Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7

Abstract

Recent studies have revealed the presence of intracellular lipid droplets in wide variety of species. In mammalian cells, there exist proteins specifically localize in lipid droplets. However, the protein profile in the droplet remains yet to be clarified. In this study, a fraction enriched with lipid droplets was isolated from a human hepatocyte cell line HuH7 using sucrose density gradient centrifugation, and 17 major proteins in the fraction were identified using nano LC-MS/MS techniques. Adipose differentiation-related protein (ADRP) was the most abundant protein in the fraction. The secondary abundant proteins were identified to be acyl-CoA synthetase 3 (ACS3) and 17beta-hydroxysteroid dehydrogenase 11 (17betaHSD11). Included in the identified proteins were five lipid-metabolizing enzymes as well as two lipid droplet-specific proteins. When HuH7 cell lysate was fractionated by a density gradient, most of 17betaHSD11 was found in the droplet-enriched fraction. In immunocytochemical analysis, 17betaHSD11 showed ring-shaped images which overlapped with those for ADRP. These results suggest that a specific set of proteins is enriched in the lipid droplet-enriched fraction and that 17betaHSD11 localizes specifically in the fraction.

Long-Chain Acyl-Coa Synthetases And Fatty Acid Channeling

“…[6, 48] ER, nuclear fraction, plasma membrane (3T3-L1 adipocytes) [12]; GLUT4 vesicle (rat adipocytes) [13]; mitochondria (PtK2 epithelial cells) [15]; ER, MAM, cytosol (rat liver) [79]; lipid droplet fraction (3T3-L1 adipocytes) [80] Triacsin C [35,81] NP_001986 ACSL3 Brain, gonads [48,82] Lipid droplet fraction (3T3-L1 adipocytes [80]; HuH7 cells [83]; CHO cells [84]; epithelial A431 cells [85]; HepG2 cells [86] Triacsin [26]; plasma membrane (mouse adipocytes) [20]; ER (multiple cell lines) [15]; mitochondria, nuclei, MAM, peroxisomes (skin fibroblasts) [1] n-dodecyl-D-maltopyranoside, …”

Mechanisms of leukocyte lipid body formation and function in inflammation

“…Although not yet characterized in leukocytes, lipid bodies from different cell types are sites of localization of lipid metabolic enzymes, including key enzymes involved in cholesterol metabolism (squalene epoxidase, 17-β-hydroxysteroid dehydrogenase, lanosterol synthase) and key enzymes of fatty acid synthesis (acetyl-coenzyme A carboxilase, NADH cytochrome b5 reductase), suggesting that both anabolic and catabolic steps in lipid metabolism may take place at lipid bodies (Brasaemle et al 2004, Fujimoto et al 2004, Liu et al 2004, Umlauf et al 2004. Proteins potentially associated with lipid trafficking including the cholesterol-binding protein caveolin and stomatin may be targeted to lipid bodies according to stimulatory conditions (Fujimoto et al 2001, Ostermeyer et al 2001, Pol et al 2001, Umlauf et al 2004.…”

Ethanol‐induced steatosis involves impairment of lipophagy, associated with reduced Dynamin2 activity

“…The hydrophobic cores of LDs are decorated with structural proteins, lipid‐synthesizing enzymes, lipases, and membrane‐trafficking proteins that regulate LD lipid storage and use. Proteins that are critical for vesicular transport, fusion, and fission have been implicated in LD interaction with other organelles, including the endoplasmic reticulum, endosomes, mitochondria, and peroxisomes for lipid exchange and metabolism 6. Under nutrient deprivation (fasting) conditions, LDs are an important source of energy, as they undergo a catabolic process called lipophagy, (a specific form of autophagy) to ultimately generate free fatty acids that undergo beta oxidation to generate energy for cellular demand 7, 8, 9…”