Physiological role of hepatic Site-1 Protease in plasma and liver lipid homeostasis.

Abstract

Understanding of lipid and lipoprotein metabolism is critical for further development of lipid-lowering strategies in humans, which has been shown to be effective in preventing atherosclerosis, hence cardiovascular disease. This thesis summarizes basic research findings of three independent yet integral aspects of lipid and lipoprotein metabolism.

Part 1: Development of assays for Lipoprotein lipase (LPL) and Endothelial lipase (EL).

LPL is the rate limiting enzyme for hydrolysis of TG-rich lipoproteins in the circulation. The activity of LPL can change depending on the physiological and nutritional state. We developed a homogeneous fluorogenic LPL activity assay, using a triglyceride analog, the EnzChek lipase substrate. The substrate does not fluoresce, owing to opposition of fluorescent and fluorescent quenching groups at the sn -1 and sn -2 positions, fluorescence becoming unquenched upon release of the sn -1 BODIPY FA derivative following hydrolysis. The assay was validated in post-heparin mouse plasma.

EL is a major negative regulator of plasma HDL levels in mice, rabbits, and most probably, humans. Although this regulatory function is critically dependent on EL’s hydrolysis of HDL phospholipids, as yet there is no phospholipase assay specific for EL in plasma. We developed such an assay for the mouse enzyme using a phospholipid-like fluorescent substrate in combination with an EL neutralizing antibody. The specificity of the assay was established using EL knockout mice and its utility demonstrated by detection of an increase in plasma EL phospholipase activity following exposure of wild-type mice to lipopolysaccharide.

Part 2: The role of hepatic Site-1Protease (S1P) in regulating plasma cholesterol in ApoB containing lipoproteins (Blp-c) levels and development of atherosclerosis.

Plasma Blp-c level is the cornerstone of current management of cardiovascular disease. S1P is the key enzyme required for activation of Sterol regulatory element binding proteins (SREBPs) that govern lipid synthesis. However, its role in regulating plasma Blp-c metabolism is unknown. A hepatic-specific knockdown (KD) of S1P using floxed S1P mouse models (S1Pf/f) and hepatic expression of Cre recombinase resulted in a 45% and 38% reduction in plasma total cholesterol and TG levels, respectively. Hepatic S1P KD had a minimal effect on plasma Blp cholesterol (Blp-c) in S1Pf/f mice, despite significantly reducing VLDL secretion. Notably, hepatic S1P KD decreased the LDL receptor (LDLR) mRNA expression by 50%. However, the reduction in LDLR protein levels was less than that of mRNA expression, especially under fed conditions. Further assessment of hepatic S1P deficiency revealed that it increased LDLR protein stability in vivo. Mechanistically, hepatic S1P KD was shown to decrease the liver and plasma levels of the protein proprotein convertase subtilisin/kexin type 9 (PCSK9), which degrades LDLR protein. This effect was more prominent in the fed condition and sufficient to account for the discordance in LDLR mRNA and protein levels. Furthermore, hepatic S1P was shown to regulate PCSK9 expression through activation of the SREBPs. In the LDLR-/- background, hepatic S1P KD significantly reduced Blp-c levels.

To test whether hepatic S1P deficiency would lower plasma Blp-c under nutrient excess conditions and would affect development of atherosclerotic lesions. We used two approaches to knockdown S1P in the liver. Short term inhibition of S1P was achieved by adenovirus mediated expression of Cre whereas adeno-associated virus mediated Cre expression was used for the long term inhibition study. We found that inhibition of hepatic S1P reduces plasma TC and TG in LDLR-/- mice fed western diet. Cholesterol and TG content in LDL fraction was considerably reduced in S1P KD mice. Metabolic analysis revealed suppression of VLDL secretion in S1P KD mice. Moreover, S1P KD in LDLR-/- mice had less atherosclerotic lesions than control mice in both models.

Part 3: The role of hepatic Site-1-Protease in regulating liver lipid homeostasis. Non alcoholic fatty liver disease is a chronic liver disease prevalent world-wide. It is strongly associated with obesity and insulin resistance. Steatosis or accumulation of TG occurs when there is an imbalance in rate of import or synthesis of fatty acids by hepatocytes and the rate of export or catabolism. These excess fatty acids are converted to triglycerides and stored in the cytoplasm, leading to steatosis. S1P can potentially regulate SREBPs, Activating transcription factor (ATF6) and cyclic AMP response element binding protein-H (CREBH), all transcription factors involved in hepatic lipid metabolism. Here, we investigated the effect of S1P deficiency on high fat diet induced hepatic steatosis. Inhibition of hepatic S1P in mice liver blocked activation of SREBP, ATF6 and CREBH. Our study provides the first definitive evidence that S1P is the in vivo protease for ATF6 and CREBH. S1P KD mice accumulated less TG in the livers compared to the controls. Metabolic analysis revealed that de novo lipid synthesis was reduced and hepatic FFA uptake was suppressed in S1P KD mice. Notably, hepatic S1P deficiency robustly reduced HFD induced body weight gain. In addition, S1P deficient mice had lower fasting glucose levels and were more insulin sensitive.

In summary, our studies demonstrate that hepatic S1P is a valid target for treating metabolic diseases like hyperlipidaemia, atherosclerosis and non alcoholic fatty liver disease.