Role of cholesterol metabolism in hepatic steatosis and glucose tolerance

The liver is the central organ for lipid, lipoprotein and glucose homeostasis, thus hepatic metabolic disturbances can predispose individuals to develop cardiometabolic disorders (CMD) such as atherosclerotic cardiovascular diseases (ASCVD), type 2 diabetes mellitus (T2DM), and nonalcoholic fatty liver disease (NAFLD).The overall aim of this thesis was to expand the knowledge on how genetic and pharmacological interventions on hepatic and intestinal cholesterol metabolism could affect the pathophysiology of CMD.

Papers I and II: Acyl-Coenzyme A:cholesterol acyltransferase 2 (ACAT2, encoded by the Soat2 gene) is exclusively expressed in hepatocytes and enterocytes and catalyzes the biosynthesis of cholesteryl esters from cholesterol and long-chain fatty acids. Previous studies in mice model have shown that loss of ACAT2 activity protects from atherosclerosis, diet-induced hypercholesterolemia, and dietary cholesterol-driven hepatic steatosis. Here, we aimed to dissect the potential molecular mechanisms by which genetic depletion of Soat2 could affect the pathophysiology of hepatic steatosis and insulin sensitivity, independently of dietary regimens. We found that depletion of Soat2 significantly reduces hepatic steatosis and improves glucose tolerance, independently of high levels of cholesterol in the diet. We proposed the downregulation of hepatic de novo lipogenesis; DNL (lipid synthesis from glucose), GLUT2 membrane protein and Cd36 mRNA levels, as main mechanisms by which Soat2 depletion improves CMD. Dampening induction of CIDEC/FSP27 mRNA and protein levels in the severe fatty liver is another potential mechanism. Thus, cholesterol esterification by ACAT2 seems to be linked to hepatic steatosis and glucose homeostasis. Taken together, our study strongly supports ACAT2 inhibition as a promising target to treat CMD.

Papers III and IV: Ezetimibe and simvastatin inhibit cholesterol absorption and cholesterol synthesis, respectively. Adding ezetimibe to simvastatin therapy has been shown to result in an additional absolute risk reduction of death from ASCVD, particularly among patients with T2DM (IMPROVE-IT trial). In Paper III, we aimed to investigate the potential positive effects of cholesterol absorption and/or cholesterol synthesis inhibition on remnant particles, the binding to arterial proteoglycans (PG), and biliary lipid compositions as well as hepatic sterol regulatory element-binding protein 2 (SREBP2) target genes. Combined therapy resulted in athero-protective changes on remnant and apoB-lipoprotein particles, and on the affinity for arterial PG. In Paper IV, we aimed to further characterize the effects by the addition of ezetimibe to simvastatin therapy on the hepatic transcriptional signature to uncover potential beneficial responses on different metabolic pathways in humans. We identified a total of 260 reliable genes to be altered during the different treatments. Gene ontology and pathways analysis displayed involvement of the combined therapy in classical antibody-mediated complement activation. In view of individual genes, adding ezetimibe to simvastatin seems to affect the predisposition to hepatic steatosis and NAFLD, and improve the glucose tolerance; however functional validation in bigger cohorts is needed.

Collectively, our data might explain the decrease of ASCVD events reported in the IMPROVE-IT and SHARP trials, especially in T2DM patients. Hence, we propose the addition of ezetimibe to simvastatin therapy as an optimal intervention for lipid disorders characterized by elevated remnant-cholesterol (such as T2DM) to improve the outcome of CMD.

List of scientific papers

I. Ahmed O, Pramfalk C, Pedrelli M, Olin M, Steffensen KR, Eriksson M, Parini P. Genetic depletion of Soat2 diminishes hepatic steatosis via genes regulating de novo lipogenesis and by GLUT2 protein in female mice. Dig Liver Dis. 2018.
https://doi.org/10.1016/j.dld.2018.12.007

II. Pramfalk C, Ahmed O, Härdfeldt J, Pedrelli M, Vedin LL, Steffensen KR, Eriksson M, Parini P. Genetic depletion of the Soat2 gene improves glucose tolerance by reducing hepatic steatosis in male mice. [Manuscript]

III. Ahmed O, Littmann K, Gustafsson U, Pramfalk C, Öörni K, Larsson L, Minniti M E, Sahlin S, Camejo G, Parini P, Eriksson M. Ezetimibe in combination with simvastatin reduces remnant-cholesterol without affecting biliary lipid concentrations in gallstone patients. Journal of the American Heart Association. 2018; 7:e009876.
https://doi.org/10.1161/JAHA.118.009876

IV. Ahmed O, Mukarram AK, Pirazzini C, Marasco E, Minniti M E, Gustafsson U, Sahlin S, Pramfalk C, Garagnani P, Daub CO, Eriksson M, Parini P. Hepatic transcriptional response to combination of ezetimibe with simvastatin in gallstone patients. [Manuscript]