Abstract
Objective: To investigate the hepatic and serum lipid profile, and to examine the gene expression of the START family of intracellular lipid-trafficking proteins in liver tissues derived from lean (Fa/?) and obese (fa/fa) Zucker rodents, a model of leptin receptor deficiency and genetic obesity. To investigate the functional role of cholesterol-trafficking StarD proteins on the lipid metabolism of McA-RH7777 cells.Methods: Glycaemia, body weight, serum and hepatic lipids were measured from animals aged three to six months. Gene expressions of the START family (Stardl-dl5) and protein expression of the cholesterol-binding StarD proteins were profiled by quantitative-PCR and immunoblotting, respectively; all values were normalized to GAPDH. Stably transfected McArdle cell lines overexpressing cholesterol-binding StarD proteins were generated, and siRNA-mediated transient gene silencing was employed to study the effects of reduced expression of StarD3. The lipidation of ApoA-I with [3H]cholesterol and the synthesis and secretion of radiolabeled cholesterol, fatty acids, triglycerides and cholesteryl esters from radiolabeled precursors ([3H]glycerol, [l4C]acetate and [14C]oleate) monitored (2h). The genetic profile of McArdle cells overexpressing StarD3 was
determined by RT2-qPCR array.
Results: Obese male Zucker rats accumulated greater quantities of hepatic triacylglycerol and phospholipid than obese female rats, while obese female rats exhibited a more profound hyperlipidaemia. Protein expressions of StarD3 and StarD4 were repressed in obese animals compared to lean controls, and the expression of StarD5 was repressed by obesity in female rats. Notably, protein levels of StarD3 and StarD4 were higher in female, compared with male rodents, while the abundance of StarD5 was higher in males than females. Linear regression analyses revealed gendered expression of Stard3, Stard4, Stard5, Stard8, Stardl2 and Stardl3 to be inversely correlated with hepatic and/or serum lipid concentrations. Overexpression of StarD3 and StarD4 in McArdle cells increased the lipidation of exogenous ApoA-I and StarD3 increased the incorporation of de novo fatty acids into triglycerides and cholesteryl esters. Knockdown of StarD3 reduced the synthesis of cholesterol and cholesteryl esters, and repressed the incorporation of [14C]acetate into the triglycerides pool. Overexpression of StarD 1 and StarD5 increased the incorporation of exogenously supplied preformed fatty acids into the triglycerides pool. No increase in the cellular lipid mass was measured in any of the stable cell lines.
Conclusions: Genetic obesity was found to alter the expression of multiple StarD lipid-trafficking, and may therefore impact on the efficiency of hepatic lipid transport, a key issue in diabetic dyslipidaemia and non-alcoholic fatty liver disease. Hepatic overexpression of StarD3 and StarD4 increased lipidation of ApoA-I, suggesting that increased hepatic levels of these proteins might enhance the levels of circulating HDL. Increased expression of StarD 1 and StarD5 was found to promote the incorporation of [l4C]oleate into triglycerides, possibly by favoring the activity of DGAT-1. Increased hepatic levels of StarD 1 and StarD5 might reduce the intracellular levels of DAG, limiting the progression of insulin resistance triggered by fatty acids and DAG accumulation, but also increase the risk of developing steatosis and dyslipidaemia.
| Date of Award | 2014 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Annette Graham (Supervisor) & Sharron Dolan (Supervisor) |