Fighting cardiometabolic disease : validation of new experimental models and therapeutic targets

Abstract: The prevalence of cardiometabolic diseases (CMD) such as atherosclerotic cardiovascular diseases, type 2 diabetes mellitus and nonalcoholic fatty liver disease (NAFLD), has grown dramatically during the last decades. Hence, massive research efforts are allocated to identify the pathophysiological mechanisms and new therapeutic targets for these morbidities. However, the data gained from preclinical studies using in vitro cellular or in vivo animal models are not always clinically translatable. The overall aim of this thesis was to develop and characterize new experimental models relevant to the human condition with respect to liver and lipoprotein metabolism, and to use these models to validate new therapeutic targets to treat CMD. Various strains of mice, genetically altered or unaltered, are extensively used to study human CMD. However, major species differences limit the human translatability of animal models. In Papers I and II we thoroughly characterized the lipoprotein and liver metabolism of liver- humanized mice (LHM), a promising preclinical model to study human hepatic metabolism. To generate LHM, immunocompromised Fah/Rag2/Il2rg-triple knockout mice on the nonobese diabetic background are repopulated with human hepatocytes. Cholesterol lipoprotein profiles of LHM showed a human-like pattern, shifting the cholesterol transport into low-density lipoprotein (LDL) rather than in high-density lipoprotein particles. The humanization of lipoprotein profiles does not require cholesteryl ester transfer protein, and was instead determined by higher levels of apolipoprotein B100 in the circulation, as a result of lower hepatic mRNA editing and LDL receptor expression, and higher levels of circulating proprotein convertase subtilisin/kexin type 9. As a consequence, LHM lipoproteins bind to human aortic proteoglycans in a pattern similar to human lipoproteins, which entails the potential use of LHM as a model for studies of atherosclerosis. A human-like bile acid metabolism was also observed in LHM, with higher levels of glycine-conjugated bile acids and taurodeoxycholic acid, and lower levels of mouse-specific tauromuricholic acids. However, an altered enterohepatic signaling in LHM results in abnormal bile acid synthesis. We also investigated the response to pharmacological and dietary stimuli in LHM. When treated with the liver X receptor (LXR) agonist GW3965, LHM mimicked the negative lipid outcomes seen in the first human trial of LXR stimulation, and thus allowed the characterization of the hepatic effects at a molecular level. To induce CMD in mouse models, challenge with high-fat/high-sucrose diet (HFHSD) is often used. However, LHM appeared to be resistant to HFHSD. We also present the preliminary results on the development of sever hepatic steatosis and atherosclerosis after feeding LHM with a high-fat/high- fructose/high-cholesterol diet. Taken together, these results indicate LHM as an interesting translatable model of human hepatic and lipoprotein metabolism. Because several metabolic parameters displayed donor dependency, LHM may also be used for studies of personalized medicine. Human hepatocyte-like cell lines (such as HepG2, Huh7 and Huh7.5 cells) are also widely used in preclinical research to study CMD. However, these cell lines exhibit major differences compared with human hepatocytes in vivo. For instance, hepatocytes in vivo only express sterol-O acyltransferase (SOAT) 2, whereas both SOAT1 and SOAT2 are found in HepG2, Huh7 and Huh7.5 cells. SOAT1 and SOAT2 catalyze the formation of cholesteryl esters, but only SOAT2 determines the amount of CE secreted in apolipoprotein B-containing lipoproteins. Therefore, in Paper III we used the clustered regularly interspaced short palindromic repeats (CRISPR) technology to knock out SOAT1 in HepG2 and Huh7.5 cells. Moreover, culturing HepG2 cells with medium supplemented with human instead of fetal bovine serum dramatically improves the lipid and lipoprotein metabolism. Hence, unedited and SOAT2-only cells were cultured with either fetal bovine or human serum to assess whether the combination of SOAT1-KO with culturing with human serum could additionally improve the phenotype of HepG2 and Huh7.5 cells. SOAT2-only-HepG2 cells exhibited higher levels of cholesterol, triglycerides and apolipoprotein B in the medium compared with unedited HepG2 cells. Further increase was seen when culturing SOAT2-only-HepG2 cells with human serum. Opposite effects were instead found in SOAT2-only-Huh7.5 cells. This study shows that SOAT1 expression in hepatocyte-like cells contributes to the distorted phenotype observed in HepG2 and Huh7.5 cells. SOAT2-only-HepG2 cells cultured with human serum represent an improved model for studies of human hepatic lipid metabolism. Inhibition of the lipid droplet-associated gene cell death-inducing DFFA-like effector c (CIDEC) has been proposed as a therapeutic strategy for hepatic steatosis and NAFLD. Hence, in Paper IV we knocked out CIDEC in HepG2 cells using the CRISPR technology in order to study its potential role as therapeutic target for hepatic steatosis/NAFLD. Knockout of CIDEC in HepG2 cells was accompanied by changes in the expression of several mediators of lipid metabolism. Nonetheless, the intracellular levels of cholesterol and triglycerides were not affected. Future studies will elucidate the role of CIDEC in hepatic lipid and carbohydrate metabolism and its potential as a therapeutic target for hepatic steatosis. Collectively, these results highlight LHM and SOAT2-only-HepG2 cells cultured with human serum as new preclinical models that greatly improve the translatability into humans compared with the commonly used in vivo and in vitro models.