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

Abstract

Hepatic steatosis is characterized by the accumulation of lipid droplets (LDs), which are composed of a neutral lipid core surrounded by a phospholipid monolayer embedded with many proteins. Although the LD-associated proteome has been investigated in multiple tissues and organisms, the dynamic changes in the murine LD-associated proteome in response to obesity and hepatic steatosis have not been studied. We characterized the hepatic LD-associated proteome of C57BL/6J male mouse livers following high-fat feeding using isobaric tagging for relative and absolute quantification. Of the 1,520 proteins identified with a 5% local false discovery rate, we report a total of 48 proteins that were increased and 52 proteins that were decreased on LDs in response to high-fat feeding. Most notably, ribosomal and endoplasmic reticulum proteins were increased and extracellular and cytosolic proteins were decreased in response to high-fat feeding. Additionally, many proteins involved in fatty acid catabolism or xenobiotic metabolism were enriched in the LD fraction following high-fat feeding. In contrast, proteins involved in glucose metabolism and liver X receptor or retinoid X receptor activation were decreased on LDs of high-fat-fed mice. This study provides insights into unique biological functions of hepatic LDs under normal and steatotic conditions.

Keywords: liver; nutrition; obesity; proteomics; β-oxidation.

Fig. 1.

Liver morphology and purification of hepatic LDs. A: Hematoxylin and eosin-stained liver sections from mice fed the ND or HFD. B: Equal amounts of protein from the postnuclear supernatant (PNS) and LD fractions were analyzed by Western blotting with antibodies against protein markers of LD (PLIN2) or common contaminating organelles such as ER (BIP), mitochondria (COX IV), and cytosol (ATPCL).

Fig. 2.

Enriched pathway analysis of hepatic LD-associated proteome. A: Top five enriched biological function categories as assigned by IPA. The P value range represents the lowest and highest enrichment within the subcategories. B: The top thirty enriched canonical pathways as assigned by IPA. The pathways are ranked left to right by – log (P value). The line graph represents the ratio of proteins from our dataset that map to the pathway divided by the total number of molecules in the same pathway. Pathways with fewer than three mapped proteins were discarded. C: The distribution of all 1,520 LD-associated proteins (ALL) versus the 849 novel mammalian LD-associated proteins (NOVEL) in a subset of GO terms as analyzed using STRAP.

Fig. 3.

Enriched pathway analysis of hepatic LD-associated proteome regulated by diet. A: Enriched biological function categories for proteins increased and decreased on LDs in response to high-fat feeding as assigned by IPA. The P value range represents the lowest and highest enrichment within the subcategories. Categories containing fewer than two assigned proteins were discarded. B: The enriched canonical pathways as assigned by IPA. The blue bars represent increased pathways and the green bars represent decreased pathways. The line graph represents the ratio of proteins from our dataset that map to the pathway divided by the total number of molecules in the same pathway. Pathways with fewer than two proteins, P > 0.01, or with enrichment in both increased and decreased datasets were discarded. C: The distribution of increased or decreased proteins in a subset of cellular component GO terms as analyzed using STRAP.

Fig. 4.

High-fat feeding results in an enriched of ACSL1 and CPT2 on LDs. Single plane image of a liver tissue section from a ND- and HFD-fed mouse stained for DAPI (A, E, K, O) or immunolabeled for PLIN2 (B, F, L, P), ACSL1 (C, G), or CPT2 (M, Q) or their respective merged images (D, H, N, R). I, J: Magnified from (H). S, T: Magnified from (R).

Fig. 5.

LD-associated proteome network. Only proteins found in the LD-associated proteome dataset are shown. Similar colors represent similar location and/or functional groups.