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 (Fal?) and obese (jaIJa) 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-dI5) and protein expression of the cholesterol-binding StarD proteins were profiled by quantitative-PCR and immunoblotting, respectively; all values were nOlmalized 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 eH]cholesterol and the synthesis and secretion of radiolabeled cholesterol, fatty acids, triglycerides and cholesteryl esters from radiolabeled precursors (eH]glycerol, [,4C]acetate and ['4C]0Ieate) 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 Stard13 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 [,4C]acetate into the triglycerides pool. Overexpression of StarDI 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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:654761 |
| Date | January 2014 |
| Creators | Soffientini, Ugo |
| Publisher | Glasgow Caledonian University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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