Abstract
BackgroundNicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide adenine dinucleotide (NAD) levels are crucial for liver function. The saturated fatty acid palmitate and the unsaturated fatty acid oleate are the main free fatty acids in adipose tissue and human diet. We asked how these fatty acids affect cell survival, NAMPT and NAD levels in HepG2 cells and primary human hepatocytes.MethodsHepG2 cells were stimulated with palmitate (0.5mM), oleate (1mM) or a combination of both (0.5mM/1mM) as well as nicotinamide mononucleotide (NMN) (0.5 mM) or the specific NAMPT inhibitor FK866 (10nM). Cell survival was measured by WST-1 assay and Annexin V/propidium iodide staining. NAD levels were determined by NAD/NADH Assay or HPLC. Protein and mRNA levels were analysed by Western blot analyses and qPCR, respectively. NAMPT enzyme activity was measured using radiolabelled 14C–nicotinamide. Lipids were stained by Oil red O staining.ResultsPalmitate significantly reduced cell survival and induced apoptosis at physiological doses. NAMPT activity and NAD levels significantly declined after 48h of palmitate. In addition, NAMPT mRNA expression was enhanced which was associated with increased NAMPT release into the supernatant, while intracellular NAMPT protein levels remained stable. Oleate alone did not influence cell viability and NAMPT activity but ameliorated the negative impact of palmitate on cell survival, NAMPT activity and NAD levels, as well as the increased NAMPT mRNA expression and secretion. NMN was able to normalize intracellular NAD levels but did not ameliorate cell viability after co-stimulation with palmitate. FK866, a specific NAMPT inhibitor did not influence lipid accumulation after oleate-treatment.ConclusionsPalmitate targets NAMPT activity with a consequent cellular depletion of NAD. Oleate protects from palmitate-induced apoptosis and variation of NAMPT and NAD levels. Palmitate-induced cell stress leads to an increase of NAMPT mRNA and accumulation in the supernatant. However, the proapoptotic action of palmitate seems not to be mediated by decreased NAD levels.
Highlights
Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide adenine dinucleotide (NAD) levels are crucial for liver function
Palmitate-induced cell death is rescued by oleate To investigate the impact of palmitate and oleate on cell survival, HepG2 cells were incubated with palmitate (0.5 mM), oleate (1 mM) and a combination of both (0.5 mM/1 mM)
Since NAMPT expression and NAD concentrations had been shown to be downregulated during the development of fatty liver disease [9] and NAMPT activity has been implicated in the regulation of lipid metabolism in the liver [22], we examined whether a decreased NAMPT activity and NAD levels would lead to higher lipid accumulation
Summary
Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide adenine dinucleotide (NAD) levels are crucial for liver function. The saturated fatty acid palmitate and the unsaturated fatty acid oleate are the main free fatty acids in adipose tissue and human diet. We asked how these fatty acids affect cell survival, NAMPT and NAD levels in HepG2 cells and primary human hepatocytes. Less is known about the regulation of hepatic NAMPT by the two main free fatty acids of human nutrition and adipose tissue, the monounsaturated fatty acid oleate (18:1) and the saturated fatty acid palmitate (16:0) [15, 16]. We investigated the impact of palmitate and oleate on NAMPT-mediated NAD biosynthesis in HepG2 cells and primary human hepatocytes. Hepatic NAD levels seemed to have no effect on hepatocyte survival or fat accumulation
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