Adipose differentiation-related protein is a reliable lipid droplet marker in alcoholic fatty liver of rats

Background: Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that coats cytoplasmic lipid droplets. The present study evaluated whether alcohol feeding enhances ADRP expression and whether ADRP is a lipid droplet marker in alcoholic fatty liver of rats. Because medium-chain triglycerides (MCT) reduce alcoholic hepatosteatosis, their effects on ADRP were also evaluated. Methods: Fatty liver was induced in rats by the consumption of the Lieber-DeCarli alcohol liquid diet with or without replacement of long-chain triglycerides (LCT) by MCT (32% of calories). Immunohistochemical staining for ADRP was performed in formalin-fixed, paraffin-embedded liver sections. ADRP immunostaining was quantified by image analysis. Triacylglycerol was measured chemically. ADRP mRNA and protein were analyzed by real-time polymerase chain reaction and western blot, respectively. Double staining technique was performed to distinguish ADRP from glycogen in hepatocytes. Results: Alcohol feeding for 21 days increased ADRP staining in the centrilobular and mid zonal regions of the liver lobules coincident with fat deposition in the liver. Replacing LCT in the alcohol diet with MCT diminished ADRP immunostaining in parallel with reduced steatosis. MCT also attenuated the up-regulation of ADRP mRNA and protein after alcohol. In steatotic hepatocytes ADRP selectively stained the surface of macrovesicular and microvesicular lipid droplets. ADRP immunostaining quantitatively correlated with hepatic triacylglycerol levels, validating ADRP as a reliable lipid droplet marker. Compared with hematoxylin and eosin stains, ADRP was more sensitive in detecting microvesicular lipid droplets. ADRP immunostaining also distinguished lipid droplets from glycogen, as demonstrated by double staining for ADRP and glycogen. Conclusions: Alcohol induction of fatty liver enhances ADRP expression and MCT oppose the alcohol effects. ADRP is a reliable and sensitive marker for lipid droplets in alcoholic fatty liver. ADRP immunostaining permits quantification of fatty change in hepatocytes and can be used as an ancillary technique in assessing the efficacy of diets or drugs against hepatosteatosis.