Dynamic Regulation of Hepatic Lipid Droplet Properties by Diet

Abstract

Cytoplasmic lipid droplets (CLD) are organelle-like structures that function in neutral lipid storage, transport and metabolism through the actions of specific surface-associated proteins. Although diet and metabolism influence hepatic CLD levels, how they affect CLD protein composition is largely unknown. We used non-biased, shotgun, proteomics in combination with metabolic analysis, quantitative immunoblotting, electron microscopy and confocal imaging to define the effects of low- and high-fat diets on CLD properties in fasted-refed mice. We found that the hepatic CLD proteome is distinct from that of CLD from other mammalian tissues, containing enzymes from multiple metabolic pathways. The hepatic CLD proteome is also differentially affected by dietary fat content and hepatic metabolic status. High fat feeding markedly increased the CLD surface density of perilipin-2, a critical regulator of hepatic neutral lipid storage, whereas it reduced CLD levels of betaine-homocysteine S-methyltransferase, an enzyme regulator of homocysteine levels linked to fatty liver disease and hepatocellular carcinoma. Collectively our data demonstrate that the hepatic CLD proteome is enriched in metabolic enzymes, and that it is qualitatively and quantitatively regulated by diet and metabolism. These findings implicate CLD in the regulation of hepatic metabolic processes, and suggest that their properties undergo reorganization in response to hepatic metabolic demands.

Quantitative analysis of the murine lipid droplet-associated proteome during diet-induced hepatic steatosis

“…However, the peroxisomal marker protein, catalase, was not depleted in the LD fraction. Although the LD fraction is largely free of contaminant proteins, the sucrose centrifugation method to isolate LD does yield some contaminant proteins, as has been reported in other LD proteomic studies (14)(15)(16).…”

“…ACSL1 has been identifi ed to activate long chain fatty acids and selectively channel them to mitochondrial ␤ -oxidation (43)(44)(45), and has also been shown to be present on LDs, in addition to its primary location on other organelles ( 13,15 ). CPT2 is an inner mitochondrial membrane protein important for fatty acid transport and activation.…”

“…Recently, the human hepatic LD proteome has been described for the fi rst time in NAFLD subjects ( 14 ). A few studies have examined the murine hepatic LD proteome ( 11,15 ); however, changes in the LD proteome have not been quantifi ed in a chronic animal model of steatosis. In the present study, we have quantifi ed the hepatic LD-associated proteome and characterized its changes in response to diet-induced development of hepatic steatosis.…”

“…The proteins identifi ed through this method are proteins that are associated with the LD fraction and are not necessarily bona-fi de LD proteins confi rmed to reside primarily or exclusively on the LD and, thus, will be referred to as LD-associated proteins. For comparison, a list of the LDassociated proteins identifi ed by previous proteomic studies of mammalian cells and tissues was compiled ( 10,11,15,16,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42). To decrease the likelihood of similar isoforms preventing a protein match, we used the UniRef50, which merges proteins of similar sequence to one UniRef entry.…”

Differential Roles of Cell Death-inducing DNA Fragmentation Factor-α-like Effector (CIDE) Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes

“…These proteins play important roles in regulating the size and function of LDs (14). Perilipin1/2/3/4/5 (Plin1/2/3/4/5) are LD-associated proteins in mammalian cells (15).…”