Chronic glucocorticoid (GC) excess (Cushing’s syndrome, pharmacotherapy) causes metabolic and cardiovascular disease. This might be predicted from the known metabolic (dyslipidaemia, insulin resistance/hyperglycaemia) and hypertensive effects of chronically elevated GC levels. Intracellular GC levels within target tissues are controlled by 11β-hydroxysteroid dehydrogenases. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1, encoded by Hsd11b1) is an enzyme that, in intact cells and in vivo, converts inert GCs (cortisone in humans, and 11- dehydrocorticosterone in mice and rats) into their active forms (cortisol and corticosterone, respectively). Consequently, 11β-HSD1 amplifies intracellular GC levels. Additionally, 11β-HSD1 is also involved in the metabolism of 7-oxysterols; it catalyses the reduction of 7-ketocholesterol (7-KC) to 7β-hydroxycholesterol (7β- HC). 7-KC may inhibit cholesterol biosynthesis through its ability to inhibit cleavage/processing of sterol regulatory element binding protein-2 (SREBP-2), the key regulator of cholesterol synthesis. Alteration of cholesterol homeostasis is a major risk factor for cardiovascular disease. Improvement of metabolic syndrome and attenuation of atherosclerosis are observed in susceptible rodent models with 11β- HSD1 deficiency or inhibition. Conversely, pilot data showed decreased levels of 7- KC as well as increased levels of cleaved SREBP-2 protein (the transcriptionally active form) in liver of mice with hepatic 11β-HSD1 overexpression (LOE mice), suggesting increased cholesterol biosynthesis. It was hypothesised that hepatic 11β- HSD1 promotes cholesterol biosynthesis through hepatic induction of SREBP-2 target genes in the cholesterol biosynthetic pathway. The hypothesis was tested in adult, male LOE and wild-type C57BL/6 mice. In mice fed a standard chow diet, hepatic levels of mRNA encoding hydroxymethylglutarylcoenzyme A (HMG-CoA) reductase and HMG-CoA synthase, SREBP-2 targets in the cholesterol biosynthetic pathway, did not differ between genotypes. Compared to chow, a cholesterol-rich ‘Western’ diet (WD) decreased hepatic levels of mRNA encoding SREBP-2, HMG-CoA reductase and HMG-CoA synthase in wild-type as well as in LOE mice. These data imply that LOE mice show a normal physiological response with respect to cholesterol synthesis when challenged with cholesterol-rich diet, and, contrary to the hypothesis, liver 11β-HSD1 does not increase cholesterol biosynthesis via elevated expression of mRNAs encoding hepatic cholesterol biosynthetic enzymes. The liver X receptors (LXR) are well-known as sensors of oxysterols and regulators of genes involved in processes that decrease total body cholesterol levels i.e. reverse cholesterol transport and cholesterol excretion into bile. Cholesterol is the precursor to oxysterol LXR ligands. It was predicted that liver overexpression of 11β-HSD1 leads to activation of LXRα (the isoform with dominant roles in reverse cholesterol transport and whole-body cholesterol homeostasis) and its downstream targets involved in cholesterol efflux and excretion, in response to increased intracellular cholesterol levels. Indeed, levels of Lxrα mRNA were increased in livers of WD-fed LOE mice compared to wild-type mice on the same diet. There was no evidence for increased cholesterol clearance through bile acid synthesis in LOE mice as indicated by unchanged levels of hepatic Cyp7a1 mRNA between LOE and wild-type mice. However, consistent with being direct targets of LXRα, increased Abcg5 and Abcg8 mRNA levels were observed in livers of WD-fed LOE mice compared to WD-fed wild-type mice. These results corroborate findings in chow-fed LOE mice. Moreover, these data suggest that LOE mice ‘sense’ intracellular cholesterol excess and respond to it by increasing cholesterol efflux into the biliary lumen for excretion, thereby supporting a role for hepatic 11β-HSD1 in promoting biliary cholesterol secretion. To assess the effect(s) of hepatic 11β-HSD1 deficiency on cholesterol homeostasis as well as evaluate the importance of liver 11β-HSD1 in metabolic syndrome, liver-specific 11β-HSD1 knockout (LKO) mice were generated by crossing “floxed” Hsd11b1 mice with Alb-Cre transgenic mice in which Cre expression is restricted to hepatocytes. In liver of LKO mice, 11β-HSD1 mRNA, protein and enzyme activity were reduced by >80%, with no differences in 11β-HSD1 protein levels in kidney, adipose tissue or muscle between LKO and floxed Hsd11b1 littermate controls. These results indicate liver-specificity of Hsd11b1 knockdown in these mice. Body weight and weights of liver, adipose tissue, adrenal, muscle, kidney and brain were unaltered by liver-specific 11β-HSD1 deficiency on a standard chow diet. These mice were subject to a 14-week high fat (HF) diet, which typically causes metabolic syndrome in control but not globally 11β-HSD1 deficient mice. In HF-fed LKO mice, weights of the subcutaneous and epididymal fat depots were decreased compared to HF-fed control mice, resulting in an overall decrease in total white adipose tissue weight. Although no differences were observed in subcutaneous adipocyte hypertrophy between HF-fed LKO and control mice in a small number of samples tested, the above finding suggests that liver 11β-HSD1 influences adiposity and that liver-specific deficiency of 11β-HSD1 may reduce diet-induced adiposity. In terms of cholesterol homeostasis, no differences were observed in hepatic levels of mRNAs encoding cholesterol biosynthetic enzymes as well as those encoding enzymes/transporters for cholesterol catabolism/excretion between LKO and control mice, on either chow or HF diet. In summary, these data do not support a role for hepatic 11β-HSD1 in cholesterol synthesis. However, my evidence suggests that increased hepatic 11β-HSD1 promotes hepatobiliary cholesterol secretion. Finally, knockdown of liver 11β-HSD1, combined with HF feeding, reduces adiposity, suggesting that hepatic 11β-HSD1 may play a key role in adipose tissue lipogenesis/lipolysis and/or lipid storage, and that liver-specific 11β-HSD1 inhibition (or deficiency) may be advantageous in diet-induced obesity. Data presented in this thesis contribute to the understanding of the role of hepatic 11β-HSD1 in cholesterol homeostasis and metabolic syndrome.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:716612 |
| Date | January 2016 |
| Creators | Manwani, Kajal |
| Contributors | Chapman, Karen ; Seckl, Jonathan |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/22058 |
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