Amitriptyline inhibits nonalcoholic steatohepatitis and atherosclerosis induced by high-fat diet and LPS through modulation of sphingolipid metabolism

We reported previously that increased acid sphingomyelinase (ASMase)-catalyzed hydrolysis of sphingomyelin, which leads to increases in ceramide and sphingosine 1 phosphate (S1P), played a key role in the synergistic upregulation of proinflammatory cytokines by palmitic acid (PA), a major saturated fatty acid, and lipopolysaccharide (LPS) in macrophages. Since macrophages are vital players in nonalcoholic steatohepatitis (NASH) and atherosclerosis, we assessed the effect of ASMase inhibition on NASH and atherosclerosis cooperatively induced by high-PA-containing high-fat diet (HP-HFD) and LPS in LDL receptor-deficient (LDLR−/−) mice. LDLR−/− mice were fed HP-HFD, injected with low dose of LPS and treated with or without the ASMase inhibitor amitriptyline. The neutral sphingomyelinase inhibitor GW4869 was used as control. Metabolic study showed that both amitriptyline and GW4869 reduced glucose, lipids, and insulin resistance. Histological analysis and Oil Red O staining showed that amitriptyline robustly reduced hepatic steatosis while GW4869 had modest effects. Interestingly, immunohistochemical study showed that amitriptyline, but not GW4869, strongly reduced hepatic inflammation. Furthermore, results showed that both amitriptyline and GW4869 attenuated atherosclerosis. To elucidate the underlying mechanisms whereby amitriptyline inhibited both NASH and atherosclerosis, but GW4869 only inhibited atherosclerosis, we found that amitriptyline, but not GW4869, downregulated proinflammatory cytokines in macrophages. Finally, we found that inhibition of sphingosine 1 phosphate production is a potential mechanism whereby amitriptyline inhibited proinflammatory cytokines. Collectively, this study showed that amitriptyline inhibited NASH and atherosclerosis through modulation of sphingolipid metabolism in LDLR−/− mice, indicating that sphingolipid metabolism in macrophages plays a crucial role in the linkage of NASH and atherosclerosis.