Excess adipose tissue associates with insulin resistance, high levels of triglyceride-rich VLDL, low levels of HDL and hepatic steatosis. The impact of obesity on lipid transport within the liver is unknown. This study investigated expression of cholesterol-trafficking proteins, mitochondrial StarD1 and endosomal StarD3, their relationship with dyslipidaemia and steatosis in genetically obese rats, and their functions in lipid metabolism in rat McArdle RH-7777 hepatoma cells.Heterozygous Zucker rats (Fa/fa) were purchased from Harlan Laboratories (Bicester, UK). Male and female, lean and obese rats were terminally anaesthetized at 4 months, using pentobarbital sodium (100mg/kg, I.P); blood was collected by cardiac puncture, and plasma levels of glucose and lipids determined [1]. The study was approved by the institution’s Animal Ethics and Welfare Committee, and procedures performed according to the UK Animals (Scientific Procedures) Act, 1986. Gene and protein expression in liver samples and stably transfected cell lines were determined by Q-PCR and immunoblotting; radiolabelled precursors were used to measure lipid synthesis and secretion, and lipidation of exogenous apolipoprotein (apo) A-I [2-4].There was no change in hepatic expression of StarD1 mRNA in obese rats, compared (cf.) with lean rats; low levels of StarD1 protein were difficult to detect by immunoblotting. Hepatic levels of StarD3 mRNA (48%; p<0.01; n=4) decreased in obese male cf. lean male rats. Levels of StarD3 protein decreased in obese male (11.65-fold; p<0.01) and obese female (2.31-fold; p<0.001) rats, cf. lean controls, linking StarD3 with hepatic storage or export of lipids. Overexpression (OE) of StarD1 (10-fold; p<0.05) increased [3H]glycerol (0.25mM) and [14C]oleate (0.7mM) incorporation (2h) into cellular triglycerides by 2.3-fold (p<0.05) and 1.4-fold (p<0.05). StarD3 OE (1.3-fold; p<0.05) increased synthesis of triacyl[3H]glycerol by 2.0-fold (p<0.05), and [14C]acetate (0.5mM; 2h) incorporation into the same pool by 1.8-fold (p<0.05) cf.EV. OE of StarD3, but not StarD1, increased lipidation of apoA-I (10μg/ml; 24h) by 1.7-fold (p<0.01) cf. EV. Finally, StarD3 OE significantly (p<0.01) altered expression of genes which impact on hepatic insulin resistance, inducing Ppargcla (8.6-fold), Cyp2e1 (2.8-fold), Nr1h4 (2.4-fold), G6pc (5.9-fold) and Irs1 (2.6-fold), and repressing Scl2a1 (2.8-fold), Igfbp1 (2.1-fold), Casp3 (2.1-fold) and Serpine 1 (3.8-fold) cf. EV. In conclusion, targeting StarD3 may increase circulating levels of HDL and protect the liver against lipotoxicity; loss of hepatic expression of this protein, induced by genetic obesity, may contribute to the pathogenesis of steatosis.