IDDF2023-ABS-0114 Hepatocyte SphK2 maintains the homeostasis of lipid metabolism in NAFLD

Background

Non-alcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver injury without alcohol and other definite liver injury factors, which is characterized by diffuse hepatic steatosis and ballooning injury, accompanied by obesity and insulin resistance. However, there are none FDA-approved therapeutic options for NAFLD treatment. Sphingosine kinases (SphKs), SphK1 and SphK2, are key rate-limiting enzyme that catalyzes the phosphorylation of sphingosine to sphingosine 1-phosphate (S1P). However, the roles of SphK2 in the hepatic lipid metabolism of NAFLD have not been elucidated.

Methods

Wild-type (SphK2-Flox), hepatocyte-specific SphK2 knockout (SphK2-HKO) mice and SphK2 overexpression mice induced by adeno-associated virus (AAV-SphK2) injection were fed with high-fat diet (HFD) for 20 weeks to induce NAFLD. Lipid metabolomics was used to analyze and screen the differential metabolites significantly affected by hepatocyte SphK2. In addition, coIP, homology modeling and molecular docking were used to construct the three-dimensional structure of proteins, and observe the interaction and binding sites between proteins. The SphK2-Flox and mutant plasmids were transfected and the ubiquitination assay was performed.

Results

Hepatocyte SphK2 deficiency markedly aggravated hepatic lipid deposition and steatosis, hepatocellular injury and insulin resistance (IDDF2023-ABS-0114 Figure 1. Hepatocyte specific SphK2 knockout exacerbates HFD induced hepatic steatosis) and promoted intrahepatic triglyceride (TG) deposition induced by high-fat diet (IDDF2023-ABS-0114 Figure 2. Hepatocyte specific SphK2 knockout promotes TG deposition induced by HFD diet). In contrast, SphK2 overexpression markedly reversed the lipid metabolism disorder induced by a high-fat diet (IDDF2023-ABS-0114 Figure 3 Hepatocyte specific SphK2 overexpression improves HFD induced hepatic steatosis). Mechanistically, hepatocyte SphK2 competes with Constitutive Photomorphogenic Protein 1 (COP1) via its H335 to occupy the VP motif of ATGL to prevent COP1 from binding to adipose triglyceride lipase (ATGL), thereby inhibiting ubiquitination degradation of ATGL and up-regulating the protein expression of ATGL (IDDF2023-ABS-0114 Figure 4 SphK2 competes with COP1 to inhibit ubiquitination degradation of ATGL).

Conclusions

These data demonstrated that hepatocyte SphK2 competitively binds ATGL with COP1, inhibits ATGL ubiquitination, and accelerates TG degradation in hepatocytes, thereby maintaining the homeostasis of lipid metabolism and improving NAFLD. Targeting hepatocyte SphK2 activation is a promising therapeutic strategy for NAFLD treatment.