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

Abstract

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.

The role of sphingolipids in endoplasmic reticulum stress

“…Furthermore, imipramine, an ASMase inhibitor, also improved ethanol-induced hepatic steatosis [107]. Similarly, amitriptyline, which is another ASMase inhibitor, robustly reduced hepatic steatosis and hepatic inflammation in high-palmitic acid-containing-HFD-fed mice treated with lipopolysaccharides, while GW4869, which is a NSMase inhibitor [108], had modest effects [109]. Collectively, these studies suggest the possibility of using ASMase inhibitors as therapeutic agents for hepatic steatosis.…”

“…In addition, treating MIN6 cells with GW4869, a specific NSMase inhibitor [108], decreased both palmitate-induced apoptosis and ER stress, which suggests that a loss of sphingomyelin in the ER is a key event for initiating b-cell lipotoxicity [113]. Indeed, inhibition of either ASMase or NSMase improved glucose, cholesterol, triglycerides and insulin resistance in high-palmitic acid-containing HFD-fed mice [109], which suggests the potential of SMase inhibitors as therapeutic agents for diabetes.…”

Acid Sphingomyelinase, a Lysosomal and Secretory Phospholipase C, Is Key for Cellular Phospholipid Catabolism

“…Its downregulation was reported to alleviate vascular endothelial insulin resistance in diabetic rats [152] and improve vascular dysfunction in diabetic mice [153]. Amitriptyline inhibits nonalcoholic steatohepatitis and atherosclerosis as induced by high-fat diet and lipopolysaccharide in mice [154]. The survival of cultured oligodendrocytes treated with glutamate was enhanced by the downregulation of ASM-expression as well as by blocking ASM activity [155].…”

Involvement of Ceramides in Non-Alcoholic Fatty Liver Disease (NAFLD) Atherosclerosis (ATS) Development: Mechanisms and Therapeutic Targets

“…The inhibition of nSMase2 suppress inflammation by two mechanisms: a short time process implicating Nrf2 (nuclear factor [erythroid-derived 2]-like 2 or) that plays as an anti-inflammatory response, and long-term action, by decreasing the production of pro-inflammatory ceramides [124]. The pharmacological or genetic inhibition of nSMase2 was not associated with plasma lipoprotein changes, but with the direct effect on the arterial wall [122].…”

“…To elucidate the role of nSMase2 in atherogenesis, researchers explored a genetic double mutant mouse model deficient in both nSMase2 activity and ApoE deficient and a pharmacological model of long-term inhibition of nSMase2 by GW4869 in ApoE deficient mice [121]. The results show that the genetic deficiency of nSMase2 or its pharmacological inhibition by GW4869, significantly reduced the size of atherosclerotic areas, and the accumulation of macrophages, by 68% in mice treated with GW4869 compared to 49% control mice [122] Additionally, the deficiency or inhibition of nSMase2 activity resulted in a significant decrease in plasma ceramide levels, particularly in 24:1, 22:0, and 24:0 ceramide levels [122,123]. The inhibition of nSMase2 suppress inflammation by two mechanisms: a short time process implicating Nrf2 (nuclear factor [erythroid-derived 2]-like 2 or) that plays as an anti-inflammatory response, and long-term action, by decreasing the production of pro-inflammatory ceramides [124].…”