From THE DEPARTMENT OF BIOSCIENCES AND NUTRITION Karolinska Institutet, Stockholm, Sweden LXR REGULATION OF HEPATIC GENE EXPRESSION AND PITUITARY DEPENDENT HORMONES

The liver X receptors (LXRs), members of the nuclear receptor superfamily, are ligand activated transcription factors involved in the regulation of genes with functions in cholesterol-, lipidand glucose metabolism. LXRs are considered potential drug targets to treat metabolic disorders, however, unwanted side effects, as revealed from rodent studies, include increased lipogenesis in the liver and increased plasma triglyceride levels. In this respect, it is of utmost importance to carry out studies also in human cell systems. In addition to LXR, growth hormone (GH), a peptide hormone originating from the pituitary and acting through its cognate cell surface receptor, has effects on cholesterol, lipid and glucose metabolism and a cross talk between LXR and GH signalling is conceivable. The GH secretion pattern is sexually dimorphic resulting in sex characteristic expression of many GH target genes in the liver and further characterization of hepatic genes with regard to dependency on sex specific GH secretion will help to understand liver physiology and molecular mechanisms. Furthermore, whether LXR activation affects hypothalamo-pituitaryhormonal axes had not been fully elucidated and was considered of interest to explore. In Paper I, we characterized the akr1b7 gene expression in rat liver as female specific and dependent on the female GH secretion pattern. AKR1B7 is a protein involved in detoxification of lipid peroxidationand steroidogenesis byproducts. The effect of GH was exerted directly on the hepatocyte but required ongoing protein synthesis, as shown in studies on primary rat hepatocytes in culture. Akr1b7 is in mice described as an LXR target gene. Surprisingly, treatment of rat hepatocytes with the synthetic LXR agonist T0901317 did not induce akr1b7, while co-treatment with GH and T0901317 resulted in reduced GH induced akr1b7 expression. T0901317 also interfered with the GH induced expression of CYP2C12, a female specific liver enzyme, suggesting that LXR activation interferes with signalling of the female GH pattern in regulation of female specific liver genes. In Paper II, we used human primary hepatocytes and the synthetic LXR ligand GW3965 to identify human LXR target genes by microarray. LXR-regulation of genes playing important roles in intermediary metabolism was confirmed by qRTPCR and data suggested that LXR-activation renders the liver less responsive to insulin. Comparative experiments on human and rat hepatocytes uncovered major species differences with bearings on the LXRs as drug targets in humans; the adipocyte differentiation related protein (ADFP), a protein strongly correlated with hepatic lipid storage, was induced in human but not in rat hepatocytes. This indicates a higher risk for development of hepatic steatosis in human than in rat livers upon pharmacological LXR targeting. Overall, there is an urgent need for experimental models based on human basis. In Paper III, we studied the effects of the LXR agonist T0901317 on hypothalamo-pituitary-hormonal axes in male rats. After one week of exposure the 24-hour GH secretion profile was marginally affected, just a small reduction in peak frequency and number was detected. On the other hand, T0901317 treatment resulted in decreased T3 levels, and T4 levels tended to increase, resulting in a significantly changed T3/T4 ratio. The underlying cause could be decreased hepatic conversion of T4 to T3, the active thyroid hormone, as deiodinase 1 expression in liver was decreased after LXR activation. Hypothyroidism has metabolic consequences for the individual and must be considered as an unacceptable side effect of pharmacological LXR agonism if occurring also in human. In Paper IV, the molecular mechanism of LXR regulation of the human ADFP gene, identified as an LXR target gene in Paper II, was studied in human hepatocytes. A difference in activation of the gene with two commonly used synthetic LXR agonists, GW3965 and T0901317, was evident; while GW3965 induced the gene, T0901317 did not. Consistent with gene expression data, ChIP assays showed recruitment of GW3965-activated LXR to the 3’-UTR containing a putative LXRE and to the promoter region, while T0901317-activated LXR was not recruited to these regions. Luciferase reporter assays confirmed LXR responsiveness of the putative LXRE. Interestingly, both LXR ligands induced the construct, demonstrating the importance of chromatin structure and suggesting the formation of a chromatin loop in the regulation of the human ADFP gene by certain LXR agonists. In conclusion, the studies presented in this thesis reveal important species differences in response to LXR activation. Furthermore, ligandand gene specific effects are demonstrated as well as LXR effects on pituitary hormone axes. In addition, we show cross talk between LXR and GH signalling. This urgently calls for experimental models based on human basis and refined strategies in developing LXR agonists for treatment of metabolic disorders.