Hepatic lipid droplet biology: getting to the root of fatty liver

Abstract

Hepatic steatosis is defined by the accumulation of lipid droplets (LDs). Once thought to be only inert energy storage depots, LDs are increasingly recognized as organelles that have important functions in hepatocytes beyond lipid storage. The lipid and protein composition of LDs is highly dynamic and influences their intrinsic metabolism and signaling properties, which ultimately links them to the changes in hepatic function. This concise review will highlight recent discoveries in LD biology and highlight unique aspects of hepatic LDs and their role in liver disease.

LD Biogenesis and Growth

Hepatic LDs are comprised of a core containing primarily triacylglycerol (TAG) and sterol esters surrounded by a phospholipid (PL) monolayer (1). The synthesis of these LD components requires a complex network of proteins with acyl-CoA synthetase, acyltransferase and phosphatase activities. Although many details remain to be defined, the prominent model for de novo LD biogenesis is that LDs form from a growing lipid lens within the lipid bilayer of the endoplasmic reticulum (ER) membrane and, once mature, bud to form an independent organelle; fat-storage inducible transmembrane proteins (FIT) and seipin play critical roles in this process (2). In addition to their presence on the ER, many proteins, or isoforms thereof, involved in TAG and PL synthesis, are also found on the LD (3). These proteins colocalize on select LDs suggesting that there are distinct populations of LDs that differ in their protein composition and ability to expand (3). The PL comprising the monolayer membrane appears to play an important role in LD expansion from de novo synthesis as well as coalescence or transfer of lipids from preexisting LDs. Specifically, phosphatidylcholine, the predominant PL in LDs, acts as surfactant to prevent coalescence of LDs (4). Additionally, the LD protein cell death-inducing DFFA-like effector c (CIDEC) influences LD size through its ability to promote lipid exchange between LDs (5).

LD catabolism

In addition to supplying fatty acids (FAs) for oxidation, LDs also provide substrate for VLDL synthesis. Numerous hepatic lipases have been identified that selectively channel hydrolyzed FAs between these pathways. Adipose triglyceride lipase (ATGL/PNPLA2) is a major hepatic lipase that catalyzes the initial step in TAG catabolism. ATGL selectively partitions hydrolyzed FAs to β-oxidation without influencing very low density lipoprotein (VLDL) secretion (6). The effects of ATGL on FA oxidation may in part be due peroxisome proliferator activated receptor-α activation, which is mediated via sirtuin 1, indicating an important signaling role for LDs (7). ATGL activity is highly regulated through interactions with other proteins such as the activators comparative gene identification-58 (CGI-58) and pigment epithelial derived factor and inhibitors G0/G1 switch gene 2, fas associated factor 2, CIDEC, perilipin (PLIN) 2 and 5 (8). In contrast to the role of ATGL in supplying FAs for oxidation, carboxylesterase 3 (CES3) appears to be the major lipase involved in supplying lipids for VLDL secretion. Hepatic ablation of CES3 reduces VLDL secretion without a significant increase in steatosis, which may be partially due to increased FA oxidation (9). Another member of the CIDE family, CIDEB, also appears to play an essential role in lipid trafficking and VLDL biogenesis (10).

Apart from the above lipase-catalyzed lipid breakdown another LD degradation process known as lipophagy has recently garnered attention (11). In this process, the autophagic machinery targets LDs to allow for degradation by lysosomal lipases. For example, the use of chemical inhibitors of autophagy or knockdown of autophagosome specific genes in hepatocytes increases TAG content and LD size (11). Recent evidence has highlighted a role of the small GTPase, RAB7, as a key protein involved in hepatic lipophagy. RAB7 is upregulated in response to nutrient deprivation and mediates interactions between LDs with multivesicular bodies and lysosomes (12). Although this field is rapidly expanding, much remains to be learned about the mechanisms through which the autophagic machinery recognizes hepatic LDs, how specific populations of LDs are targeted and how degradation products of lipophagy are selectively channeled within hepatocytes. Additionally, autophagy is dysregulated in numerous liver diseases [see (13) for a more comprehensive review], but its role in disease etiology is still under investigation.

Dysregulation of LDs and Disease

Non-alcoholic fatty liver disease (NAFLD)

Given the importance of LD proteins in regulating LD accumulation, it is important to understand how LD proteins change in models of liver disease. Numerous studies have characterized the LD proteome in various tissues and cell types, but only recently has the hepatic LD been explored. Using a qualitative proteomic approach, the hepatic LD proteome was characterized in humans with NAFLD (14) and in mice (15). These studies reveal that the majority of proteins on LDs are not involved in neutral lipid metabolism, but serve other functions such as cell signaling, membrane trafficking, and metabolism of steroids, proteins and carbohydrates as examples. Although the importance of these proteins in the development of steatosis or linking NAFLD to its comorbidities remains to be determined, they provide critical insights into the complexity of LD biology. As an example, 17β-hydroxysteroid dehydrogenase-13, an oxidoreductase enzyme, was identified on LDs and found to be increased in human NAFLD (14). Moreover, overexpressing this enzyme in hepatocytes promotes TAG accumulation via increased de novo lipogenesis suggestive of a etiological role in NAFLD (14). More targeted and quantitative approaches have revealed that numerous members of the PLIN family are altered during NAFLD. PLIN1, which is the major adipocyte PLIN member, is undetectable in normal livers, but is expressed in humans with NAFLD (16,17); its contribution to NAFLD pathology is unknown. PLIN2, 3 and 5 are also elevated in fatty livers of humans and their ablation alleviates steatosis (18–20).

A sequence polymorphism in patatin-like phospholipase domain-containing protein 3 (PNPLA3) is the genetic factor most strongly associated with NAFLD (21). The I148M mutation in PNPLA3 results in hepatic TAG accumulation especially in response to high carbohydrate diets, which drive PNPLA3 expression (22). This mutation also results in the sequestration of PNPLA3 on LDs (23), although the functional significance of this is unknown. Surprisingly, the mechanism through which PNPLA3 causes steatosis is still under debate. There is evidence that the I148M mutation reduces the lipolytic activity of PNPLA3 and, as a result, induces steatosis (24). However, additional data suggests the mutation is a gain of function of PNPLA3’s lysophoshatidic acid acyltransferase activity leading to increased TAG synthesis (25). Finally, more recent data have suggested that PNPLA3 may play a role in FA-selective remodeling of TAG resulting in altered TAG content and composition (26). Regardless of its mechanism of action, the I148M variant does not cause hepatic insulin resistance despite increased steatosis; although commonly correlated, emerging evidence suggests that steatosis and insulin resistance are not exclusive (27).. Given the prevalence of this mutation (15–50% across ethnicities) and its impact on NAFLD, deciphering the means through which it facilitates NAFLD is essential to facilitate tailored therapeutic options for those individuals carrying this allele.

Non-alcoholic steatohepatitis (NASH)

Numerous lines of evidence are now emerging that suggest a role for LD proteins in the progression to NASH. Several studies have identified proteins known to antagonize lipolysis including PLIN1 and 2 and CIDEC to be increased in NASH (28–30). Liver-specific ablation of CGI-58 leads to NASH and fibrosis through a reduction in hydrolytic activity (31). Additionally, the PNPLA3 variant, which has been linked to decreased lipolysis, also promotes the progression of NAFLD to NASH (32). Despite a potential reduction in lipolysis contributing to NASH, studies in other tissues suggest that lipolysis may promote inflammation. For example, in adipocytes, CIDEC regulates inflammatory responses to LDs through its direct interaction and modulation of nuclear factor of activated T cells 5, a transcription factor that promotes inflammation (33). In addition, numerous enzymes involved in eicosanoid biosynthesis are present on LDs in inflammatory cell types (34). Taken together, these data suggest that LDs may play a direct role in regulating transcriptional control of inflammation and in supplying inflammatory lipid mediators, but the role of lipolysis and inflammation in the liver requires further investigation.

The lipid composition of LDs may also contribute to NASH progression. Free cholesterol in the form of crystals is present within LDs in mice and human subjects with NASH, but is absent in subjects with simple steatosis (35). In support of a pathological role for LD cholesterol, administration of cholesterol lowering drugs to mice reduces LD cholesterol crystals and resolves NASH, whereas, supplementing a high fat diet with cholesterol promotes crystals and the progression to NASH (36,37). It appears that infiltration of Kupffer cells is the primary cause of the increased inflammation and progression to NASH (35) although the mechanisms linking LD cholesterol crystals to Kupffer cell activation has yet to be determined.

Hepatitis C Virus (HCV)

HCV requires LDs for dissemination of infectious progeny. The core protein in HCV anchors to the ER membrane and, upon peptidase cleavage, localizes to LDs (38). The movement of core to LD requires diacylglycerol transferase 1, but not diacylglycerol transferase 2, suggesting a specific pool of LDs is involved (39). Additional work has shown that HCV core protein displaces PLIN2 on LDs resulting in a redistribution of LDs around the ER near the periphery of the nucleus perhaps to increase contact with sites of replication (40); RAB18 also appears to contribute to LD relocation more proximal to the ER (41). Conversely, PLIN3 interaction with HCV is required for targeting HCV to LDs and protecting viral replicon proteins from autophagosomal degradation (42). HCV appears to induce steatosis primarily through its anti-lipolytic effects mediated via the inhibition of ATGL activity (43). Collectively, available data support LDs and LD proteins as critical regulators of HCV replication.

Summary

Lipid droplets are the defining characteristic of hepatic steatosis and play a much larger role than lipid storage. Emerging functions in cell signaling and disease development have provided novel insights into their roles as important cellular organelles. We are only in the infancy of understanding factors that regulate the lipidome and proteome of LDs, or sub-populations of LDs, and the consequences of these alterations. However, advancing our understanding of LD biology is critical to define dietary or pharmaceutical options to prevent or treat NAFLD and it complications.

Figure 1. Overview of hepatic LD metabolism.

LDs are formed within the lipid bilayer of the ER and are subsequently budded, a process involving FIT proteins and seipin. LDS can then grow due to various TAG and PL synthetic enzymes present on the LD surface. CIDEC plays an important role in mediating the transfer of lipid content between LDs resulting in the enlargement of select LDs. LDs are catabolized through RAB7-mediated autophagy and ATGL-catalyzed lipolysis, both of which appear to channel downstream FA to β-oxidation. Additionally, CES3 and CIDEB are important for the hydrolysis and repacking of cytosolic LDs in the ER leading to VLDL biosynthesis.

List of abbreviations

LD

lipid droplets

TAG

triacylglycerol

PL

phospholipid

ER

endoplasmic reticulum

FIT

fat-storage inducible transmembrane proteins

CIDE

cell death-inducing DFFA-like effector

FA

fatty acid

ATGL

adipose triglyceride lipase

VLDL

very low density lipoprotein

CGI-58

comparative gene identification-58

PLIN

perilipin

CES3

carboxylesterase 3

NAFLD

non-alcoholic fatty liver disease

PNPLA3

patatin-like phospholipase domain-containing protein 3

NASH

non-alcoholic steatohepatitis

HCV

hepatitis C virus