Gregor Lorbek

Cytochrome P450s in the synthesis of cholesterol and bile acids – from mouse models to human diseases

The present review describes the transgenic mouse models that have been designed to evaluate the functions of the cytochrome P450s involved in cholesterol and bile acid synthesis, as well as their link with disease. The knockout of cholesterogenic Cyp51 is embrionally lethal, with symptoms of Antley–Bixler syndrome occurring in mice, whereas the evidence for this association is conflicting in humans. Disruption of Cyp7a1 from classic bile acid synthesis in mice leads to either increased postnatal death or a milder phenotype with elevated serum cholesterol. The latter is similar to the case in humans, where CYP7A1 mutations associate with high plasma low‐density lipoprotein and hepatic cholesterol content, as well as deficient bile acid excretion. Disruption of Cyp8b1 from an alternative bile acid pathway results in the absence of cholic acid and a reduced absorption of dietary lipids; however, the human CYP8B1 polymorphism fails to explain differences in bile acid composition. Unexpectedly, apparently normal Cyp27a1−/− mice still synthesize bile acids that originate from the compensatory pathway. In humans, CYP27A1 mutations cause cerebrotendinous xanthomatosis, suggesting that only mice can compensate for the loss of alternative bile acid synthesis. In line with this, Cyp7b1 knockouts are also apparently normal, whereas human CYP7B1 mutations lead to a congenital bile acid synthesis defect in children or spastic paraplegia in adults. Mouse knockouts of the brain‐specific Cyp46a1 have reduced brain cholesterol excretion, whereas, in humans, CYP46A1 polymorphisms associate with cognitive impairment. At present, cytochrome P450 family 39 is poorly characterized. Despite important physiological differences between humans and mice, mouse models prove to be an invaluable tool for understanding the multifactorial facets of cholesterol and bile acid‐related disorders.

Identification of Natural RORγ Ligands that Regulate the Development of Lymphoid Cells

Mice deficient in the nuclear hormone receptor RORγt have defective development of thymocytes, lymphoid organs, Th17 cells, and type 3 innate lymphoid cells. RORγt binds to oxysterols derived from cholesterol catabolism, but it is not clear whether these are its natural ligands. Here, we show that sterol lipids are necessary and sufficient to drive RORγt-dependent transcription. We combined overexpression, RNAi, and genetic deletion of metabolic enzymes to study RORγ-dependent transcription. Our results are consistent with the RORγt ligand(s) being a cholesterol biosynthetic intermediate (CBI) downstream of lanosterol and upstream of zymosterol. Analysis of lipids bound to RORγ identified molecules with molecular weights consistent with CBIs. Furthermore, CBIs stabilized the RORγ ligand-binding domain and induced coactivator recruitment. Genetic deletion of metabolic enzymes upstream of the RORγt-ligand(s) affected the development of lymph nodes and Th17 cells. Our data suggest that CBIs play a role in lymphocyte development potentially through regulation of RORγt.

Sex Differences in the Hepatic Cholesterol Sensing Mechanisms in Mice

Cholesterol is linked to many multifactorial disorders, including different forms of liver disease where development and severity depend on the sex. We performed a detailed analysis of cholesterol and bile acid synthesis pathways at the level of genes and metabolites combined with the expression studies of hepatic cholesterol uptake and transport in female and male mice fed with a high-fat diet with or without cholesterol. Lack of dietary cholesterol led to a stronger response of the sterol sensing mechanism in females, resulting in higher expression of cholesterogenic genes compared to males. With cholesterol in the diet, the genes were down-regulated in both sexes; however, males maintained a more efficient hepatic metabolic flux through the pathway. Females had higher content of hepatic cholesterol but this was likely not due to diminished excretion but rather due to increased synthesis and absorption. Dietary cholesterol and sex were not important for gallbladder bile acids composition. Neither sex up-regulated Cyp7a1 upon cholesterol loading and there was no compensatory up-regulation of Abcg5 or Abcg8 transporters. On the other hand, females had higher expression of the Ldlr and Cd36 genes. These findings explain sexual dimorphism of cholesterol metabolism in response to dietary cholesterol in a high-fat diet in mice, which contributes to understanding the sex-basis of cholesterol-associated liver diseases.

Lessons from Hepatocyte-Specific Cyp51 Knockout Mice: Impaired Cholesterol Synthesis Leads to Oval Cell-Driven Liver Injury

We demonstrate unequivocally that defective cholesterol synthesis is an independent determinant of liver inflammation and fibrosis. We prepared a mouse hepatocyte-specific knockout (LKO) of lanosterol 14α-demethylase (CYP51) from the part of cholesterol synthesis that is already committed to cholesterol. LKO mice developed hepatomegaly with oval cell proliferation, fibrosis and inflammation, but without steatosis. The key trigger was reduced cholesterol esters that provoked cell cycle arrest, senescence-associated secretory phenotype and ultimately the oval cell response, while elevated CYP51 substrates promoted the integrated stress response. In spite of the oval cell-driven fibrosis being histologically similar in both sexes, data indicates a female-biased down-regulation of primary metabolism pathways and a stronger immune response in males. Liver injury was ameliorated by dietary fats predominantly in females, whereas dietary cholesterol rectified fibrosis in both sexes. Our data place defective cholesterol synthesis as a focus of sex-dependent liver pathologies.

Cholesterol and Inflammation at the Crossroads of Non-Alcoholic Fatty Liver Disease (NAFLD) and Atherogenesis

Non-alcoholic fatty liver disease (NAFLD) encompasses a variety of diseases ranging from simple steatosis or fatty liver through non-alcoholic steatohepatisis (NASH) to fibrosis that can eventually lead to irreversible cirrhosis. NASH was first described by Ludwig et al. (Ludwig et al., 1980) in a number of patients who reported no “significant” alcohol intake but whose liver histology resembled that of alcoholic liver disease. Today the term NAFLD is used either when referring to a full spectrum of liver disease or when the aetiology of the disease is unknown, excluding secondary causative factors such as excessive alcohol intake, hepatotoxic drugs, metabolic/genetic and other liver diseases (for instance autoimmune or viral hepatitis) (Treeprasertsuk et al., 2011). The occurrence of NAFLD has been persistently increasing in parallel with the concerning worldwide epidemic of obesity and diabetes and is expected to rise in the future (Portincasa et al., 2005). In the Western countries NAFLD is already becoming the most common cause of liver disease with the estimates of prevalence being between 17 and 33 % in the general population (McCullough, 2005) and rising as high as 90 % in morbidly obese individuals (Machado et al., 2006). NASH, the most severe and clinically significant form of NAFLD, is less common and is expected to be present between 5.7 and 17 % of the population (McCullough, 2005), again the numbers increase in the morbidly obese (up to 37 %) (Machado et al., 2006). Although NAFLD can occur in lean patients, the majority of the patients are overweight or obese (McCullough, 2005). Of particular concern, especially in the view of the future disease burden, is the presence of NAFLD in children and adolescents. For instance, the prevalence of obesity among US children has tripled in just one decade, rising from 5 % in 1980 to 15 % in 1990 and is currently around 17 %(Centers for Disease Control and Prevention CDC, 2011). The estimates of NAFLD prevalence in childhood have risen accordingly and are already between 2.6 and 9.6 % among the general pediatric population (Pacifico et al., 2010), reaching 68 % in obese children (Fu et al., 2011). Most cases of NAFLD arise in the detrimental environment of various metabolic disorders commonly known as the metabolic syndrome. The disease is strongly associated with insulin resistance, hypertension, glucose intolerance, central obesity and dyslipidemia and is thus recognized as the hepatic manifestation of the metabolic syndrome (Marchesini et al., 2005). Simple steatosis is largely benign and has a good prognosis. Nevertheless, a significant

Disrupting Hepatocyte Cyp51 from Cholesterol Synthesis Leads to Progressive Liver Injury in the Developing Mouse and Decreases RORC Signalling

Development of mice with hepatocyte knockout of lanosterol 14α-demethylase (HCyp51−/−) from cholesterol synthesis is characterized by the progressive onset of liver injury with ductular reaction and fibrosis. These changes begin during puberty and are generally more aggravated in the knockout females. However, a subgroup of (pre)pubertal knockout mice (runts) exhibits a pronounced male prevalent liver dysfunction characterized by downregulated amino acid metabolism and elevated Casp12. RORC transcriptional activity is diminished in livers of all runt mice, in correlation with the depletion of potential RORC ligands subsequent to CYP51 disruption. Further evidence for this comes from the global analysis that identified a crucial overlap between hepatic Cyp51−/− and Rorc−/− expression profiles. Additionally, the reduction in RORA and RORC transcriptional activity was greater in adult HCyp51−/− females than males, which correlates well with their downregulated amino and fatty acid metabolism. Overall, we identify a global and sex-dependent transcriptional de-regulation due to the block in cholesterol synthesis during development of the Cyp51 knockout mice and provide in vivo evidence that sterol intermediates downstream of lanosterol may regulate the hepatic RORC activity.

Hidden Disease Susceptibility and Sexual Dimorphism in the Heterozygous Knockout of Cyp51 from Cholesterol Synthesis

We examined the genotype-phenotype interactions of Cyp51+/− mice carrying one functional allele of lanosterol 14α-demethylase from cholesterol biosynthesis. No distinct developmental or morphological abnormalities were observed by routine visual inspection of Cyp51+/− and Cyp51+/+ mice and fertility was similar. We further collected a large data-set from female and male Cyp51+/− mice and controls fed for 16 weeks with three diets and applied linear regression modeling. We used 3 predictor variables (genotype, sex, diet), and 39 response variables corresponding to the organ characteristics (7), plasma parameters (7), and hepatic gene expression (25). We observed significant differences between Cyp51+/− and wild-type mice in organ characteristics and blood lipid profile. Hepatomegaly was observed in Cyp51+/− males, together with elevated total and low-density lipoprotein cholesterol. Cyp51+/− females fed high-fat, high-cholesterol diet were leaner and had elevated plasma corticosterone compared to controls. We observed elevated hepatocyte apoptosis, mitosis and lipid infiltration in heterozygous knockouts of both sexes. The Cyp51+/− females had a modified lipid storage homeostasis protecting them from weight-gain when fed high-fat high-cholesterol diet. Malfunction of one Cyp51 allele therefore initiates disease pathways towards cholesterol-linked liver pathologies and sex-dependent response to dietary challenge.