Inhibition of de novo ceramide synthesis by Sirtuin-1 improves beta-cell function and glucose metabolism in type 2 diabetes.

BACKGROUND: Obesity and type 2 diabetes (T2D) are major risk factors for cardiovascular diseases (CVD). Dysregulated pro-apoptotic ceramide synthesis reduces ß-cell insulin secretion, thereby promoting hyperglycemic states which may manifest as T2D. Pro-apoptotic ceramides modulate insulin sensitivity and glucose tolerance while being linked to poor cardiovascular outcomes. Sirtuin-1 (SIRT1) is a NAD + - dependent deacetylase that protects against pancreatic ß-cell dysfunction; however, systemic levels are decreased in obese T2D mice and may promote pro-apoptotic ceramide synthesis and hyperglycemia. Herein, we aimed to assess the effects of restoring circulating SIRT1 levels to prevent metabolic imbalance in obese and diabetic mice. METHODS AND RESULTS: Circulating SIRT1 levels were reduced in obese diabetic mice (db/db) as compared to age-matched non-diabetic db/+ controls. Restoration of SIRT1 plasma levels with recombinant murine SIRT1 for 4-weeks prevented body weight gain, improved glucose tolerance, insulin sensitivity and vascular function in mice models of obesity and T2D. Untargeted lipidomics revealed that SIRT1 restored insulin-secretory function of ß-cells by reducing synthesis and accumulation of pro-apoptotic ceramides. Molecular mechanisms involved direct binding to and deacetylation of Toll-like receptor 4 (TLR4) by SIRT1 in ß-cells thereby decreasing the rate limiting enzymes of sphingolipid synthesis SPTLC1/2 via AKT/NF-κB. Among T2D patients, those with high baseline plasma levels of SIRT1 prior to metabolic surgery displayed restored ß-cell function (HOMA2- ß) and were more likely to have T2D remission during follow-up. CONCLUSION: Acetylation of TLR4 promotes ß-cell dysfunction via ceramide synthesis in T2D, which is blunted by systemic SIRT1 replenishment. Hence, restoration of systemic SIRT1 may provide a novel therapeutic strategy to counteract toxic ceramide synthesis and mitigate cardiovascular complications of T2D.

Genetic deletion of hepatic NCOR1 protects from atherosclerosis by promoting alternative bile acid-metabolism and sterol excretion.

BACKGROUND: The nuclear receptor corepressor 1 (NCOR1) plays an important role in the regulation of gene expression in immunometabolic conditions by connecting chromatin-modifying enzymes, coregulators and transcription factors. NCOR1 has been shown to be involved in cardiometabolic diseases. Recently, we demonstrated that the deletion of macrophage NCOR1 aggravates atherosclerosis by promoting CD36-triggered foam cell formation via PPARG derepression. PURPOSE: Since NCOR1 modulates the function of several key regulators involved in hepatic lipid and bile acid metabolism, we hypothesized that its deletion in hepatocytes alters lipid metabolism and atherogenesis. METHODS: To test this hypothesis, we generated hepatocyte-specific Ncor1 knockout mice on a Ldlr-/- background. Besides assessing the progression of the disease in thoracoabdominal aortae en face, we analyzed hepatic cholesterol and bile acid metabolism at expression and functional levels. RESULTS: Our data demonstrate that liver-specific Ncor1 knockout mice on an atherosclerosis-prone background develop less atherosclerotic lesions than controls. Interestingly, under chow diet, plasma cholesterol levels of liver-specific Ncor1 knockout mice were slightly higher compared to control, but strongly reduced compared to control mice after feeding them an atherogenic diet for 12 weeks. Moreover, the hepatic cholesterol content was decreased in liver-specific Ncor1 knockout compared to control mice. Our mechanistic data revealed that NCOR1 reprograms the synthesis of bile acids towards the alternative pathway, which in turn reduce bile hydrophobicity and enhances fecal cholesterol excretion. CONCLUSIONS: Our data suggest that hepatic Ncor1 deletion in mice decreases atherosclerosis development by reprograming bile acid metabolism and enhancing fecal cholesterol excretion.

Carotid Plaque Composition and the Importance of Non-Invasive in Imaging Stroke Prevention.

Luminal stenosis has been the standard feature for the current management strategies in patients with atherosclerotic carotid disease. Histological and imaging studies show considerable differences between plaques with identical degrees of stenosis. They indicate that specific plaque characteristics like Intraplaque hemorrhage, Lipid Rich Necrotic Core, Plaque Inflammation, Thickness and Ulceration are responsible for the increased risk of ischemic events. Intraplaque hemorrhage is defined by the accumulation of blood components within the plaque, Lipid Rich Necrotic Core is composed of macrophages loaded with lipid, Plaque Inflammation is defined as the process of atherosclerosis itself and Plaque thickness and Ulceration are defined as morphological features. Advances in imaging methods like Magnetic Resonance Imaging, Ultrasound, Computed Tomography and Positron Emission Tomography have enabled a more detailed characterization of the plaque, and its vulnerability is linked to these characteristics, changing the management of these patients based only on the degree of plaque stenosis. Studies like Rotterdam, ARIC, PARISK, CAPIAS and BIOVASC were essential to evaluate and prove the relevance of these characteristics with cerebrovascular symptoms. A better approach for the prevention of stroke is needed. This review summarizes the more frequent carotid plaque features and the available validation from recent studies with the latest evidence.

Modulating Sirtuin Biology and Nicotinamide Adenine Diphosphate Metabolism in Cardiovascular Disease-From Bench to Bedside.

Sirtuins (SIRT1-7) comprise a family of highly conserved deacetylases with distribution in different subcellular compartments. Sirtuins deacetylate target proteins depending on one common substrate, nicotinamide adenine diphosphate (NAD+), thus linking their activities to the status of cellular energy metabolism. Sirtuins had been linked to extending life span and confer beneficial effects in a wide array of immune-metabolic and cardiovascular diseases. SIRT1, SIRT3, and SIRT6 have been shown to provide protective effects in various cardiovascular disease models, by decreasing inflammation, improving metabolic profiles or scavenging oxidative stress. Sirtuins may be activated collectively by increasing their co-substrate NAD+. By supplementing NAD+ precursors, NAD+ boosters confer pan-sirtuin activation with protective cardiometabolic effects in the experimental setting: they improve endothelial dysfunction, protect from experimental heart failure, hypertension and decrease progression of liver steatosis. Different precursor molecules were applied ranging from nicotinamide (NAM), nicotinamide mononucleotide (NMN) to nicotinamide riboside (NR). Notably, not all experimental results showed protective effects. Moreover, the results are not as striking in clinical studies as in the controlled experimental setting. Species differences, (lack of) genetic heterogeneity, different metabolic pathways, dosing, administration routes and disease contexts may account for these challenges in clinical translation. At the clinical scale, caloric restriction can reduce the risks of cardiovascular disease and raise NAD+ concentration and sirtuin expression. In addition, antidiabetic drugs such as metformin or SGLT2 inhibitors may confer cardiovascular protection, indirectly via sirtuin activation. Overall, additional mechanistic insight and clinical studies are needed to better understand the beneficial effects of sirtuin activation and NAD+ boosters from bench to bedside.

Deletion of fibroblast activation protein provides atheroprotection.

AIMS: Fibroblast activation protein (FAP) is upregulated at sites of tissue remodelling including chronic arthritis, solid tumours, and fibrotic hearts. It has also been associated with human coronary atherosclerotic plaques. Yet, the causal role of FAP in atherosclerosis remains unknown. To investigate the cause-effect relationship of endogenous FAP in atherogenesis, we assessed the effects of constitutive Fap deletion on plaque formation in atherosclerosis-prone apolipoprotein E (Apoe) or low-density lipoprotein receptor (Ldlr) knockout mice. METHODS AND RESULTS: Using en face analyses of thoraco-abdominal aortae and aortic sinus cross-sections, we demonstrate that Fap deficiency decreased plaque formation in two atherosclerotic mouse models (-46% in Apoe and -34% in Ldlr knockout mice). As a surrogate of plaque vulnerability fibrous cap thickness was used; it was increased in Fap-deficient mice, whereas Sirius red staining demonstrated that total collagen content remained unchanged. Using polarized light, atherosclerotic lesions from Fap-deficient mice displayed increased FAP targets in terms of enhanced collagen birefringence in plaques and increased pre-COL3A1 expression in aortic lysates. Analyses of the Stockholm Atherosclerosis Gene Expression data revealed that FAP expression was increased in human atherosclerotic compared to non-atherosclerotic arteries. CONCLUSIONS: Our data provide causal evidence that constitutive Fap deletion decreases progression of experimental atherosclerosis and increases features of plaque stability with decreased collagen breakdown. Thus, inhibition of FAP expression or activity may not only represent a promising therapeutic target in atherosclerosis but appears safe at the experimental level for FAP-targeted cancer therapies.

Role of the Nuclear Receptor Corepressor 1 (NCOR1) in Atherosclerosis and Associated Immunometabolic Diseases.

Atherosclerotic cardiovascular disease is part of chronic immunometabolic disorders such as type 2 diabetes and nonalcoholic fatty liver disease. Their common risk factors comprise hypertension, insulin resistance, visceral obesity, and dyslipidemias, such as hypercholesterolemia and hypertriglyceridemia, which are part of the metabolic syndrome. Immunometabolic diseases include chronic pathologies that are affected by both metabolic and inflammatory triggers and mediators. Important and challenging questions in this context are to reveal how metabolic triggers and their downstream signaling affect inflammatory processes and vice-versa. Along these lines, specific nuclear receptors sense changes in lipid metabolism and in turn induce downstream inflammatory and metabolic processes. The transcriptional activity of these nuclear receptors is regulated by the nuclear receptor corepressors (NCORs), including NCOR1. In this review we describe the function of NCOR1 as a central immunometabolic regulator and focus on its role in atherosclerosis and associated immunometabolic diseases.

The NO-donor MPC-1011 stimulates angiogenesis and arteriogenesis and improves hindlimb ischemia via a cGMP-dependent pathway involving VEGF and SDF-1α.

BACKGROUND AND AIMS: Peripheral arterial disease (PAD) is an important cause of morbidity and mortality with little effective medical treatment currently available. Nitric oxide (NO) is crucially involved in organ perfusion, tissue protection and angiogenesis. METHODS: We hypothesized that a novel NO-donor, MPC-1011, might elicit vasodilation, angiogenesis and arteriogenesis and in turn improve limb perfusion, in a hindlimb ischemia model. Hindlimb ischemia was induced by femoral artery ligation in Sprague-Dawley rats, which were randomized to receive either placebo, MPC-1011, cilostazol or both, up to 28 days. Limb blood flow was assessed by laser Doppler imaging. RESULTS: After femoral artery occlusion, limb perfusion in rats receiving MPC-1011 alone or in combination with cilostazol was increased throughout the treatment regimen. Capillary density and the number of arterioles was increased only with MPC-1011. MPC-1011 improved vascular remodeling by increasing luminal diameter in the ischemic limb. Moreover, MPC-1011 stimulated the release of proangiogenic cytokines, including VEGF, SDF1α and increased tissue cGMP levels, reduced platelet activation and aggregation, potentiated proliferation and migration of endothelial cells which was blunted in the presence of soluble guanylyl cyclase inhibitor LY83583. In MPC-1011-treated rats, Lin-/CD31+/CXCR4+ cells were increased by 92.0% and Lin-/VEGFR2+/CXCR4+ cells by 76.8% as compared to placebo. CONCLUSIONS: Here we show that the NO donor, MPC-1011, is a specific promoter of angiogenesis and arteriogenesis in a hindlimb ischemia model in an NO-cGMP-VEGF- dependent manner. This sets the basis to evaluate and confirm the efficacy of such therapy in a clinical setting in patients with PAD and impaired limb perfusion.

Implications of NAD+ boosters in translational medicine.

Nicotinamide adenine dinucleotide (NAD+ ) is an essential metabolite in energy metabolism as well as a co-substrate in biochemical reactions such as protein deacylation, protein ADP-ribosylation and cyclic ADP-ribose synthesis mediated by sirtuins, poly (ADP-ribose) polymerases (PARPs) and CD38. In eukaryotic cells, NAD+ is synthesized through three distinct pathways, which offer different strategies to modulate the bioavailability of NAD+ . The therapeutic potential of dietarily available NAD+ boosters preserving the NAD+ pool has been attracting attention after the discovery of declining NAD+ levels in ageing model organisms as well as in several age-related diseases, including cardiometabolic and neurodegenerative diseases. Here, we review the recent advances in the biology of NAD+ , including the salubrious effects of NAD+ boosters and discuss their future translational strategies.

Sirt6 deletion in bone marrow-derived cells increases atherosclerosis - Central role of macrophage scavenger receptor 1.

AIMS: Sirtuin 6 (Sirt6) is a NAD+-dependent deacetylase that plays a key role in DNA repair, inflammation and lipid regulation. Sirt6-null mice show severe metabolic defects and accelerated aging. Macrophage-foam cell formation via scavenger receptors is a key step in atherogenesis. We determined the effects of bone marrow-restricted Sirt6 deletion on foam cell formation and atherogenesis using a mouse model. METHODS AND RESULTS: Sirt6 deletion in bone marrow-derived cells increased aortic plaques, lipid content and macrophage numbers in recipient Apoe-/- mice fed a high-cholesterol diet for 12 weeks (n = 12-14, p < .001). In RAW macrophages, Sirt6 overexpression reduced oxidized low-density lipoprotein (oxLDL) uptake, Sirt6 knockdown enhanced it and increased mRNA and protein levels of macrophage scavenger receptor 1 (Msr1), whereas levels of other oxLDL uptake and efflux transporters remained unchanged. Similarly, in human primary macrophages, Sirt6 knockdown increased MSR1 protein levels and oxLDL uptake. Double knockdown of Sirt6 and Msr1 abolished the increase in oxLDL uptake observed upon Sirt6 single knockdown. FACS analyses of macrophages from aortic plaques of Sirt6-deficient bone marrow-transplanted mice showed increased MSR1 protein expression. Double knockdown of Sirt6 and the transcription factor c-Myc in RAW cells abolished the increase in Msr1 mRNA and protein levels; c-Myc overexpression increased Msr1 mRNA and protein levels. CONCLUSIONS: Loss of Sirt6 in bone marrow-derived cells is proatherogenic; hereby macrophages play an important role given a c-Myc-dependent increase in MSR1 protein expression and an enhanced oxLDL uptake in human and murine macrophages. These findings assign endogenous SIRT6 in macrophages an important atheroprotective role.

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.

Mouse Models for Atherosclerosis Research-Which Is My Line?

Atherosclerosis is one of the primary causes of cardiovascular disease and mortality. This chronic immunometabolic disease evolves during decades in humans and encompasses different organs and immune cell types, as well as local and systemic processes that promote the progression of the disease. The most frequently used animal model to study these atherogenic processes and inter-organ crosstalk in a short time frame are genetically modified mouse models. Some models have been used throughout the last decades, and some others been developed recently. These models have important differences in cholesterol and lipoprotein metabolism, reverse cholesterol transport pathway, obesity and diabetes as well as inflammatory processes. Therefore, the disease develops and progresses differently in the various mouse models. Since atherosclerosis is a multifaceted disease and many processes contribute to its progression, the choice of the right mouse model is important to study specific aspects of the disease. We will describe the different mouse models and provide a roadmap to facilitate current and future atherosclerosis researchers to choose the right model depending on their scientific question.

Fas cell surface death receptor controls hepatic lipid metabolism by regulating mitochondrial function.

Nonalcoholic fatty liver disease is one of the most prevalent metabolic disorders and it tightly associates with obesity, type 2 diabetes, and cardiovascular disease. Reduced mitochondrial lipid oxidation contributes to hepatic fatty acid accumulation. Here, we show that the Fas cell surface death receptor (Fas/CD95/Apo-1) regulates hepatic mitochondrial metabolism. Hepatic Fas overexpression in chow-fed mice compromises fatty acid oxidation, mitochondrial respiration, and the abundance of mitochondrial respiratory complexes promoting hepatic lipid accumulation and insulin resistance. In line, hepatocyte-specific ablation of Fas improves mitochondrial function and ameliorates high-fat-diet-induced hepatic steatosis, glucose tolerance, and insulin resistance. Mechanistically, Fas impairs fatty acid oxidation via the BH3 interacting-domain death agonist (BID). Mice with genetic or pharmacological inhibition of BID are protected from Fas-mediated impairment of mitochondrial oxidation and hepatic steatosis. We suggest Fas as a potential novel therapeutic target to treat obesity-associated fatty liver and insulin resistance.Hepatic steatosis is a common disease closely associated with metabolic syndrome and insulin resistance. Here Item et al. show that Fas, a member of the TNF receptor superfamily, contributes to mitochondrial dysfunction, steatosis development, and insulin resistance under high fat diet.

Impaired SUMOylation of nuclear receptor LRH-1 promotes nonalcoholic fatty liver disease.

Hepatic steatosis is caused by metabolic imbalances that could be explained in part by an increase in de novo lipogenesis that results from increased sterol element binding protein 1 (SREBP-1) activity. The nuclear receptor liver receptor homolog 1 (LRH-1) is an important regulator of intermediary metabolism in the liver, but its role in regulating lipogenesis is not well understood. Here, we have assessed the contribution of LRH-1 SUMOylation to the development of nonalcoholic fatty liver disease (NAFLD). Mice expressing a SUMOylation-defective mutant of LRH-1 (LRH-1 K289R mice) developed NAFLD and early signs of nonalcoholic steatohepatitis (NASH) when challenged with a lipogenic, high-fat, high-sucrose diet. Moreover, we observed that the LRH-1 K289R mutation induced the expression of oxysterol binding protein-like 3 (OSBPL3), enhanced SREBP-1 processing, and promoted de novo lipogenesis. Mechanistically, we demonstrated that ectopic expression of OSBPL3 facilitates SREBP-1 processing in WT mice, while silencing hepatic Osbpl3 reverses the lipogenic phenotype of LRH-1 K289R mice. These findings suggest that compromised SUMOylation of LRH-1 promotes the development of NAFLD under lipogenic conditions through regulation of OSBPL3.

Inhibiting poly ADP-ribosylation increases fatty acid oxidation and protects against fatty liver disease.

BACKGROUND & AIMS: To date, no pharmacological therapy has been approved for non-alcoholic fatty liver disease (NAFLD). The aim of the present study was to evaluate the therapeutic potential of poly ADP-ribose polymerase (PARP) inhibitors in mouse models of NAFLD. METHODS: As poly ADP-ribosylation (PARylation) of proteins by PARPs consumes nicotinamide adenine dinucleotide (NAD+), we hypothesized that overactivation of PARPs drives NAD+ depletion in NAFLD. Therefore, we assessed the effectiveness of PARP inhibition to replenish NAD+ and activate NAD+-dependent sirtuins, hence improving hepatic fatty acid oxidation. To do this, we examined the preventive and therapeutic benefits of the PARP inhibitor (PARPi), olaparib, in different models of NAFLD. RESULTS: The induction of NAFLD in C57BL/6J mice using a high-fat high-sucrose (HFHS)-diet increased PARylation of proteins by PARPs. As such, increased PARylation was associated with reduced NAD+ levels and mitochondrial function and content, which was concurrent with elevated hepatic lipid content. HFHS diet supplemented with PARPi reversed NAFLD through repletion of NAD+, increasing mitochondrial biogenesis and ß-oxidation in liver. Furthermore, PARPi reduced reactive oxygen species, endoplasmic reticulum stress and fibrosis. The benefits of PARPi treatment were confirmed in mice fed with a methionine- and choline-deficient diet and in mice with lipopolysaccharide-induced hepatitis; PARP activation was attenuated and the development of hepatic injury was delayed in both models. Using Sirt1hep-/- mice, the beneficial effects of a PARPi-supplemented HFHS diet were found to be Sirt1-dependent. CONCLUSIONS: Our study provides a novel and practical pharmacological approach for treating NAFLD, fueling optimism for potential clinical studies. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is now considered to be the most common liver disease in the Western world and has no approved pharmacological therapy. PARP inhibitors given as a treatment in two different mouse models of NAFLD confer a protection against its development. PARP inhibitors may therefore represent a novel and practical pharmacological approach for treating NAFLD.