Mitochondrial function in liver cells is resistant to perturbations in NAD⁺ salvage capacity

Supplementation with NAD precursors such as nicotinamide riboside (NR) has been shown to enhance mitochondrial function in the liver and to prevent hepatic lipid accumulation in high-fat diet (HFD)-fed rodents. Hepatocyte-specific knockout of the NAD⁺-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT) reduces liver NAD⁺ levels, but the metabolic phenotype of Nampt-deficient hepatocytes in mice is unknown. Here, we assessed Nampt's role in maintaining mitochondrial and metabolic functions in the mouse liver. Using the Cre-LoxP system, we generated hepatocyte-specific Nampt knockout (HNKO) mice, having a 50% reduction of liver NAD⁺ levels. We screened the HNKO mice for signs of metabolic dysfunction following 60% HFD feeding for 20 weeks ± NR supplementation and found that NR increases hepatic NAD⁺ levels without affecting fat mass or glucose tolerance in HNKO or WT animals. High-resolution respirometry revealed that NR supplementation of the HNKO mice did not increase state III respiration, which was observed in WT mice following NR supplementation. Mitochondrial oxygen consumption and fatty-acid oxidation were unaltered in primary HNKO hepatocytes. Mitochondria isolated from whole-HNKO livers had only a 20% reduction in NAD⁺, suggesting that the mitochondrial NAD⁺ pool is less affected by HNKO than the whole-tissue pool. When stimulated with tryptophan in the presence of [¹⁵N]glutamine, HNKO hepatocytes had a higher [¹⁵N]NAD⁺ enrichment than WT hepatocytes, indicating that HNKO mice compensate through de novo NAD⁺ synthesis. We conclude that NAMPT-deficient hepatocytes can maintain substantial NAD⁺ levels and that the Nampt knockout has only minor consequences for mitochondrial function in the mouse liver.