Abstract Body (Do not enter title and authors here): Background: Atherosclerotic cardiovascular disease (ASCVD) stands as the foremost cause of mortality and disability among adults worldwide. Despite non-alcoholic fatty liver disease (NAFLD) has been well-characterized as an independent risk factor of atherosclerosis (AS), the precise underlying mechanisms remain poorly elucidated. We recently identified N-methyl-2-pyridone-5-carboxamide (2PY), the main end product of nicotinamide in human, as a pro-steatotic metabolite markedly elevated in hepatic steatosis conditions. The pathological increase in plasma 2PY levels has been intricately linked to an escalated susceptibility to cardiovascular issues.
Research Questions: We hypothesized that 2PY might be a potential mediator bridging the association between NAFLD and the progression of AS.
Goals: To elucidate the role and mechanistic insights of 2PY in the crosslink between hepatic steatosis and AS.
Methods: Mass spectrometry was conducted to determine the plasma levels of 2PY in Apolipoprotein E-deficient (ApoE-/-) mice. Five-week-old male ApoE-/- mice fed a standard laboratory diet (SLD) or a western diet (WD) were intraperitoneally injected with 2PY or its vehicle for 10 weeks. Parameters of systemic metabolism, hepatic steatosis and AS were evaluated. Human THP-1 macrophages were used to investigate the effect of 2PY on oleic acid-induced foam cell formation. Lipidomic and transcriptomic analysis were performed to identify the alterations of lipids and genes in THP-1 macrophages upon 2PY treatment.
Results: The expression of hepatic aldehyde oxidase 1, a key metabolic enzyme involved in 2PY metabolism, showed upregulation in the steatotic liver, resulting in the excess 2PY in WD-induced ApoE-/- mice. The plasma concentration of 2PY directly correlated with plaque area and lipid deposition in mice aortas. Furthermore, our study demonstrated that administration of 2PY aggravated lipid metabolic dysbiosis, hepatic steatosis and AS progression in mice fed either a SLD or a WD. In vitro, supplementation of 2PY dose-dependently increased intracellular lipid content in THP-1 macrophages. Mechanistically, 2PY upregulated ELOVL Fatty Acid Elongase 6 (ELOVL6) expression and induced lipid metabolic reprogram in macrophages, thereby facilitating foam cell formation.
Conclusion: Our study indicates 2PY as a pro-atherogenic metabolite that mediates the association between NAFLD and elevated ASCVD risk, thus providing mechanistic insight into the pathogenesis of AS.