LAUSR

Simple Summary Alcohol-associated liver disease is a major healthcare problem worldwide and is the third leading cause of preventable deaths in the US. Hepatic steatosis is the earliest manifestation of chronic alcohol misuse, characterized by accumulation of specialized fat storing organelles called lipid droplets (LDs). Our previous studies reported that the alcohol-induced increase in the number and size of LDs is attenuated by simultaneous treatment with the methyl group donor, betaine. In this study, we examined alcohol ± betaine-induced changes in the LD lipidome with respect to their size. Untargeted lipidomic analyses of the three different-sized hepatic LD fractions revealed higher phospholipids, cholesteryl esters, diacylglycerols, ceramides, and hexosylceramides in each fraction isolated from livers of ethanol-fed rats compared with the corresponding fractions of pair-fed controls. Betaine supplementation significantly attenuated the ethanol-induced LD lipidomic changes. We conclude that ethanol-induced changes in the hepatic LD lipidome may stabilize larger-sized LDs during steatosis development. Furthermore, betaine supplementation could effectively reduce the size and dynamics of LDs to attenuate alcohol-associated hepatic steatosis. Abstract The earliest manifestation of alcohol-associated liver disease is hepatic steatosis, which is characterized by fat accumulation in specialized organelles called lipid droplets (LDs). Our previous studies reported that alcohol consumption elevates the numbers and sizes of LDs in hepatocytes, which is attenuated by simultaneous treatment with the methyl group donor, betaine. Here, we examined changes in the hepatic lipidome with respect to LD size and dynamics in male Wistar rats fed for 6 weeks with control or ethanol-containing liquid diets that were supplemented with or without 10 mg betaine/mL. At the time of sacrifice, three hepatic LD fractions, LD1 (large droplets), LD2 (medium-sized droplets), and LD3 (small droplets) were isolated from each rat. Untargeted lipidomic analyses revealed that each LD fraction of ethanol-fed rats had higher phospholipids, cholesteryl esters, diacylglycerols, ceramides, and hexosylceramides compared with the corresponding fractions of pair-fed controls. Interestingly, the ratio of phosphatidylcholine to phosphatidylethanolamine (the two most abundant phospholipids on the LD surface) was lower in LD1 fraction compared with LD3 fraction, irrespective of treatment; however, this ratio was significantly lower in ethanol LD fractions compared with their respective control fractions. Betaine supplementation significantly attenuated the ethanol-induced lipidomic changes. These were mainly associated with the regulation of LD surface phospholipids, ceramides, and glycerolipid metabolism in different-sized LD fractions. In conclusion, our results show that ethanol-induced changes in the hepatic LD lipidome likely stabilizes larger-sized LDs during steatosis development. Furthermore, betaine supplementation could effectively reduce the size and dynamics of LDs to attenuate alcohol-associated hepatic steatosis.