lesion progression in atherosclerotic patients.

Endothelial Mineralocorticoid Receptors Contribute to Vascular Inflammation in Atherosclerosis in a Sex-Specific Manner.

OBJECTIVE: MR (mineralocorticoid receptor) activation is associated with cardiovascular ischemia in humans. This study explores the role of the MR in atherosclerotic mice of both sexes and identifies a sex-specific role for endothelial cell (EC)-MR in vascular inflammation. Approach and Results: In the AAV-PCSK9 (adeno-associated virus-proprotein convertase subtilisin/kexin type 9) mouse atherosclerosis model, MR inhibition attenuated vascular inflammation in males but not females. Further studies comparing male and female littermates with intact MR or EC-MR deletion revealed that although EC-MR deletion did not affect plaque size in either sex, it reduced aortic arch inflammation specifically in male mice as measured by flow cytometry. Moreover, MR-intact females had larger plaques but were protected from vascular inflammation compared with males. Intravital microscopy of the mesenteric vasculature demonstrated that EC-MR deletion attenuated TNFα (tumor necrosis factor α)-induced leukocyte slow rolling and adhesion in males, while females exhibited fewer leukocyte-endothelial interactions with no additional effect of EC-MR deletion. These effects corresponded with decreased TNFα-induced expression of the endothelial adhesion molecules ICAM-1 (intercellular adhesion molecule-1) and E-selectin in males with EC-MR deletion compared with MR-intact males and females of both genotypes. These observations were also consistent with MR and estrogen regulation of ICAM-1 transcription and E-selectin expression in primary cultured mouse ECs and human umbilical vein ECs. CONCLUSIONS: In male mice, EC-MR deletion attenuates leukocyte-endothelial interactions, plaque inflammation, and expression of E-selectin and ICAM-1, providing a potential mechanism by which the MR promotes vascular inflammation. In females, plaque inflammation and leukocyte-endothelial interactions are decreased relative to males and EC-MR deletion is not protective.

Mineralocorticoid receptor antagonism induces browning of white adipose tissue through impairment of autophagy and prevents adipocyte dysfunction in high-fat-diet-fed mice.

The mineralocorticoid receptor (MR) controls adipocyte function, but its role in the conversion of white adipose tissue (WAT) into thermogenic fat has not been elucidated. We investigated responses to the MR antagonists spironolactone (spiro; 20 mg/kg/d) and drospirenone (DRSP; 6 mg/kg/d) in C57BL/6 mice fed a high-fat (HF) diet for 90 d. DRSP and spiro curbed HF diet-induced impairment in glucose tolerance, and prevented body weight gain and white fat expansion. Notably, either MR antagonist induced up-regulation of brown adipocyte-specific transcripts and markedly increased protein levels of uncoupling protein 1 (UCP1) in visceral and inguinal fat depots when compared with the HF diet group. Positron emission tomography and magnetic resonance spectroscopy confirmed acquisition of brown fat features in WAT. Interestingly, MR antagonists markedly reduced the autophagic rate both in murine preadipocytes in vitro (10(-5) M) and in WAT depots in vivo, with a concomitant increase in UCP1 protein expression. Moreover, the autophagy repressor bafilomycin A1 (10(-8) M) mimicked the effect of MR antagonists, increasing UCP1 protein expression in primary preadipocytes. Hence, we showed that adipocyte MR regulates brown remodeling of WAT through a modulation of autophagy. These results provide a rationale for the use of MR antagonists to prevent the adverse metabolic consequences of adipocyte dysfunction.

A Novel Mix of Polyphenols and Micronutrients Reduces Adipogenesis and Promotes White Adipose Tissue Browning via UCP1 Expression and AMPK Activation.

Background: Obesity is a pandemic disease characterized by excessive severe body comorbidities. Reduction in fat accumulation represents a mechanism of prevention, and the replacement of white adipose tissue (WAT) with brown adipose tissue (BAT) has been proposed as one promising strategy against obesity. In the present study, we sought to investigate the ability of a natural mixture of polyphenols and micronutrients (A5+) to counteract white adipogenesis by promoting WAT browning. Methods: For this study, we employed a murine 3T3-L1 fibroblast cell line treated with A5+, or DMSO as control, during the differentiation in mature adipocytes for 10 days. Cell cycle analysis was performed using propidium iodide staining and cytofluorimetric analysis. Intracellular lipid contents were detected by Oil Red O staining. Inflammation Array, along with qRT-PCR and Western Blot analyses, served to measure the expression of the analyzed markers, such as pro-inflammatory cytokines. Results: A5+ administration significantly reduced lipids' accumulation in adipocytes when compared to control cells (p < 0.005). Similarly, A5+ inhibited cellular proliferation during the mitotic clonal expansion (MCE), the most relevant stage in adipocytes differentiation (p < 0.0001). We also found that A5+ significantly reduced the release of pro-inflammatory cytokines, such as IL-6 and Leptin (p < 0.005), and promoted fat browning and fatty acid oxidation through increasing expression levels of genes related to BAT, such as UCP1 (p < 0.05). This thermogenic process is mediated via AMPK-ATGL pathway activation. Conclusion: Overall, these results demonstrated that the synergistic effect of compounds contained in A5+ may be able to counteract adipogenesis and then obesity by inducing fat browning.

Efficacy and safety of finerenone for treatment of diabetic kidney disease: current knowledge and future perspective.

INTRODUCTION: Diabetic kidney disease (DKD) represents a leading cause of morbidity and mortality in subjects with diabetes and develops in more than one third of diabetic patients. Steroidal mineralocorticoid receptor antagonists (MRAs - eplerenone and spironolactone) reduce mortality in patients with heart failure with reduced ejection fraction (HFrEF). However, in clinical practice the use of steroidal MRAs is limited by the significant risk of hyperkalemia, especially in patients with impaired renal function. Finerenone, a novel nonsteroidal MRA, shows a higher selectivity and binding affinity for mineralocorticoid receptor (MR) compared to steroidal MRAs and has been shown to reduce chronic kidney disease (CKD) progression and cardiovascular mortality in patients with CKD and T2DM. AREAS COVERED: This review summarizes the current evidence on efficacy and safety of finerenone in the treatment of patients with CKD and T2DM, and discusses its mechanisms of action investigated in preclinical studies. EXPERT OPINION: Pharmacological properties of finerenone and its unique tissue distribution are responsible for a lower risk of hyperkalemia. Therefore, finerenone represents a valuable therapeutic tool in patients with CKD/diabetic kidney disease (DKD). Recent studies have shown that finerenone delays the progression of CKD and reduce cardiovascular events in patients with DKD, highlighting its safety and efficacy in this high-risk population.

SIRT5 Inhibition Induces Brown Fat-Like Phenotype in 3T3-L1 Preadipocytes.

Brown adipose tissue (BAT) activity plays a key role in regulating systemic energy. The activation of BAT results in increased energy expenditure, making this tissue an attractive pharmacological target for therapies against obesity and type 2 diabetes. Sirtuin 5 (SIRT5) affects BAT function by regulating adipogenic transcription factor expression and mitochondrial respiration. We analyzed the expression of SIRT5 in the different adipose depots of mice. We treated 3T3-L1 preadipocytes and mouse primary preadipocyte cultures with the SIRT5 inhibitor MC3482 and investigated the effects of this compound on adipose differentiation and function. The administration of MC3482 during the early stages of differentiation promoted the expression of brown adipocyte and mitochondrial biogenesis markers. Upon treatment with MC3482, 3T3-L1 adipocytes showed an increased activation of the AMP-activated protein kinase (AMPK), which is known to stimulate brown adipocyte differentiation. This effect was paralleled by an increase in autophagic/mitophagic flux and a reduction in lipid droplet size, mediated by a higher lipolytic rate. Of note, MC3482 increased the expression and the activity of adipose triglyceride lipase, without modulating hormone-sensitive lipase. Our findings reveal that SIRT5 inhibition stimulates brown adipogenesis in vitro, supporting this approach as a strategy to stimulate BAT and counteract obesity.

Cellular models for understanding adipogenesis, adipose dysfunction, and obesity.

White adipose tissue (WAT) is no longer considered a depot for energy storage in the form of triglycerides, but is a secretory organ that releases factors, known as adipokines, capable of regulating several physiological processes. Alteration of WAT function with subsequent dysfunctional expression and secretion of adipokines plays a key role in the pathogenesis of obesity, diabetes, and other metabolic diseases. For this reason, a deeper understanding of the molecular mechanisms regulating adipocyte function is deemed necessary for planning strategies to treat and prevent obesity and its metabolic complications. This review examines cell culture models currently available for studying adipocyte biology. We focus on advantages, disadvantages and main differences between established preadipocyte cell lines and primary preadipocyte cultures. We revise protocols used to promote adipocyte differentiation and mature adipocytes dedifferentiation into preadipocytes. Finally, we briefly describe co-cultures of adipocytes with other cell types and three-dimensional adipocyte culture systems. These models allow investigation of cell-cell interactions with the cross-talk physiologically occurring between adipocytes and other cell types residing within or outside adipose tissue.

Mineralocorticoid Receptors in Metabolic Syndrome: From Physiology to Disease.

Over the past decade, several studies have shown that activity of extra-renal mineralocorticoid receptors (MR) regulates vascular tone, adipogenesis, adipose tissue function, and cardiomyocyte contraction. In mice, abnormal activation of MR in the vasculature and in adipose tissue favors the occurrence of several components of the metabolic syndrome (MetS), such as hypertension, obesity, and glucose intolerance. Accordingly, high levels of aldosterone are associated with obesity and MetS in humans, suggesting that altered activation of aldosterone-MR system in extra-renal tissues leads to profound metabolic dysfunctions. In this context, in addition to the classical indications for heart failure and hypertension, MR antagonists (MRAs) nowadays represent a promising approach to tackle cardiovascular and metabolic disorders occurring in the MetS.

Neuroendocrine and Metabolic Effects of Low-Calorie and Non-Calorie Sweeteners.

Since excessive sugar consumption has been related to the development of chronic metabolic diseases prevalent in the western world, the use of sweeteners has gradually increased worldwide over the last few years. Although low- and non-calorie sweeteners may represent a valuable tool to reduce calorie intake and prevent weight gain, studies investigating the safety and efficacy of these compounds in the short- and long-term period are scarce and controversial. Therefore, future studies will need to elucidate the potential beneficial and/or detrimental effects of different types of sweeteners on metabolic health (energy balance, appetite, body weight, cardiometabolic risk factors) in healthy subjects and patients with diabetes, obesity and metabolic syndrome. In this regard, the impact of different sweeteners on central nervous system, gut hormones and gut microbiota is important, given the strong implications that changes in such systems may have for human health. The aim of this narrative review is to summarize the current evidence for the neuroendocrine and metabolic effects of sweeteners, as well as their impact on gut microbiota. Finally, we briefly discuss the advantages of the use of sweeteners in the context of very-low calorie ketogenic diets.

Design and Rationale of the ERA-CVD Consortium PREMED-CAD-Precision Medicine in Coronary Artery Disease.

Cardiovascular diseases (CVDs) comprise 45% of all deaths in Europe and causes 3.9 million deaths annually. Coronary artery disease (CAD) which includes myocardial infarction (MI) represents the most common form of CVD. A relevant proportion of MI cases seems preventable since reports claim that up to two-thirds of these patients exhibit symptoms suggestive for MI within 12 months prior to the acute MI event. An early identification of these at-risk subjects is necessary to manage an early and efficient treatment during the ischemic phase. The aim of the PRecision MEDicine in Coronary Artery Disease (PREMED-CAD) consortium is to apply a system medicine approach towards studying and identifying an ischemia specific 'biomarker signature' that improves the identification of individuals 'at-risk' for acute MI. The consortium will take an interdisciplinary and translational approach integrating knowledge from CAD epidemiology, imaging, bioinformatics, statistics and molecular biology, as well as existing phenotypic, blood-based and clinical biomarker data of distinct CAD and subclinical MI phenotypes. This biomarker signature will be validated through atherosclerosis-prone mouse models and human cohorts. The validated signature will be translated in a real-world clinical setting using an ongoing clinical trial comprising patients with subclinical ischemia. The aim of the knowledge obtained from this project is to aid in early MI detection and reduce the mortality and morbidity rate in these at-risk MI individuals.

Altered Tregs Differentiation and Impaired Autophagy Correlate to Atherosclerotic Disease.

Atherosclerosis is a progressive vascular disease representing the primary cause of morbidity and mortality in developed countries. Formerly, atherosclerosis was considered as a mere passive accumulation of lipids in blood vessels. However, it is now clear that atherosclerosis is a complex and multifactorial disease, in which the involvement of immune cells and inflammation play a key role. A variety of studies have shown that autophagy-a cellular catalytic mechanism able to remove injured cytoplasmic components in response to cellular stress-may be proatherogenic. So far, in this context, its role has been investigated in smooth muscle cells, macrophages, and endothelial cells, while the function of this catabolic protective process in lymphocyte functionality has been overlooked. The few studies carried out so far, however, suggested that autophagy modulation in lymphocyte subsets may be functionally related to plaque formation and development. Therefore, in this research, we aimed at better clarifying the role of lymphocyte subsets, mainly regulatory T cells (Tregs), in human atherosclerotic plaques and in animal models of atherosclerosis investigating the contribution of autophagy on immune cell homeostasis. Here, we investigate basal autophagy in a mouse model of atherosclerosis, apolipoprotein E (ApoE)-knockout (KO) mice, and we analyze the role of autophagy in driving Tregs polarization. We observed defective maturation of Tregs from ApoE-KO mice in response to tumor growth factor-ß (TGFß). TGFß is a well-known autophagy inducer, and Tregs maturation defects in ApoE-KO mice seem to be related to autophagy impairment. In this work, we propose that autophagy underlies Tregs maturation, advocating that the study of this process in atherosclerosis may open new therapeutic strategies.

Adipocyte Mineralocorticoid Receptor.

Mineralocorticoid receptor (MR) has been recently identified in adipose tissue, where its excessive activation contributes to several metabolic derangements often observed in obesity and metabolic syndrome. Recent findings support the existence of a bidirectional cross-talk between adipose tissue and adrenal glands, contributing to obesity-related hyperaldosteronism and subsequent adipocyte MR excessive activation. In this regard, MR pharmacological blockade has led to prevention of weight gain and metabolic benefits in murine models of genetic or diet-induced obesity. However, there is still a lack of knowledge on the potential metabolic effects of MR antagonists in clinical settings. Hence, larger clinical studies are deemed necessary to clarify the role of MR antagonism in obesity and metabolic syndrome in humans.

Mineralocorticoid Receptor and Aldosterone-Related Biomarkers of End-Organ Damage in Cardiometabolic Disease.

The mineralocorticoid receptor (MR) was first identified as a blood pressure regulator, modulating renal sodium handling in response to its principal ligand aldosterone. The mineralocorticoid receptor is also expressed in many tissues other than the kidney, such as adipose tissue, heart and vasculature. Recent studies have shown that MR plays a relevant role in the control of cardiovascular and metabolic function, as well as in adipogenesis. Dysregulation of aldosterone/MR signaling represents an important cause of disease as high plasma levels of aldosterone are associated with hypertension, obesity and increased cardiovascular risk. Aldosterone displays powerful vascular effects and acts as a potent pro-fibrotic agent in cardiovascular remodeling. Mineralocorticoid receptor activation regulates genes involved in vascular and cardiac fibrosis, calcification and inflammation. This review focuses on the role of novel potential biomarkers related to aldosterone/MR system that could help identify cardiovascular and metabolic detrimental conditions, as a result of altered MR activation. Specifically, we discuss: (1) how MR signaling regulates the number and function of different subpopulations of circulating and intra-tissue immune cells; (2) the role of aldosterone/MR system in mediating cardiometabolic diseases induced by obesity; and (3) the role of several MR downstream molecules as novel potential biomarkers of cardiometabolic diseases, end-organ damage and rehabilitation outcome.

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice.

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

Minor role of mature adipocyte mineralocorticoid receptor in high fat induced obesity.

Obesity is a major risk factor that contribute to the development of cardiovascular disease and type 2 diabetes. Mineralocorticoid receptor (MR) expression is increased in the adipose tissue of obese patients and several studies provide evidence that MR pharmacological antagonism improves glucose metabolism in genetic and diet-induced mouse models of obesity. In order to investigate whether the lack of adipocyte MR is sufficient to explain these beneficial metabolic effects, we generated a mouse model with inducible adipocyte-specific deletion of Nr3c2 gene encoding MR (adipo-MRKO). We observed a significant, yet not complete, reduction of Nr3c2 transcript and MR protein expression in subcutaneous and visceral adipose depots of adipo-MRKO mice. Notably, only mature adipocyte fraction lacks MR, whereas the stromal vascular fraction maintains normal MR expression in our mouse model. Adipo-MRKO mice fed a 45% high fat diet for 14 weeks did not show any significant difference in body weight and fat mass compared to control littermates. Glucose and insulin tolerance tests revealed that mature adipocyte MR deficiency did not improve insulin sensitivity in response to a metabolic homeostatic challenge. Accordingly, no significant changes were observed in gene expression profile of adipogenic and inflammatory markers in adipose tissue of adipo-MRKO mice. Moreover, pharmacological MR antagonism in mature primary murine adipocytes, which differentiated ex vivo from wild-type mice, did not display any effect on adipokine expression. Taken together, these data demonstrate that the depletion of MR in mature adipocytes displays a minor role in diet-induced obesity and metabolic dysfunctions.

Essential role of ICAM-1 in aldosterone-induced atherosclerosis.

OBJECTIVE: Elevated aldosterone is associated with increased risk of atherosclerosis complications, whereas treatment with mineralocorticoid receptor (MR) antagonists decreases the rate of cardiovascular events. Here we test the hypothesis that aldosterone promotes early atherosclerosis by modulating intercellular adhesion molecule-1 (ICAM-1) expression and investigate the molecular mechanisms by which aldosterone regulates ICAM-1 expression. METHODS AND RESULTS: Apolipoprotein-E (ApoE)-/- mice fed an atherogenic diet and treated with aldosterone for 4weeks showed increased vascular expression of ICAM-1, paralleled by enhanced atherosclerotic plaque size in the aortic root. Moreover, aldosterone treatment resulted in increased plaque lipid and inflammatory cell content, consistent with an unstable plaque phenotype. ApoE/ICAM-1 double knockout (ApoE-/-/ICAM-1-/-) littermates were protected from the aldosterone-induced increase in plaque size, lipid content and macrophage infiltration. Since aldosterone is known to regulate ICAM-1 transcription via MR in human endothelial cells, we explored MR regulation of the ICAM-1 promoter. Luciferase reporter assays performed in HUVECs using deletion constructs of the human ICAM-1 gene promoter showed that a region containing a predicted MR-responsive element (MRE) is required for MR-dependent transcriptional regulation of ICAM-1. CONCLUSIONS: Pro-atherogenic effects of aldosterone are mediated by increased ICAM-1 expression, through transcriptional regulation by endothelial MR. These data enhance our understanding of the molecular mechanism by which MR activation promotes atherosclerosis complications.

Inhibition of Vascular c-Jun N-Terminal Kinase 2 Improves Obesity-Induced Endothelial Dysfunction After Roux-en-Y Gastric Bypass.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) reduces obesity-associated comorbidities and cardiovascular mortality. RYGB improves endothelial dysfunction, reducing c-Jun N-terminal kinase (JNK) vascular phosphorylation. JNK activation links obesity with insulin resistance and endothelial dysfunction. Herein, we examined whether JNK1 or JNK2 mediates obesity-induced endothelial dysfunction and if pharmacological JNK inhibition can mimic RYGB vascular benefits. METHODS AND RESULTS: After 7 weeks of a high-fat high-cholesterol diet, obese rats underwent RYGB or sham surgery; sham-operated ad libitum-fed rats received, for 8 days, either the control peptide D-TAT or the JNK peptide inhibitor D-JNKi-1 (20 mg/kg per day subcutaneous). JNK peptide inhibitor D-JNKi-1 treatment improved endothelial vasorelaxation in response to insulin and glucagon-like peptide-1, as observed after RYGB. Obesity increased aortic phosphorylation of JNK2, but not of JNK1. RYGB and JNK peptide inhibitor D-JNKi-1 treatment blunted aortic JNK2 phosphorylation via activation of glucagon-like peptide-1-mediated signaling. The inhibitory phosphorylation of insulin receptor substrate-1 was reduced, whereas the protein kinase B/endothelial NO synthase pathway was increased and oxidative stress was decreased, resulting in improved vascular NO bioavailability. CONCLUSIONS: Decreased aortic JNK2 phosphorylation after RYGB rapidly improves obesity-induced endothelial dysfunction. Pharmacological JNK inhibition mimics the endothelial protective effects of RYGB. These findings highlight the therapeutic potential of novel strategies targeting vascular JNK2 against the severe cardiovascular disease associated with obesity.

Cellular mechanisms of MR regulation of adipose tissue physiology and pathophysiology.

In addition to the well-documented expression and activity of the mineralocorticoid receptor (MR) in the kidney, in the last decade research on MR has also revealed its important role in regulating functions of extrarenal tissues, including adipose tissue, where MR is involved in adipocyte fundamental processes such as differentiation, autophagy and adipokine secretion. MR expression is increased in adipose tissue of murine models of obesity and in obese human subjects, suggesting that over-activation of the mineralocorticoid signaling leads to dysfunctional adipocyte and associated metabolic disorders. Notably, pharmacological blockade of MR prevents metabolic dysfunctions observed in obese mice and suggests a potential therapeutic use of MR antagonists in the treatment of obesity and metabolic syndrome. However, the molecular pathways affected by MR blockade have been poorly investigated. This review summarizes the functions of MR in the adipocyte, discusses potential signaling pathways mediating MR action, and describes post-translational modifications regulating its activity.