Final Report Summary - VISFATIN (RELEVANCE OF DIETARY FATTY ACIDS AND VITAMIN B3 FOR MANAGING VISFATIN-MEDIATED DYSLIPIDEMIA AND INFLAMMATION IN OBESITY)
Visfatin/NAMPT, initially discovered as an adipocyte cytokine, has been recently implicated in the pathogenesis of various cardiovascular disorders. During the fellow´s postdoctoral research period at the Department of Vascular Pathology, University of Maastricht (PIEF-GA-2008-221836), she initiated studies about the role of NAMPT on inflammatory responses modulation and redox homeostasis, apoptosis, and immune processes regulation. In the Instituto de la Grasa (CSIC), we set out to address the role of iNAMPT in the genesis and development of atherosclerosis and its effect on the metabolic syndrome.
The well-known changes in lifestyle habits including physical inactivity and over nutrition have led to striking adverse effects on public health (e.g. obesity, diabetes, and metabolic syndrome) over recent decades. Herein, we addressed the functional implications of dysregulated NAMPT expression on atherosclerotic plaque development, the underlying pathophysiology in a majority of cardiovascular disease. We demonstrate that patients with symptomatic disease showed a profound upregulation of NAMPT gene expression in peripheral blood mononuclear cells (PBMCs). Moreover, NAMPT was also upregulated in human atherosclerotic samples obtained during carotid endarterectomy; and, in fact, the relative gene expression levels of NAMPT increased progressively from early stable lesions to advanced and ruptured plaques.
To deepen knowledge into this evidence, we generated chimeric LDLR-/- mice overexpressing human iNAMPT (iNAMPThi) in the hematopoietic compartment by reconstitution of irradiated recipient mice with PGK-NAMPT lentivirus–infected bone marrow. It was interesting to note that hematopoietic iNAMPT overexpression did impede diet–induced atherosclerosis in the animals. We observed a significant decrease in plaque burden and a delay in plaque advance in the aortic roots of the iNAMPT chimera when compared with control chimera. Indeed, iNAMPThi chimera did not progress beyond a fatty streak stage, which was characterized by aortic lesions with a low content of macrophages and a marginal size of necrotic core. The common pathway of apoptosis by means of caspase-3 cleavage sites was also minimally stimulated in the aortic roots of the iNAMPThi chimera. Our findings also suggest that the effect of iNAMPT on plaque development involved the transition of macrophages into an anti-inflammatory phenotype, suggesting that iNAMPT could influence the hallmark of atherosclerosis.
iNAMPT chimera featured a marked shift of the monocyte subset towards patrolling CD11b+Ly6Clo (anti-inflammatory) in blood. Bone marrow monocytes were slightly increased while circulating monocyte numbers were lower, suggestive of stromal sequestration of Ly6Chi (pro-inflammatory) monocytes, albeit that subset specific effects on cell turnover cannot be excluded. Vice versa, administration of the NAMPT inhibitor FK866 boosted resident bone marrow monocyte release and circulating (Ly6Chi) monocyte numbers. The apparent stromal Ly6Chi retention in iNAMPT overexpression led us to study CCR2 (a chemokine which mediates monocyte migration) function, which was shown to control stromal release of this subset. Indeed iNAMPThi BMDMs had almost ablated CCR2 expression and migratory response. The quenched CCR2 expression and the inhibited migratory response were PPARgamma dependent; as they did not occur in LysM-Cre PPARgammaflox/flox BMDMs (cells with reduced expression of PPARgamma). We show that BMDMs of the iNAMPThi chimera displayed hyporeactivity to M1–type (pro-inflammatory) ligands and polarization to an M2 (anti-inflammatory) phenotype, and that PPARgamma–deficient BMDMs, even overexpressing iNAMPT, recovered their classical inflammatory gene transcription pattern, which identified the critical role of the iNAMPT–PPARgamma axis for phenotypic plasticity of macrophages into M2 skewing. This phenotype switch via PPARgamma activation.
Collectively, we demonstrate that hematopoietic iNAMPT confers protection against high fat diet–induced atherosclerosis. Furthermore, we show that this may well be linked to an increased stromal retention of pro-inflammatory monocytes, to the ensuing attenuation of monocyte migratory response. These effects of iNAMPT required the functional presence of PPARgamma, which appears not only to be activated but also to be induced by iNAMPT.
Thus, our studies establish for the first time, the importance of the iNAMPT–PPARgamma axis in the control of monocyte homeostasis in the context of inflammatory disorders such as atherosclerosis. We suggest a novel and complex interaction between iNAMPT and PPARgamma that could represent a target for therapy in atherosclerosis and related inflammatory disorders.
In parallel, we undertook in vivo human studies of dietary fatty acids on NAMPT and its relation with postprandial inflammation and atherosclerosis in the setting of metabolic syndrome, framed within the Spanish National Plan Project (AGL2011-29008). At this point, we are at the end of the in vivo studies with the analysis and statistics of all the data that we have available now.
From 2011-2012 we focus in the recruitment of healthy subjects and patients with metabolic syndrome, to elucidate the influence of olive oil (MUFA), butter (SFA) or fish oil (omega-3 PUFA) meals supplemented by vitamin B3 on postprandial NAMPT modulation and its involvement on leukocyte inflammatory response in subjects with metabolic syndrome.
We have selected 20 male volunteers with metabolic syndrome, diagnosed following 2004 American Heart Association/Updated NCEP definition criteria and 20 healthy male volunteers. We used a postprandial test with mixed high-fat emulsions containing 72% fat, 22% carbohydrate, and 6% protein (patented by us). Dietary fats [refined olive oil (MUFA), butter (SFA) and a mixture of fish oil with vegetable oils (PUFA)] provided ratios of MUFA to SFA ranging from 0.5 to 7.0 and the corresponding amount of omega-3 PUFA in the case of the meal containing fish oil. Together with corresponding high-fat emulsion, all the subjects received a vitamin B3 supplement (2 g/m2 body surface area). The subjects were attended in the Clinic Experimental Research Unit for Vascular Risk at the University Hospital Virgen del Rocio (UHVR, Seville) on 4 separate occasions. Following the ingestion of the meals, blood samples were collected at several time-points (t = 0, 2, 3, 4, and 6 h). Different empiric indices of postprandial beta-cell function and insulin sensitivity are being determined.
To complete the in vivo studies we undertook in vitro studies using triglycerol-rich lipoproteins (TRL), monocytes and neutrophils isolated from volunteers. Postprandial TRL were immediately isolated from blood samples at the postprandial peak by ultracentrifugation. TRL from the individuals in each group were mixed and analysed and the pools used for cell culture studies. Monocytes and neutrophils were also isolated from fasting and postprandial blood samples. Plasma NAMPT, NAD+, and NADH levels, nicotinic acid and its metabolites, and several inflammatory chemokines and inflammatory cytokines are under analysis by measuring with ELISA kits. Amongst others biochemical determinations, glucose, insulin, TG, and non-esterified fatty acids (NEFA) levels are being assessed. Glucose, insulin, C-peptide, triglyceride, and NEFA levels in plasma at several time-points (t = 0, 2, 3, 4, and 6 h) using routine biochemical procedures.
We are also investigating whether NAMPT expression and the inflammatory profile is altered in circulating monocytes and neutrophils throughout the postprandial state after the ingestion of the high-fat emulsion supplemented with vitamin B3, which has not been explored yet. Herein, we isolated circulating monocytes and neutrophils at fasting and postprandially. Monocytes and neutrophils activation markers by FACS analysis are in progress. Inflammatory markers are being measured in on fasting and postprandial monocytes and neutrophils at transcriptional (real time RT-PCR) and protein (i.e. Western blot and FACS) levels.
Data processed so far offer important novel insights on the relationship among dietary fatty acids, NAD+ metabolism, and metabolic syndrome.
Additionally to this research, and in order to deepen the contribution of NAMPT to the MUFA, SFA or omega-3 PUFA diets supplemented with vitamin B3 on inflammation, an in vivo animal experimentation is taking place at the Institute of Biomedicine of Seville (IBIS). To approach this point animal studies are running based on B6.Cg-Lepob LDLRtm1Her/J mouse model. An useful model in obesity, diabetes, dyslipidemia, and atherosclerosis. Animals are placed now on diets, which contains (refined olive oil, 21% MUFA) Mediterranean-type diet, (butter, 21% SFA) Western-type diet (21% EPA + DHA) Fish oil-supplementation diet, and normal chow diet). All the groups receive a vitamin B3 supplement (1%) in drink water. After diet, blood will be drawn via saphenous vein to determine lipids and NAD+ levels using routine biochemical procedures. Moreover, NAMPT levels will be measure by ELISA assay. Inflammatory cytokines will also be measured on plasma by Bio-Plex Pro Cytokine, Chemokine. Work will be finished by the end of the actual project running in the group.
Societal relevance is that we will be able to identify new targets that can be affected by NAMPT and vitamin B3 associated and can lead to the prevention of endemic obesity in western society. The European Union has embraced olive oil as the oil of choice and is investing more than 35 million euros to promote consumption in its member states. The results of the project leads to evidence based nutritional advice with the ultimate goals to establish a credible basis for health claims, elucidate the biochemical structure-function relationship of the naturally occurring compounds in olive oil, and prevent diet-related diseases such as CVD. Furthermore, vitamin B3 is one of the oldest lipid-modulating agents, yet new discoveries regarding its mechanisms of action have wide implications for combination therapy. In this lane, both vitamin B3 and statins have demonstrated clinical trial evidence of reducing cardiovascular events and mortality. The rationale for combination vitamin B3 and a natural lipid-lowering agent such as olive oil is powerful. There is therefore an imperious need for new research to determine whether combination therapy of vitamin B3 and olive oil results in cardiovascular event reduction, this will contribute to the use of these nutraceuticals as a natural alternative to conventional drug therapy and healthcare and its rising cost.
The well-known changes in lifestyle habits including physical inactivity and over nutrition have led to striking adverse effects on public health (e.g. obesity, diabetes, and metabolic syndrome) over recent decades. Herein, we addressed the functional implications of dysregulated NAMPT expression on atherosclerotic plaque development, the underlying pathophysiology in a majority of cardiovascular disease. We demonstrate that patients with symptomatic disease showed a profound upregulation of NAMPT gene expression in peripheral blood mononuclear cells (PBMCs). Moreover, NAMPT was also upregulated in human atherosclerotic samples obtained during carotid endarterectomy; and, in fact, the relative gene expression levels of NAMPT increased progressively from early stable lesions to advanced and ruptured plaques.
To deepen knowledge into this evidence, we generated chimeric LDLR-/- mice overexpressing human iNAMPT (iNAMPThi) in the hematopoietic compartment by reconstitution of irradiated recipient mice with PGK-NAMPT lentivirus–infected bone marrow. It was interesting to note that hematopoietic iNAMPT overexpression did impede diet–induced atherosclerosis in the animals. We observed a significant decrease in plaque burden and a delay in plaque advance in the aortic roots of the iNAMPT chimera when compared with control chimera. Indeed, iNAMPThi chimera did not progress beyond a fatty streak stage, which was characterized by aortic lesions with a low content of macrophages and a marginal size of necrotic core. The common pathway of apoptosis by means of caspase-3 cleavage sites was also minimally stimulated in the aortic roots of the iNAMPThi chimera. Our findings also suggest that the effect of iNAMPT on plaque development involved the transition of macrophages into an anti-inflammatory phenotype, suggesting that iNAMPT could influence the hallmark of atherosclerosis.
iNAMPT chimera featured a marked shift of the monocyte subset towards patrolling CD11b+Ly6Clo (anti-inflammatory) in blood. Bone marrow monocytes were slightly increased while circulating monocyte numbers were lower, suggestive of stromal sequestration of Ly6Chi (pro-inflammatory) monocytes, albeit that subset specific effects on cell turnover cannot be excluded. Vice versa, administration of the NAMPT inhibitor FK866 boosted resident bone marrow monocyte release and circulating (Ly6Chi) monocyte numbers. The apparent stromal Ly6Chi retention in iNAMPT overexpression led us to study CCR2 (a chemokine which mediates monocyte migration) function, which was shown to control stromal release of this subset. Indeed iNAMPThi BMDMs had almost ablated CCR2 expression and migratory response. The quenched CCR2 expression and the inhibited migratory response were PPARgamma dependent; as they did not occur in LysM-Cre PPARgammaflox/flox BMDMs (cells with reduced expression of PPARgamma). We show that BMDMs of the iNAMPThi chimera displayed hyporeactivity to M1–type (pro-inflammatory) ligands and polarization to an M2 (anti-inflammatory) phenotype, and that PPARgamma–deficient BMDMs, even overexpressing iNAMPT, recovered their classical inflammatory gene transcription pattern, which identified the critical role of the iNAMPT–PPARgamma axis for phenotypic plasticity of macrophages into M2 skewing. This phenotype switch via PPARgamma activation.
Collectively, we demonstrate that hematopoietic iNAMPT confers protection against high fat diet–induced atherosclerosis. Furthermore, we show that this may well be linked to an increased stromal retention of pro-inflammatory monocytes, to the ensuing attenuation of monocyte migratory response. These effects of iNAMPT required the functional presence of PPARgamma, which appears not only to be activated but also to be induced by iNAMPT.
Thus, our studies establish for the first time, the importance of the iNAMPT–PPARgamma axis in the control of monocyte homeostasis in the context of inflammatory disorders such as atherosclerosis. We suggest a novel and complex interaction between iNAMPT and PPARgamma that could represent a target for therapy in atherosclerosis and related inflammatory disorders.
In parallel, we undertook in vivo human studies of dietary fatty acids on NAMPT and its relation with postprandial inflammation and atherosclerosis in the setting of metabolic syndrome, framed within the Spanish National Plan Project (AGL2011-29008). At this point, we are at the end of the in vivo studies with the analysis and statistics of all the data that we have available now.
From 2011-2012 we focus in the recruitment of healthy subjects and patients with metabolic syndrome, to elucidate the influence of olive oil (MUFA), butter (SFA) or fish oil (omega-3 PUFA) meals supplemented by vitamin B3 on postprandial NAMPT modulation and its involvement on leukocyte inflammatory response in subjects with metabolic syndrome.
We have selected 20 male volunteers with metabolic syndrome, diagnosed following 2004 American Heart Association/Updated NCEP definition criteria and 20 healthy male volunteers. We used a postprandial test with mixed high-fat emulsions containing 72% fat, 22% carbohydrate, and 6% protein (patented by us). Dietary fats [refined olive oil (MUFA), butter (SFA) and a mixture of fish oil with vegetable oils (PUFA)] provided ratios of MUFA to SFA ranging from 0.5 to 7.0 and the corresponding amount of omega-3 PUFA in the case of the meal containing fish oil. Together with corresponding high-fat emulsion, all the subjects received a vitamin B3 supplement (2 g/m2 body surface area). The subjects were attended in the Clinic Experimental Research Unit for Vascular Risk at the University Hospital Virgen del Rocio (UHVR, Seville) on 4 separate occasions. Following the ingestion of the meals, blood samples were collected at several time-points (t = 0, 2, 3, 4, and 6 h). Different empiric indices of postprandial beta-cell function and insulin sensitivity are being determined.
To complete the in vivo studies we undertook in vitro studies using triglycerol-rich lipoproteins (TRL), monocytes and neutrophils isolated from volunteers. Postprandial TRL were immediately isolated from blood samples at the postprandial peak by ultracentrifugation. TRL from the individuals in each group were mixed and analysed and the pools used for cell culture studies. Monocytes and neutrophils were also isolated from fasting and postprandial blood samples. Plasma NAMPT, NAD+, and NADH levels, nicotinic acid and its metabolites, and several inflammatory chemokines and inflammatory cytokines are under analysis by measuring with ELISA kits. Amongst others biochemical determinations, glucose, insulin, TG, and non-esterified fatty acids (NEFA) levels are being assessed. Glucose, insulin, C-peptide, triglyceride, and NEFA levels in plasma at several time-points (t = 0, 2, 3, 4, and 6 h) using routine biochemical procedures.
We are also investigating whether NAMPT expression and the inflammatory profile is altered in circulating monocytes and neutrophils throughout the postprandial state after the ingestion of the high-fat emulsion supplemented with vitamin B3, which has not been explored yet. Herein, we isolated circulating monocytes and neutrophils at fasting and postprandially. Monocytes and neutrophils activation markers by FACS analysis are in progress. Inflammatory markers are being measured in on fasting and postprandial monocytes and neutrophils at transcriptional (real time RT-PCR) and protein (i.e. Western blot and FACS) levels.
Data processed so far offer important novel insights on the relationship among dietary fatty acids, NAD+ metabolism, and metabolic syndrome.
Additionally to this research, and in order to deepen the contribution of NAMPT to the MUFA, SFA or omega-3 PUFA diets supplemented with vitamin B3 on inflammation, an in vivo animal experimentation is taking place at the Institute of Biomedicine of Seville (IBIS). To approach this point animal studies are running based on B6.Cg-Lepob LDLRtm1Her/J mouse model. An useful model in obesity, diabetes, dyslipidemia, and atherosclerosis. Animals are placed now on diets, which contains (refined olive oil, 21% MUFA) Mediterranean-type diet, (butter, 21% SFA) Western-type diet (21% EPA + DHA) Fish oil-supplementation diet, and normal chow diet). All the groups receive a vitamin B3 supplement (1%) in drink water. After diet, blood will be drawn via saphenous vein to determine lipids and NAD+ levels using routine biochemical procedures. Moreover, NAMPT levels will be measure by ELISA assay. Inflammatory cytokines will also be measured on plasma by Bio-Plex Pro Cytokine, Chemokine. Work will be finished by the end of the actual project running in the group.
Societal relevance is that we will be able to identify new targets that can be affected by NAMPT and vitamin B3 associated and can lead to the prevention of endemic obesity in western society. The European Union has embraced olive oil as the oil of choice and is investing more than 35 million euros to promote consumption in its member states. The results of the project leads to evidence based nutritional advice with the ultimate goals to establish a credible basis for health claims, elucidate the biochemical structure-function relationship of the naturally occurring compounds in olive oil, and prevent diet-related diseases such as CVD. Furthermore, vitamin B3 is one of the oldest lipid-modulating agents, yet new discoveries regarding its mechanisms of action have wide implications for combination therapy. In this lane, both vitamin B3 and statins have demonstrated clinical trial evidence of reducing cardiovascular events and mortality. The rationale for combination vitamin B3 and a natural lipid-lowering agent such as olive oil is powerful. There is therefore an imperious need for new research to determine whether combination therapy of vitamin B3 and olive oil results in cardiovascular event reduction, this will contribute to the use of these nutraceuticals as a natural alternative to conventional drug therapy and healthcare and its rising cost.