Proteome of skeletal muscle lipid droplet reveals association with mitochondria and apolipoprotein a-I

The lipid droplet (LD) is a universal organelle governing the storage and turnover of neutral lipids. Mounting evidence indicates that elevated intramuscular triglyceride (IMTG) in skeletal muscle LDs is closely associated with insulin resistance and Type 2 Diabetes Mellitus (T2DM). Therefore, the identification of the skeletal muscle LD proteome will provide some clues to dissect the mechanism connecting IMTG with T2DM. In the present work, we identified 324 LD-associated proteins in mouse skeletal muscle LDs through mass spectrometry analysis. Besides lipid metabolism and membrane traffic proteins, a remarkable number of mitochondrial proteins were observed in the skeletal muscle LD proteome. Furthermore, imaging by fluorescence microscopy and transmission electronic microscopy (TEM) directly demonstrated that mitochondria closely adhere to LDs in vivo. Moreover, our results revealed for the first time that apolipoprotein A-I (apo A-I), the principal apolipoprotein of high density lipoprotein (HDL) particles, was also localized on skeletal muscle LDs. Further studies verified that apo A-I was expressed endogenously by skeletal muscle cells. In conclusion, we report the protein composition and characterization of skeletal muscle LDs and describe a novel LD-associated protein, apo A-I.     

Identification of the major functional proteins of prokaryotic lipid droplets

Storage of cellular triacylglycerols (TAGs) in lipid droplets (LDs) has been linked to the progression of many metabolic diseases in humans, and to the development of biofuels from plants and microorganisms. However, the biogenesis and dynamics of LDs are poorly understood. Compared with other organisms, bacteria seem to be a better model system for studying LD biology, because they are relatively simple and are highly efficient in converting biomass to TAG. We obtained highly purified LDs from Rhodococcus sp. RHA1, a bacterium that can produce TAG from many carbon sources, and then comprehensively characterized the LD proteome. Of the 228 LD-associated proteins identified, two major proteins, ro02104 and PspA, constituted about 15% of the total LD protein. The structure predicted for ro02104 resembles that of apolipoproteins, the structural proteins of plasma lipoproteins in mammals. Deletion of ro02104 resulted in the formation of supersized LDs, indicating that ro02104 plays a critical role in cellular LD dynamics. The putative α helix of the ro02104 LD-targeting domain (amino acids 83-146) is also similar to that of apolipoproteins. We report the identification of 228 proteins in the proteome of prokaryotic LDs, identify a putative structural protein of this organelle, and suggest that apolipoproteins may have an evolutionarily conserved role in the storage and trafficking of neutral lipids.     

ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6

Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs.     

Identification and characterization of associated with lipid droplet protein 1: A novel membrane-associated protein that resides on hepatic lipid droplets

Alcoholic and nonalcoholic liver steatosis and steatohepatitis are characterized by the massive accumulation of lipid droplets (LDs) in the cytosol of hepatocytes. Although LDs are ubiquitous and dynamic organelles found in the cells of a wide range of organisms, little is known about the mechanisms and sites of LD biogenesis. To examine the participation of these organelles in the pathophysiological disorders of steatotic livers, we used a combination of mass spectrometry (matrix-assisted laser desorption ionization-time of flight and LC-MS electrospray) and Western blot analysis to study the composition of LDs purified from rat liver after a partial hepatectomy. Fifty proteins were identified. Adipose differentiation-related protein was the most abundant, but other proteins such as calreticulin, TIP47, Sar1, Rab GTPases, Rho and actin were also found. In addition, we identified protein associated with lipid droplets I ALDI (tentatively named Associated with LD protein 1), a novel protein widely expressed in liver and kidney corresponding to the product of 0610006F02Rik (GI:27229118). Our results show that, upon lipid loading of the cells, ALDI translocates from the endoplasmic reticulum into nascent LDs and indicate that ALDI may be targeted to the initial lipid deposits that eventually form these droplets. Moreover, we used ALDI expression studies to view other processes related to these droplets, such as LD biogenesis, and to analyze LD dynamics. In conclusion, here we report the composition of hepatic LDs and describe a novel bona fide LD-associated protein that may provide new insights into the mechanisms and sites of LD biogenesis.     

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

Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions.     

Creb-Pgc1α pathway modulates the interaction between lipid droplets and mitochondria and influences high fat diet-induced changes of lipid metabolism in the liver and isolated hepatocytes of yellow catfish

Although the crucial role of lipid droplets (LDs), mitochondria (MT) and their interactions in regulating lipid metabolism are well accepted, the mechanism of LDs-MT interactions in high fat diet (HFD)-induced changes of lipid metabolism remains unknown. Thus, this study was conducted to determine the mechanism of LDs-MT interactions in HFD-induced changes of lipid accumulation. We found that HFD not only up-regulated the expression of key proteins linked with TAG biosynthesis, but also increased the expression of proteins involved in lipolysis and fatty acid (FA) oxidation in LDs, including Rab32 (the only Rab protein associated with the MT). FA-induced LDs accumulation coincided with increased mitochondrial biogenesis, suggesting the potential LDs-MT interaction in hepatocytes after FA incubation. Also, FA incubation markedly increased the localization of Rab32 into LDs and MT, which confirmed the LDs-MT interaction and indicated the involvement of Rab32 in LDs-MT interaction following FA incubation. Inhibitors of Creb-Pgc1α pathway significantly blocked the localization of Rab32 into LDs and MT, and significantly reduced FA-induced LDs lipolysis by targeting Atgl and Plin5. Meanwhile, the FA-enhanced LDs accumulation, and mitochondrial biogenesis, fusion and oxidation were also significantly repressed. These indicated the regulatory role of Creb-Pgc1α in Rab32-mediated LDs-MT interactions and lipolysis after FA incubation. Taken together, these results revealed a novel mechanism of HFD- and FA-induced LDs-MT interactions in regulating hepatic LDs lipolysis, which provided new insight into the crosstalk between LDs-MT interaction and their potential role in HFD-induced hepatic steatosis.     

Reduction of TIP47 improves hepatic steatosis and glucose homeostasis in mice

Lipid droplets in the liver are coated with the perilipin family of proteins, notably adipocyte differentiation-related protein (ADRP) and tail-interacting protein of 47 kDa (TIP47). ADRP is increased in hepatic steatosis and is associated with hyperlipidemia, insulin resistance, and glucose intolerance. We have shown that reducing ADRP in the liver via antisense oligonucleotide (ASO) treatment attenuates steatosis and improves insulin sensitivity and glucose tolerance. We hypothesized that TIP47 has similar effects on hepatic lipid and glucose metabolism. We found that TIP47 mRNA and protein levels were increased in response to a high-fat diet (HFD) in C57BL/6J mice. TIP47 ASO treatment decreased liver TIP47 mRNA and protein levels without altering ADRP levels. Low-dose TIP47 ASO (15 mg/kg) and high-dose TIP47 ASO (50 mg/kg) decreased triglyceride content in the liver by 35% and 52%, respectively. Liver histology showed a drastic reduction in hepatic steatosis following TIP47 ASO treatment. The high dose of TIP47 ASO significantly blunted hepatic triglyceride secretion, improved glucose tolerance, and increased insulin sensitivity in liver, adipose tissue, and muscle. These findings show that TIP47 affects hepatic lipid and glucose metabolism and may be a target for the treatment of nonalcoholic fatty liver and related metabolic disorders.     

Dynamic Regulation of Hepatic Lipid Droplet Properties by Diet

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.     

RGC-32 Deficiency Protects against Hepatic Steatosis by Reducing Lipogenesis

Hepatic steatosis is associated with insulin resistance and metabolic syndrome because of increased hepatic triglyceride content. We have reported previously that deficiency of response gene to complement 32 (RGC-32) prevents high-fat diet (HFD)-induced obesity and insulin resistance in mice. This study was conducted to determine the role of RGC-32 in the regulation of hepatic steatosis. We observed that hepatic RGC-32 was induced dramatically by both HFD challenge and ethanol administration. RGC-32 knockout (RGC32^−/−) mice were resistant to HFD- and ethanol-induced hepatic steatosis. The hepatic triglyceride content of RGC32^−/− mice was decreased significantly compared with WT controls even under normal chow conditions. Moreover, RGC-32 deficiency decreased the expression of lipogenesis-related genes, sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase, and stearoyl-CoA desaturase 1 (SCD1). RGC-32 deficiency also decreased SCD1 activity, as indicated by decreased desaturase indices of the liver and serum. Mechanistically, insulin and ethanol induced RGC-32 expression through the NF-κB signaling pathway, which, in turn, increased SCD1 expression in a SREBP-1c-dependent manner. RGC-32 also promoted SREBP-1c expression through activating liver X receptor. These results demonstrate that RGC-32 contributes to the development of hepatic steatosis by facilitating de novo lipogenesis through activating liver X receptor, leading to the induction of SREBP-1c and its target genes. Therefore, RGC-32 may be a potential novel drug target for the treatment of hepatic steatosis and its related diseases.     

Proteomic Analysis of Lipid Droplets from Caco-2/TC7 Enterocytes Identifies Novel Modulators of Lipid Secretion

In enterocytes, the dynamic accumulation and depletion of triacylglycerol (TAG) in lipid droplets (LD) during fat absorption suggests that cytosolic LD-associated TAG contribute to TAG-rich lipoprotein (TRL) production. To get insight into the mechanisms controlling the storage/secretion balance of TAG, we used as a tool hepatitis C virus core protein, which localizes onto LDs, and thus may modify their protein coat and decrease TRL secretion. We compared the proteome of LD fractions isolated from Caco-2/TC7 enterocytes expressing or not hepatitis C virus core protein by a differential proteomic approach (isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with liquid chromatography and tandem mass spectrometry). We identified 42 proteins, 21 being involved in lipid metabolism. Perilipin-2/ADRP, which is suggested to stabilize long term-stored TAG, was enriched in LD fractions isolated from Caco-2/TC7 expressing core protein while perilipin-3/TIP47, which is involved in LD synthesis from newly synthesized TAG, was decreased. Endoplasmic reticulum-associated proteins were strongly decreased, suggesting reduced interactions between LD and endoplasmic reticulum, where TRL assembly occurs. For the first time, we show that 17β-hydroxysteroid dehydrogenase 2 (DHB2), which catalyzes the conversion of 17-keto to 17 β-hydroxysteroids and which was the most highly enriched protein in core expressing cells, is localized to LD and interferes with TAG secretion, probably through its capacity to inactivate testosterone. Overall, we identified potential new players of lipid droplet dynamics, which may be involved in the balance between lipid storage and secretion, and may be altered in enterocytes in pathological conditions such as insulin resistance, type II diabetes and obesity.     

Effects of binge ethanol on lipid homeostasis and oxidative stress in a rat model of nonalcoholic fatty liver disease

Excess fat accumulation renders the liver more vulnerable to ethanol, but it is still unclear how alcohol enhances lipid dysmetabolism and oxidative stress in a pre-existing steatosis condition. The effects produced by binge ethanol consumption in the liver of male Wistar rats fed a standard (Ctrl) or a high-fat diet HFD were compared. The liver status was checked through tissue histology and standard serum parameters. Alteration of hepatic lipid homeostasis and consequent oxidative unbalance were assessed by quantifying the mRNA expression of the lipid-regulated peroxisome proliferator-activated receptors (PPARs), of the cytochromes CYP2E1 and CYP4A1, and of some antioxidant molecules such as the metallothionein isoforms MT1 and MT2 and the enzymes catalase and superoxide dismutase. The number of adipose differentiation-related protein (ADRP)-positive lipid droplets (LDs) was evaluated by immunohistochemical staining. As a response to the double insult of diet and ethanol the rat liver showed: (1) a larger increase in fat accumulation within ADRP-positive LDs; (2) stimulation of lipid oxidation in the attempt to limit excess fat accumulation; (3) induction of antioxidant proteins (MT2, in particular) to protect the liver from the ethanol-induced overproduction of oxygen radicals. The data indicate an increased susceptibility of fatty liver to ethanol and suggest that the synergistic effect of diet and ethanol on lipid dysmetabolism might be mediated, at least in part, by PPARs and cytochromes CYP4A1 and CYP2E1.     

Lipid Droplet-associated Proteins Are Involved in the Biosynthesis and Hydrolysis of Triacylglycerol in Mycobacterium bovis Bacillus Calmette-Guérin

Mycobacteria store triacylglycerols (TGs) in the form of intracellular lipid droplets (LDs) during hypoxia-induced nonreplicating persistence. These bacteria are phenotypically drug-resistant and therefore are believed to be the cause for prolonged tuberculosis treatment. LDs are also associated with bacilli in tuberculosis patient sputum and hypervirulent strains. Although proteins bound to LDs are well characterized in eukaryotes, the identities and functions of such proteins have not been described in mycobacteria. Here, we have identified five proteins: Tgs1 (BCG3153c), Tgs2 (BCG3794c), BCG1169c, BCG1489c, and BCG1721, which are exclusively associated with LDs purified from hypoxic nonreplicating Mycobacterium bovis bacillus Calmette-Guérin (BCG). Disruption of genes tgs1, tgs2, BCG1169c, and BCG1489c in M. bovis BCG revealed that they are indeed involved in TG metabolism. We also characterized BCG1721, an essential bi-functional enzyme capable of promoting buildup and hydrolysis of TGs, depending on the metabolic state. Nonreplicating mycobacteria overexpressing a BCG1721 construct with an inactive lipase domain displayed a phenotype of attenuated TG breakdown and regrowth upon resuscitation. In addition, by heterologous expression in baker''s yeast, these mycobacterial proteins also co-localized with LDs and complemented a lipase-deficient yeast strain, indicating that neutral lipid deposition and homeostasis in eukaryotic and prokaryotic microorganisms are functionally related. The demonstrated functional role of BCG1721 to support growth upon resuscitation makes this novel LD-associated factor a potential new target for therapeutic intervention.     

Adaptive failure to high-fat diet characterizes steatohepatitis in Alms1 mutant mice

The biochemical differences between simple steatosis, a benign liver disease, and non-alcoholic steatohepatitis, which leads to cirrhosis, are unclear. Fat aussie is an obese mouse strain with a truncating mutation (foz) in the Alms1 gene. Chow-fed female foz/foz mice develop obesity, diabetes, and simple steatosis. We fed foz/foz and wildtype mice a high-fat diet. Foz/foz mice developed serum ALT elevation and severe steatohepatitis with hepatocyte ballooning, inflammation, and fibrosis; wildtype mice showed simple steatosis. Biochemical pathways favoring hepatocellular lipid accumulation (fatty acid uptake; lipogenesis) and lipid disposal (fatty acid beta-oxidation; triglyceride egress) were both induced by high-fat feeding in wildtype but not foz/foz mice. The resulting extremely high hepatic triglyceride levels were associated with induction of mitochondrial uncoupling protein-2 and adipocyte-specific fatty acid binding protein-2, but not cytochrome P4502e1 or lipid peroxidation. In this model of metabolic syndrome, transition of steatosis to steatohepatitis was associated with hypoadiponectinemia, a mediator of hepatic fatty acid disposal pathways.     

Temporal and spatial variations of lipid droplets during adipocyte division and differentiation

By capturing time-lapse images of primary stromal-vascular cells (SVCs) derived from rat mesenteric adipose tissue, we revealed temporal and spatial variations of lipid droplets (LDs) in individual SVCs during adipocyte differentiation. Numerous small LDs (a few micrometers in diameter) appeared in the perinuclear region at an early stage of differentiation; subsequently, several LDs grew to more than 10 microm in diameter and occupied the cytoplasm. We have developed a method for the fluorescence staining of LDs in living adipocytes. Time-lapse observation of the stained cells at higher magnification showed that nascent LDs (several 100 nm in diameter) grew into small LDs while moving from lamellipodia to the perinuclear region. We also found that adipocytes are capable of division and that they evenly distribute the LDs between two daughter cells. Immunofluorescence observations of LD-associated proteins revealed that such cell divisions of SVCs occurred even after LDs were coated with perilipin, suggesting that the "final" cell division during adipocyte differentiation occurs considerably later than that characterized in 3T3-L1 cells. Our time-lapse observations have provided a detailed account of the morphological changes that SVCs undergo during adipocyte division and differentiation.