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A new perspective on NAFLD: focusing on lipid droplets

Published:November 15, 2021DOI:https://doi.org/10.1016/j.jhep.2021.11.009

      Abstract

      Lipid droplets (LDs) are complex and metabolically active organelles. They are composed of a neutral lipid core surrounded by a monolayer of phospholipids and proteins. LD accumulation in hepatocytes is the distinctive characteristic of non-alcoholic fatty liver disease (NAFLD). NAFLD is a chronic, heterogeneous liver condition that can progress to liver fibrosis and hepatocellular carcinoma.
      Though recent research has improved our understanding of the mechanisms linking LDs accumulation to NAFLD progression, numerous aspects of LD biology are either poorly understood or unknown. In this review, we provide a description of several key mechanisms that contribute to LDs accumulation in the hepatocytes, favouring NAFLD progression. First, we highlight the importance of LD architecture and describe how the dysregulation of LD biogenesis leads to endoplasmic reticulum stress and inflammation. This is followed by an analysis of the causal nexus that exists between LD proteome composition and LD degradation. Finally, we describe how the increase in size of LDs causes activation of hepatic stellate cells, leading to liver fibrosis and hepatocellular carcinoma.
      We conclude that acquiring a more sophisticated understanding of LD biology will provide crucial insights into the heterogeneity of NAFLD and assist in the development of therapeutic approaches for this liver disease.

      Key words

      • Lipid droplets (LDs) are metabolically active organelles with a complex architecture. The alteration of the biogenesis, growth or degradation of LDs activates mechanisms leading to LDs accumulation in the hepatocytes.
      • Alteration of LDs biogenesis leads to endoplasmic reticulum (ER) stress and progression of NAFLD to non-alcoholic steatohepatitis (NASH).
      • Genetic factors alter LD proteome composition leading to a decrease in LD degradation favoring NAFLD development.
      • LD size and hepatocytes ballooning compresses the hepatic sinusoids leading to hypoxia and activation of hepatic stellate cells.

      1. Introduction

      Non-alcoholic fatty liver disease (NAFLD) is a chronic liver condition characterized by lipid droplet (LD) in more than 5% of the hepatocytes (
      • Mashek D.G.
      • Khan S.A.
      • Sathyanarayan A.
      • Ploeger J.M.
      • Franklin M.P.
      Hepatic lipid droplet biology: Getting to the root of fatty liver.
      ,
      • Farese Jr., R.V.
      • Walther T.C.
      Lipid droplets finally get a little R-E-S-P-E-C-T.
      ). NAFLD encompasses a spectrum of liver related pathologies wherein the first stage is characterized by simple steatosis (
      • Chalasani N.
      • al e
      The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology.
      ,
      • Calzadilla Bertot L.
      • Adams L.A.
      The Natural Course of Non-Alcoholic Fatty Liver Disease.
      ,
      • de Alwis N.M.
      • Day C.P.
      Non-alcoholic fatty liver disease: the mist gradually clears.
      ,
      • Angulo P.
      Nonalcoholic fatty liver disease.
      ). This liver condition can progress further to non-alcoholic steatohepatitis (NASH), histologically defined as hepatocyte injury due to inflammation and hepatocellular ballooning. The progression of steatosis to NASH can be followed by the development of fibrosis (in 25%-33% of people with NAFLD); liver cirrhosis (5%–15% of people with NAFLD); liver failure (38% of people after 7-10 years of the diagnosis of NAFLD); and potentially hepatocellular carcinoma (2%-5%) (
      • Goh G.B.
      • McCullough A.J.
      Natural History of Nonalcoholic Fatty Liver Disease.
      ).
      The prognosis of patients with NAFLD is difficult to determine, as NAFLD is a heterogeneous disease, (
      • Targher G.
      • Byrne C.D.
      Clinical Review: Nonalcoholic fatty liver disease: a novel cardiometabolic risk factor for type 2 diabetes and its complications.
      ,
      • Byrne C.D.
      Fatty liver: role of inflammation and fatty acid nutrition.
      ,
      • Angulo P.
      • al e
      The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD.
      ). Moreover, NAFLD is now recognized as a multisystemic disease affecting extra-hepatic organs (
      • Saiman Y.
      • Hooks R.
      • Carr R.M.
      High-Risk Groups for Non-alcoholic Fatty Liver and Non-alcoholic Steatohepatitis Development and Progression.
      ). Strong evidence lends support to the view that NAFLD increases the risk of type 2 diabetes, cardiovascular diseases, dyslipidemia, hypertension, and chronic kidney disease (
      • Byrne C.D.
      Ectopic fat, insulin resistance and non-alcoholic fatty liver disease.
      ,
      • Byrne C.D.
      • Targher G.
      NAFLD: a multisystem disease.
      ,
      • Targher G.
      • Byrne C.D.
      • Lonardo A.
      • Zoppini G.
      • Barbui C.
      Non-alcoholic fatty liver disease and risk of incident cardiovascular disease: A meta-analysis.
      ). Currently, the mechanisms behind the pathogenesis and progression of NAFLD are not completely elucidated, and hepatic inflammation is triggered by multiple stimuli including the accumulation of lipotoxic metabolites, emergence of oxidative and endoplasmic reticulum (ER) stress, and development of tissue hypoxia and sinusoidal endothelium cell dysfunction (
      • Furuta K.
      • Guo Q.
      • Pavelko K.D.
      • Lee J.H.
      • Robertson K.D.
      • Nakao Y.
      • et al.
      Lipid-induced endothelial vascular cell adhesion molecule 1 promotes nonalcoholic steatohepatitis pathogenesis.
      ).
      LDs are dynamic and metabolically active organelles that consist of a hydrophobic core of neutral lipids (predominantly triglycerides (TG) and cholesterol esters) enveloped by a phospholipid monolayer. Embedded in this monolayer are heterogeneous proteins and enzymes responsible for neutral lipid synthesis or metabolism. Liver LD accumulation is considered an adaptive response to the increased flow of free fatty acids (FAs) from the diet, adipose tissue, and hepatocyte de novo lipogenesis. Dysregulation of LD biogenesis and degradation can increase intracellular lipid accumulation and promote the activation of pathogenetic mechanisms leading to steatosis, hepatocellular inflammation and fibrosis (
      • Mashek D.G.
      Hepatic lipid droplets: A balancing act between energy storage and metabolic dysfunction in NAFLD.
      ). The hydrolysis of TG and other esterified neutral lipids from the LDs produce lipid metabolites and intermediates of TG synthesis that affect cell homeostasis inducing organelle dysfunction, cell injury, cell malfunction and death (
      • Lee Y.
      • Hirose H.
      • Ohneda M.
      • Johnson J.H.
      • McGarry J.D.
      • Unger R.H.
      Beta-cell lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese rats: impairment in adipocyte-beta-cell relationships.
      ).
      This review aims to outline the significance of LDs in the pathogenesis of NAFLD and NASH. We seek to expound the role of LDs as dynamic and metabolically active organelles in triggering hepatocyte inflammation, ballooning and fibrogenic processes by exploring their biogenesis, proteome composition and degradation.

      2. Lipid droplet biogenesis and endoplasmic reticulum stress

      The biosynthesis of LDs is controlled by the endoplasmic reticulum (ER) (Figure 1). FAs from the diet and those released from the adipose tissue after hydrolysis by lipoprotein lipase are transported into the hepatocytes to form LDs or very low-density lipoprotein (VLDL) (
      • Hodson L.
      • Gunn P.J.
      The regulation of hepatic fatty acid synthesis and partitioning: the effect of nutritional state.
      ). FAs can also be produced in the hepatocytes through a process called de novo lipogenesis (
      • Hodson L.
      • Gunn P.J.
      The regulation of hepatic fatty acid synthesis and partitioning: the effect of nutritional state.
      ). This is a metabolic mechanism regulated by transcriptional factors, such as insulin-responsive sterol regulatory element binding protein 1 (SREBP1), carbohydrate-responsive element binding protein (ChREBP), and cholesterol-induced liver X receptors activated (LXR). Once activated, these transcriptional factors enhance the expression of lipogenic genes. FAs are then transported into the ER to be converted to neutral lipids (triglycerides and cholesterol ester) that in turn are secreted as VLDL or stored as LDs (
      • Hodson L.
      • Gunn P.J.
      The regulation of hepatic fatty acid synthesis and partitioning: the effect of nutritional state.
      ,
      • Fu S.
      • Watkins S.M.
      • Hotamisligil G.S.
      The role of endoplasmic reticulum in hepatic lipid homeostasis and stress signaling.
      ). In obesity, the dysregulation of lipid metabolism causes an excess of FA production that alters adipose tissue and hepatic metabolic pathways.
      Figure thumbnail gr1
      Figure 1LD biogenesis. Lipid droplets (LDs) consist of a neutral lipid core surrounded by a phospholipid monolayer. The surface of each LD contains various proteins involved in lipid metabolism, membrane trafficking and protein degradation. LD formation begins with neutral lipid synthesis in the ER bilayer. FAs accumulate in the ER bilayer forming a lens. Eventually, this accumulation deforms the bilayer, and an LD is formed through a budding process. At this stage, LD growth is fuelled by class I and class II proteins that re-localize to the ER membrane for triglycerides synthesis. Created with BioRender.com
      FAs that enter the liver are directed to the ER for esterification. In the cytosolic and luminal side of the ER bilayer membranes, there are specific enzymes involved in the synthesis of neutral lipids (
      • Olzmann J.A.
      • Carvalho P.
      Dynamics and functions of lipid droplets.
      ). The acyl-CoA synthetase family catalyze the formation of fatty acyl-CoAs from ATP, CoA, and long-chain fatty acids. Whereas, the enzymes Acyl-CoA:cholesterol O-acyltransferases (ACAT1 and ACAT2) are responsible for the esterification of cholesterol. Diacylglycerol acyltransferases 1 and 2 (DGAT1 and DGAT2) produce TGs from cytosolic FAs. DGAT1 is found exclusively in the ER membrane and uses diacylglycerol (DAG) derived from TG lipolysis in the cytosol to re-synthesize TG in the ER lumen. DGAT2 is present in both the ER bilayer and on the surface of LDs and plays a crucial role in the synthesis of de novo TG from FAs. When the cytosolic concentration of FAs is high, DGAT2 re-localizes to the LD surface and synthesizes TGs to be incorporated into the LDs (
      • Wilfling F.
      • Wang H.
      • Haas J.T.
      • Krahmer N.
      • Gould T.J.
      • Uchida A.
      • et al.
      Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.
      ,
      • Stone S.J.
      • Levin M.C.
      • Zhou P.
      • Han J.
      • Walther T.C.
      • Farese Jr., R.V.
      The endoplasmic reticulum enzyme DGAT2 is found in mitochondria-associated membranes and has a mitochondrial targeting signal that promotes its association with mitochondria.
      ). Both DAGT1 and DAGT2 prevent accumulation of lipotoxic lipids such as DAG and FAs that would otherwise activate ER stress pathways leading to hepatocyte inflammation (
      • Wilfling F.
      • Wang H.
      • Haas J.T.
      • Krahmer N.
      • Gould T.J.
      • Uchida A.
      • et al.
      Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.
      ).
      The budding of the LD is linked to the concentration of TGs in the ER bilayer membrane. A recent study showed that an LD lens is formed from the ER bilayer when the concentration of TGs ranges between 5-10 mol% (
      • Khandelia H.
      • Duelund L.
      • Pakkanen K.I.
      • Ipsen J.H.
      Triglyceride blisters in lipid bilayers: implications for lipid droplet biogenesis and the mobile lipid signal in cancer cell membranes.
      ,
      • Duelund L.
      • Jensen G.V.
      • Hannibal-Bach H.K.
      • Ejsing C.S.
      • Pedersen J.S.
      • Pakkanen K.I.
      • et al.
      Composition, structure and properties of POPC-triolein mixtures. Evidence of triglyceride domains in phospholipid bilayers.
      ). At this concentration, lipids induce a local curvature of the ER membrane bilayer.
      This membrane curvature can be either positive, when it tends towards the cytoplasm (polar surface area) or negative, when it tends towards the ER (away from the cytoplasm) (
      • Thiam A.R.
      • Forêt L.
      The physics of lipid droplet nucleation, growth and budding.
      ,
      • Thiam A.R.
      • Farese Jr., R.V.
      • Walther T.C.
      The biophysics and cell biology of lipid droplets.
      ). The type and shape of lipids that form the ER membrane bilayer at the site of LD budding determine whether the curvature is either positive or negative. Lipids with an inverted shape (hydrophilic-head predominant) engender a positive curvature which favors the emergence of LDs towards the cytosolic side. By contrast, lipids with a cone shape (lipolytic-tale predominant), such as DAG and phosphatidylethanolamine, engender a negative curvature which promotes the embedding of LDs in the ER membrane bilayer (
      • Ben M'barek K.
      • Ajjaji D.
      • Chorlay A.
      • Vanni S.
      • Foret L.
      • Thiam A.R.
      ER Membrane Phospholipids and Surface Tension Control Cellular Lipid Droplet Formation.
      ). Crucially, in this state, proteins and enzymes cannot access LDs to regulate their neutral lipid incorporation, mobilization, or their protein composition (
      • Ben M'barek K.
      • Ajjaji D.
      • Chorlay A.
      • Vanni S.
      • Foret L.
      • Thiam A.R.
      ER Membrane Phospholipids and Surface Tension Control Cellular Lipid Droplet Formation.
      ). Therefore, embedded LDs fail to remove unfolded and misfolded proteins from the ER membrane bilayer, resulting in ER stress and inflammation (
      • Ploegh H.L.
      A lipid-based model for the creation of an escape hatch from the endoplasmic reticulum.
      ,
      • Jarc E.
      • Petan T.
      Lipid Droplets and the Management of Cellular Stress.
      ).
      Unfolded/misfolded proteins that accumulate in the ER membrane bilayer, bind to the glucose-regulated protein GRP78/BiP. Under normal physiological conditions, GRP78/BiP is bound to three ER lumen stress sensors: 1) inositol-requiring enzyme 1α (IRE1 α); 2) the double-stranded RNA-activated protein kinase-like eukaryotic initiation factor 2α kinase (PERK); and 3) activating transcription factor 6 α (ATF6 α). However, LDs embedded in the ER lumen cause the GRP78/BiP to bind to unfolded proteins and dissociate from the ER stress sensors. This mechanism causes auto-phosphorylation and activation of ER stress through an intracellular signaling pathway known as the unfolded protein response (UPR) (
      • Walter P.
      • Ron D.
      The unfolded protein response: from stress pathway to homeostatic regulation.
      ,
      • Wires E.S.
      • Trychta K.A.
      • Bäck S.
      • Sulima A.
      • Rice K.C.
      • Harvey B.K.
      High fat diet disrupts endoplasmic reticulum calcium homeostasis in the rat liver.
      ).
      ER stress causes a loss of ER luminal Ca2+. The ER contains high concentration of Ca2+ which is maintained by three main ER Ca2+ channels and transporters: 1) the sarco/endoplasmic reticulum (Ca2++Mg2+) ATP-ase2b (SERCA2b); 2) the type 1 inositol trisphosphate receptor (InsP3R1); and 3) the store-operated Ca2+ entry (SOCE) channels (
      • Vandecaetsbeek I.
      • Vangheluwe P.
      • Raeymaekers L.
      • Wuytack F.
      • Vanoevelen J.
      The Ca2+ pumps of the endoplasmic reticulum and Golgi apparatus.
      ). High Ca2+ concentration in the ER lumen is essential for proper protein production and cell function. However, accumulation of LDs in the ER can activate protein kinase C, which in turn causes protein kinase C-mediated inhibition of SOCE (
      • Wilson C.H.
      • Ali E.S.
      • Scrimgeour N.
      • Martin A.M.
      • Hua J.
      • Tallis G.A.
      • et al.
      Steatosis inhibits liver cell store-operated Ca2⁺ entry and reduces ER Ca2⁺ through a protein kinase C-dependent mechanism.
      ) and the disruption of the ER calcium channel. Moreover, accumulation of unfolded/misfolded proteins in the ER lumen causes a disruption of Ca2+ homeostasis, with loss of ER luminal Ca2+ and consequent activation of ER stress response. This leads to lipid-inhibition of SERCA2b activity which reduces ER lumen Ca2+ uptake and increases Ca2+ efflux into the cytosol via InsP3R1. The increase in Ca2+ concentration in the cytosol has several implications for hepatocytes (
      • Arruda A.P.
      • Pers B.M.
      • Parlakgül G.
      • Güney E.
      • Inouye K.
      • Hotamisligil G.S.
      Chronic enrichment of hepatic endoplasmic reticulum-mitochondria contact leads to mitochondrial dysfunction in obesity.
      ). First, there is an increase in Ca2+ concentration in the mitochondria leading both to a decrease in FA β-oxidation and an increase in ROS production with further accumulation of LDs (
      • Arruda A.P.
      • Pers B.M.
      • Parlakgül G.
      • Güney E.
      • Inouye K.
      • Hotamisligil G.S.
      Chronic enrichment of hepatic endoplasmic reticulum-mitochondria contact leads to mitochondrial dysfunction in obesity.
      ). Second, lipid inhibition of SOCE and altered intracellular Ca2+ homeostasis increases lipid synthesis and decreases lipid hydrolysis (
      • Kim J.Y.
      • Garcia-Carbonell R.
      • Yamachika S.
      • Zhao P.
      • Dhar D.
      • Loomba R.
      • et al.
      ER Stress Drives Lipogenesis and Steatohepatitis via Caspase-2 Activation of S1P.
      ). Notably, Kim et al. recently showed that ER stress induces the production of caspase-2 (a protease with an important role in programmed cell death) that initiates a SCAP-independent (SCAP is both an escort protein and a sensor of sterol levels) SREBP1/2 activation in the ER and increases de novo lipogenesis which contributes to NAFLD progression (
      • Kim J.Y.
      • Garcia-Carbonell R.
      • Yamachika S.
      • Zhao P.
      • Dhar D.
      • Loomba R.
      • et al.
      ER Stress Drives Lipogenesis and Steatohepatitis via Caspase-2 Activation of S1P.
      ). Third, high cytosolic Ca2+ levels inhibit autophagic flux by preventing autophagosome/lysosome fusion (a process which requires a stable Ca2+ concentration (0.2-2 mM), as described further below.)
      Thus, ER stress exacerbates the accumulation of liver LDs (
      • Park H.W.
      • Park H.
      • Semple I.A.
      • Jang I.
      • Ro S.H.
      • Kim M.
      • et al.
      Pharmacological correction of obesity-induced autophagy arrest using calcium channel blockers.
      ) leading to hepatic inflammation and possibly NASH development (
      • Kim J.Y.
      • Garcia-Carbonell R.
      • Yamachika S.
      • Zhao P.
      • Dhar D.
      • Loomba R.
      • et al.
      ER Stress Drives Lipogenesis and Steatohepatitis via Caspase-2 Activation of S1P.
      ).

      3. Lipid droplet proteins and their role on steatosis

      The surface of LDs contains various proteins involved in lipid metabolism, membrane trafficking and protein degradation. These proteins can be divided into two classes depending on their location (Table 1).
      Table 1Lipid droplets proteins
      ProteinsTissue expressionFunction
      Class I: ER bilayer proteins
      PNPLA3 adiponutrin (ADPN)hepatocytes, hepatic stellate cells, and adipocytestriacylglycerol lipase protein
      DGAT2 (diacylglycerol O-acyltransferases)Liver, adipose tissues, mammary gland, testis, peripheral leukocytes, heartsynthesis of de novo TG from FAs
      ACSL3 (long-chain-fatty-acid–CoA ligase 3)Ubiquitous cytoplasmic expressionligase that activates the oxidation of complex fatty acids
      GPAT4 Glycerol-3-phosphate acyltransferase 4Ubiquitous expression hepatic TAG synthesisbiosynthesis of triglycerides from glycerol 3-phosphate
      Class II: Cytosol proteins
      CTP:phosphocholine cytidylyltransferase (CCT)Ubiquitous expressionPhosphatidylcholine synthesis
      PLIN1 (Perilipin A)White adipose tissueHormone-induced lipolysis, large LD stabilization
      PLIN2 (Adipophilin)Liver, white adipose tissue – ubiquitous expressionLD generation and stabilization CMA ATGL
      PLIN3 (TIP47)Ubiquitous expressionLD stabilization, compensation of PLIN2, PGE2 production
      PLIN4 (S3-12)White adipose tissueLD stability, adipocytes differentiation
      PLIN5 (OXPAT,MLDP, LSDP5)Cardiac muscle, skeletal muscle, brown adipose tissueLD stability, fatty acids oxidation, mitochondrial recruitment
      HIF-2αUbiquitous expressionInhibit adipose triglyceride lipase; increase PLIN2 expression
      CIDELiver and adipose tissueLD stability, LF fusion, lipid transfer

       Class I: ER bilayer proteins

      Class I proteins are localized in the ER bilayer and are characterized by the inclusion of a hydrophobic hairpin membrane-embedded domain. With this structure, these proteins can easily be translocated from the ER bilayer to the LD monolayer and back to the ER membrane via an ER-LD membrane bridge (
      • Jacquier N.
      • Choudhary V.
      • Mari M.
      • Toulmay A.
      • Reggiori F.
      • Schneiter R.
      Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae.
      ,
      • Bersuker K.
      • Olzmann J.A.
      Establishing the lipid droplet proteome: Mechanisms of lipid droplet protein targeting and degradation.
      ). This class of protein comprises TG biosynthetic enzymes such as long-chain-fatty-acid–CoA ligase 3, glycerol-3-phosphate acyltransferase 4 (
      • Quiroga I.Y.
      • Pellon-Maison M.
      • Suchanek A.L.
      • Coleman R.A.
      • Gonzalez-Baro M.R.
      Glycerol-3-phosphate acyltransferases 3 and 4 direct glycerolipid synthesis and affect functionality in activated macrophages.
      ), DGAT2 (
      • Harris C.A.
      • Haas J.T.
      • Streeper R.S.
      • Stone S.J.
      • Kumari M.
      • Yang K.
      • et al.
      DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes.
      ), and proteins involved in ubiquitin-dependent proteolysis (
      • Wilfling F.
      • Wang H.
      • Haas J.T.
      • Krahmer N.
      • Gould T.J.
      • Uchida A.
      • et al.
      Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.
      ).
      A protein involved in ubiquitin-dependent proteolysis is patatin-like phospholipase domain-containing protein 3 (PNPLA3) (Figure 2). This protein is found in hepatocytes, adipocytes, and hepatic stellate cells (HSCs). Wild-type PNPLA3 protein is degraded by the ubiquitin proteasome system and autophagy. A recent study has shown that after blocking these two degradation systems in a mouse model, wild-type PNPLA3 accumulates on LDs with consequent hepatic accumulation of TG (
      • BasuRay S.
      • Wang Y.
      • Smagris E.
      • Cohen J.C.
      • Hobbs H.H.
      Accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis.
      ). A polymorphism of PNPLA3 characterized by the substitution of isoleucine to methionine in position 148 (I148M) reduces TG hydrolase activity compared to the wild-type protein favoring LD accumulation. In addition, PNPLA3 I148M is not degraded by the ubiquitin proteasome system and autophagy, and therefore accumulates on LDs (
      • BasuRay S.
      • Wang Y.
      • Smagris E.
      • Cohen J.C.
      • Hobbs H.H.
      Accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis.
      ). The combination of reduced PNPLA3 I148M TG-hydrolase activity and the accumulation on LDs might explain the low synthesis and export of VLDL. In this scenario, it might be possible that TGs are sequestered into LDs coated with PNPLA3 I148M reducing the flux of TGs into VLDL synthesis.
      Figure thumbnail gr2
      Figure 2PNPLA3 and NAFLD. Patatin-like phospholipase domain-containing protein 3 (PNPLA3 148I) is a Class I protein found on the surface of lipid droplets (LDs). PNPLA3 148I functions as TG hydrolase, releasing FAs and glycerol from the LD core. Moreover, it is degraded by ubiquitin-dependent proteasome system (Ub) and autophagy. Notably, in presence of a substitution of isoleucine to methionine in position 148 (PNPLA3 I148M) the abovementioned processes are markedly altered. PNPLA3 I148M does not function as TG hydrolase. Moreover, it is not degraded by Ub and autophagy, accumulating instead on the surface of LDs and contributing to the formation of larger droplets. Created with BioRender.com
      Interestingly, PNPLA3 I148M is associated with the pathogenesis of NAFLD and patients expressing this polymorphism are at higher risk of developing hepatic fibrosis (
      • Dongiovanni P.
      • Donati B.
      • Fares R.
      • Lombardi R.
      • Mancina R.M.
      • Romeo S.
      • et al.
      PNPLA3 I148M polymorphism and progressive liver disease.
      ). HSCs that express the PNPLA3 I148M have less retinol but high LD content (
      • Bruschi F.V.
      • Claudel T.
      • Tardelli M.
      • Caligiuri A.
      • Stulnig T.M.
      • Marra F.
      • et al.
      The PNPLA3 I148M variant modulates the fibrogenic phenotype of human hepatic stellate cells.
      ). The PNPLA3 variant was also associated with significantly increased abundance of oleic and vaccenic acids as well as high amount of arachidonic acid, which contributes to the synthesis of proinflammatory metabolites that ultimately can activate HSCs (
      • Bruschi F.V.
      • Claudel T.
      • Tardelli M.
      • Caligiuri A.
      • Stulnig T.M.
      • Marra F.
      • et al.
      The PNPLA3 I148M variant modulates the fibrogenic phenotype of human hepatic stellate cells.
      ). Such HSCs can transition from a quiescent state to an active one, leading to a myofibroblast-like phenotype (
      • Bruschi F.V.
      • Claudel T.
      • Tardelli M.
      • Caligiuri A.
      • Stulnig T.M.
      • Marra F.
      • et al.
      The PNPLA3 I148M variant modulates the fibrogenic phenotype of human hepatic stellate cells.
      ). HSC activation leads to the release of chemotactic factors and acquisition of specific receptors, thus starting the process leading to extracellular matrix deposition and fibrosis development (
      • Bruschi F.V.
      • Claudel T.
      • Tardelli M.
      • Caligiuri A.
      • Stulnig T.M.
      • Marra F.
      • et al.
      The PNPLA3 I148M variant modulates the fibrogenic phenotype of human hepatic stellate cells.
      ). Recently, Banini et al showed that, in the presence of PNPLA3 I148M, hepatocytes undergo lipid remodeling with a decrease in polyunsaturated fatty acids and an increase in ceramides with downstream activation of inflammatory cytokines and hepatic cell injury (
      • Banini B.A.
      • D P.K.
      • Cazanave S.
      • Seneshaw M.
      • Mirshahi F.
      • Santhekadur P.K.
      • et al.
      Identification of a metabolic, transcriptomic and molecular signature of PNPLA3-mediated acceleration of steatohepatitis.
      ).

       Class II: Cytosolic proteins

      Class II proteins are localized in the cytosol and target the LD surface through a hydrophobic domain. For the present review on NAFLD pathophysiology, CTP:phosphocholine cytidylyltransferase (CCT), Perilipins (PLINs), hypoxia-inducible factor-2 (HIF-2α), and cell death-inducing DFF45-like effector (CIDE) deserve special consideration as they are associated with LDs accumulation.
      CCT is a class II protein that regulates the growth and the curvature/size of LDs by synthesizing phosphatidylcholine on the growing LD surfaces; this process favors the neutral lipid core expansion (
      • Prevost C.
      • Sharp M.E.
      • Kory N.
      • Lin Q.
      • Voth G.A.
      • Farese Jr., R.V.
      • et al.
      Mechanism and Determinants of Amphipathic Helix-Containing Protein Targeting to Lipid Droplets.
      ,
      • Bacle A.
      • Gautier R.
      • Jackson C.L.
      • Fuchs P.F.J.
      • Vanni S.
      Interdigitation between Triglycerides and Lipids Modulates Surface Properties of Lipid Droplets.
      ,
      • Krahmer N.
      • Guo Y.
      • Wilfling F.
      • Hilger M.
      • Lingrell S.
      • Heger K.
      • et al.
      Phosphatidylcholine synthesis for lipid droplet expansion is mediated by localized activation of CTP:phosphocholine cytidylyltransferase.
      ). The reduced activity of CCT causes an accumulation of LDs in the liver (
      • Cornell R.B.
      Membrane Lipids Assist Catalysis by CTP: Phosphocholine Cytidylyltransferase.
      ,
      • Ogasawara Y.
      • Cheng J.
      • Tatematsu T.
      • Uchida M.
      • Murase O.
      • Yoshikawa S.
      • et al.
      Long-term autophagy is sustained by activation of CCTβ3 on lipid droplets.
      ).
      Perilipins (PLINs) are another important class II protein. There are five genes that encode five PLIN proteins (
      • Carr R.M.
      • Ahima R.S.
      Pathophysiology of lipid droplet proteins in liver diseases.
      ,
      • Carr R.M.
      • Dhir R.
      • Mahadev K.
      • Comerford M.
      • Chalasani N.P.
      • Ahima R.S.
      Perilipin Staining Distinguishes Between Steatosis and Nonalcoholic Steatohepatitis in Adults and Children.
      ). PLIN1 is abundantly expressed in adipocytes in both white and brown adipose tissue, and its role is to promote cholesterol ester storage and triacylglycerol storage in large LDs. Although PLIN1 is mainly expressed in adipocytes, there is evidence that it is highly expressed in the liver of patients with NAFLD (
      • Straub B.K.
      • Stoeffel P.
      • Heid H.
      • Zimbelmann R.
      • Schirmacher P.
      Differential pattern of lipid droplet-associated proteins and de novo perilipin expression in hepatocyte steatogenesis.
      ). PLIN1 promotes lipid droplet formation and structural remodeling and inhibits lipolysis and autophagy (
      • Ju L.
      • Han J.
      • Zhang X.
      • Deng Y.
      • Yan H.
      • Wang C.
      • et al.
      Obesity-associated inflammation triggers an autophagy-lysosomal response in adipocytes and causes degradation of perilipin 1.
      ). Especially important in the pathogenesis and development of NAFLD, PLIN2 is a cytosolic protein mainly expressed in the liver and implicated in the development of steatosis and insulin resistance (
      • Carr R.M.
      • Peralta G.
      • Yin X.
      • Ahima R.S.
      Absence of perilipin 2 prevents hepatic steatosis, glucose intolerance and ceramide accumulation in alcohol-fed mice.
      ,
      • Imai Y.
      • Boyle S.
      • Varela G.M.
      • Caron E.
      • Yin X.
      • Dhir R.
      • et al.
      Effects of perilipin 2 antisense oligonucleotide treatment on hepatic lipid metabolism and gene expression.
      ,
      • McManaman J.L.
      • Bales E.S.
      • Orlicky D.J.
      • Jackman M.
      • MacLean P.S.
      • Cain S.
      • et al.
      Perilipin-2-null mice are protected against diet-induced obesity, adipose inflammation, and fatty liver disease.
      ,
      • Libby A.E.
      • Bales E.
      • Orlicky D.J.
      • McManaman J.L.
      Perilipin-2 Deletion Impairs Hepatic Lipid Accumulation by Interfering with Sterol Regulatory Element-binding Protein (SREBP) Activation and Altering the Hepatic Lipidome.
      ). PLIN2 coats LD membranes and reduces the access of ATGL to LDs thus preventing lipolysis (
      • Carr R.M.
      • Ahima R.S.
      Pathophysiology of lipid droplet proteins in liver diseases.
      ). Additionally, PLIN2 negatively regulates LD lipophagy as demonstrated by enhanced autophagosome formation (Figure 3) (
      • Tsai T.H.
      • Chen E.
      • Li L.
      • Saha P.
      • Lee H.J.
      • Huang L.S.
      • et al.
      The constitutive lipid droplet protein PLIN2 regulates autophagy in liver.
      ,
      • Tachibana K.
      • Kobayashi Y.
      • Tanaka T.
      • Tagami M.
      • Sugiyama A.
      • Katayama T.
      • et al.
      Gene expression profiling of potential peroxisome proliferator-activated receptor (PPAR) target genes in human hepatoblastoma cell lines inducibly expressing different PPAR isoforms.
      ,
      • Kaushik S.
      • Cuervo A.M.
      Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis.
      ). In NAFLD, PLIN2 is upregulated and is associated with hepatic accumulation of ceramides (
      • Carr R.M.
      • Peralta G.
      • Yin X.
      • Ahima R.S.
      Absence of perilipin 2 prevents hepatic steatosis, glucose intolerance and ceramide accumulation in alcohol-fed mice.
      ,
      • Imai Y.
      • Boyle S.
      • Varela G.M.
      • Caron E.
      • Yin X.
      • Dhir R.
      • et al.
      Effects of perilipin 2 antisense oligonucleotide treatment on hepatic lipid metabolism and gene expression.
      ,
      • McManaman J.L.
      • Bales E.S.
      • Orlicky D.J.
      • Jackman M.
      • MacLean P.S.
      • Cain S.
      • et al.
      Perilipin-2-null mice are protected against diet-induced obesity, adipose inflammation, and fatty liver disease.
      ,
      • Carr R.M.
      • Dhir R.
      • Yin X.
      • Agarwal B.
      • Ahima R.S.
      Temporal effects of ethanol consumption on energy homeostasis, hepatic steatosis, and insulin sensitivity in mice.
      ). A recent study identified a human PLIN2 polymorphism, Ser251Pro (rs35568725), that has important effects on PLIN2 protein structure and function. This polymorphism was found to promote LD accumulation in macrophages and human embryonic kidney 293 cells. In addition, patients with PLIN2 Ser251Pro have reduced plasma levels of triglyceride and the hepatocyte-secreted lipoprotein, very-low density lipoprotein (VLDL) (
      • Magne J.
      • Aminoff A.
      • Perman Sundelin J.
      • Mannila M.N.
      • Gustafsson P.
      • Hultenby K.
      • et al.
      The minor allele of the missense polymorphism Ser251Pro in perilipin 2 (PLIN2) disrupts an alpha-helix, affects lipolysis, and is associated with reduced plasma triglyceride concentration in humans.
      ). A recent study showed that, PLIN2 Ser251Pro is associated with NASH and that the polymorphism promotes the accumulation of more numerous and smaller LDs (
      • Faulkner C.S.
      • White C.
      • Jophlin L.L.
      A single nucleotide polymorphism of <em>PLIN2</em> is associated with nonalcoholic steatohepatitis and causes phenotypic changes in hepatocyte lipid droplets.
      ). Interestingly, in a large cohort of Italian obese subjects, the presence of the PLIN2 Ser251Pro was associated with low insulin secretion and increased insulin sensitivity after oral glucose tolerance test (
      • Sentinelli F.
      • Capoccia D.
      • Incani M.
      • Bertoccini L.
      • Severino A.
      • Pani M.G.
      • et al.
      The perilipin 2 (PLIN2) gene Ser251Pro missense mutation is associated with reduced insulin secretion and increased insulin sensitivity in Italian obese subjects.
      ). However, the role of the PLIN2 Ser251Pro is not completely understood. PLIN3 is ubiquitously expressed in tissues and localized on the surface of nascent lipid droplets. As the size of LDs increases, PLIN3 is removed and substituted by PLIN2. At present, the exact nature and function of PLIN3 is not completely understood (
      • Sztalryd C.
      • Brasaemle D.L.
      The perilipin family of lipid droplet proteins: Gatekeepers of intracellular lipolysis.
      ), however, in a pre-clinical mouse model of NAFLD, reduction of PLIN3 reduces steatosis and improves insulin sensitivity (
      • Carr R.M.
      • Patel R.T.
      • Rao V.
      • Dhir R.
      • Graham M.J.
      • Crooke R.M.
      • et al.
      Reduction of TIP47 improves hepatic steatosis and glucose homeostasis in mice.
      ). PLIN4 is highly expressed in white adipose tissue; currently little is known of the biological functions of PLIN4 and its role in the pathogenesis and development of NAFLD (
      • Nimura S.
      • Yamaguchi T.
      • Ueda K.
      • Kadokura K.
      • Aiuchi T.
      • Kato R.
      • et al.
      Olanzapine promotes the accumulation of lipid droplets and the expression of multiple perilipins in human adipocytes.
      ). PLIN5 is selectively expressed in tissues where FAs are transported to the mitochondria for oxidation, such as brown adipose tissue, cardiac and skeletal muscle (
      • Sztalryd C.
      • Brasaemle D.L.
      The perilipin family of lipid droplet proteins: Gatekeepers of intracellular lipolysis.
      ,
      • Wang C.
      • Zhao Y.
      • Gao X.
      • Li L.
      • Yuan Y.
      • Liu F.
      • et al.
      Perilipin 5 improves hepatic lipotoxicity by inhibiting lipolysis.
      ,
      • Wolins N.E.
      • Quaynor B.K.
      • Skinner J.R.
      • Schoenfish M.J.
      • Tzekov A.
      • Bickel P.E.
      S3-12, Adipophilin, and TIP47 package lipid in adipocytes.
      ).
      Figure thumbnail gr3
      Figure 3PLIN2 and NAFLD. Perilipin2 (PLIN2) is implicated in the development of NAFLD. PLIN2 coats the surface of lipid droplets (LDs). In the presence of LD accumulation, there is a concomitant increase in PLIN2 expression that inhibits the access of adipose triglycerides lipase (ATGL) to the LD core. This prevents normal lipolysis, fuelling steatosis. Created with BioRender.com
      Another LD associated protein is hypoxia-inducible factor-2 (HIF-2α) (
      • de la Rosa Rodriguez M.A.
      • Deng L.
      • Gemmink A.
      • van Weeghel M.
      • Aoun M.L.
      • Warnecke C.
      • et al.
      Hypoxia-inducible lipid droplet-associated induces DGAT1 and promotes lipid storage in hepatocytes.
      ). HIF-2α is translocated to the nucleus under hypoxia conditions. There is evidence that HIF-2α is expressed in the liver tissues of human and mice with NAFLD and NASH (
      • Gonzalez F.J.
      • Xie C.
      • Jiang C.
      The role of hypoxia-inducible factors in metabolic diseases.
      ). HIF-2α is associated with PLIN2 expression, as HIF-2α modulates lipid storage by increasing PLIN2 expression (
      • Qiu B.
      • Ackerman D.
      • Sanchez D.J.
      • Li B.
      • Ochocki J.D.
      • Grazioli A.
      • et al.
      HIF2α-Dependent Lipid Storage Promotes Endoplasmic Reticulum Homeostasis in Clear-Cell Renal Cell Carcinoma.
      ).
      The cell death-inducing DFF45-like effector (CIDE) protein family accumulates on the LD-LD contact site and creates a pore interacting with PLIN1 and Rab8a (Rab-GTPases are molecular switches controlling diverse stages of vesicle traffic) (
      • Sun Y.
      • Bilan P.J.
      • Liu Z.
      • Klip A.
      Rab8A and Rab13 are activated by insulin and regulate GLUT4 translocation in muscle cells.
      ,
      • Barneda D.
      • Planas-Iglesias J.
      • Gaspar M.L.
      • Mohammadyani D.
      • Prasannan S.
      • Dormann D.
      • et al.
      The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding amphipathic helix.
      ). The formation of this pore allows lipid transfer between LDs and their fusion into a single large LD (
      • Xu W.
      • Wu L.
      • Yu M.
      • Chen F.J.
      • Arshad M.
      • Xia X.
      • et al.
      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.
      ). Interestingly, Rab8a interacts with phosphatidylinositol 3-kinase (PI3K) enhancing the mammalian target of rapamycin (mTOR) phosphorylation that in turn increases the production of pro-inflammatory cytokines (
      • Sun Y.
      • Bilan P.J.
      • Liu Z.
      • Klip A.
      Rab8A and Rab13 are activated by insulin and regulate GLUT4 translocation in muscle cells.
      ,
      • Luo L.
      • Wall A.A.
      • Yeo J.C.
      • Condon N.D.
      • Norwood S.J.
      • Schoenwaelder S.
      • et al.
      Rab8a interacts directly with PI3Kγ to modulate TLR4-driven PI3K and mTOR signalling.
      ). PLIN1 and PI3K are recruited to the pore of the LD-LD contact site. PLIN1/PI3Kγ forms a complex with Rab8a during LD expansion which ultimately inhibits LD autophagy (
      • Ju L.
      • Han J.
      • Zhang X.
      • Deng Y.
      • Yan H.
      • Wang C.
      • et al.
      Obesity-associated inflammation triggers an autophagy-lysosomal response in adipocytes and causes degradation of perilipin 1.
      ).
      Considered jointly, the dysregulation of the LD proteome (class I and class II) causes an increased and accelerated accumulation of LDs, as well as the possible fusion between LDs or between LDs and other organelles such as ER and mitochondria. These phenomena cause the production of ROS, worsening liver steatosis and accelerating the progression of NAFLD to NASH.

      4. Lipid droplet lipolysis and autophagy

      LDs have crucial functions in the context of NAFLD. First, synthesis and storage of lipids in LDs protect the cells from cytotoxic FAs. Second, TG catabolism into FAs through either neutral lipid lipolysis (hereafter “lipolysis”) or autophagy supplies essential lipids for membrane biogenesis.
      Regarding lipolysis, this is an enzymatic process that occurs at neutral pH and involves three lipases that act sequentially to release FAs from the core of LDs. The first is the adipose triglyceride lipase (ATGL) that initiates the hydrolysis of the ester bonds in TG to form diacylglycerol and FAs. The activity of ATGL is modulated by perilipin proteins and it is facilitated by comparative gene identification-58 which exposes the lipid core of the LD to the ATGL lipase (
      • Zechner R.
      • Zimmermann R.
      • Eichmann T.O.
      • Kohlwein S.D.
      • Haemmerle G.
      • Lass A.
      • et al.
      FAT SIGNALS--lipases and lipolysis in lipid metabolism and signaling.
      ,
      • Tardelli M.
      • Bruschi F.V.
      • Trauner M.
      The Role of Metabolic Lipases in the Pathogenesis and Management of Liver Disease.
      ). Previous studies showed that liver specific ATGL deficiency improved glucose tolerance via increasing hepatic glucose utilization and reducing hepatic glucose output without reducing hepatic steatosis (
      • Kaur S.
      • Auger C.
      • Barayan D.
      • Shah P.
      • Matveev A.
      • Knuth C.M.
      • et al.
      Adipose-specific ATGL ablation reduces burn injury-induced metabolic derangements in mice.
      ,
      • Fuchs C.D.
      • Radun R.
      • Dixon E.D.
      • Mlitz V.
      • Timelthaler G.
      • Halilbasic E.
      • et al.
      Hepatocyte-specific deletion of adipose triglyceride lipase (ATGL/PNPLA2) ameliorates dietary induced steatohepatitis in mice.
      ). However, the use of Atglistatin, a chemical inhibitor of ATGL, in mice fed high-fat diet reduced adipose tissue lipolysis, with consequent reduction of FAs flux from the adipose tissue to the liver (
      • Schweiger M.
      • Romauch M.
      • Schreiber R.
      • Grabner G.F.
      • Hütter S.
      • Kotzbeck P.
      • et al.
      Pharmacological inhibition of adipose triglyceride lipase corrects high-fat diet-induced insulin resistance and hepatosteatosis in mice.
      ). This was accompanied by a decrease in liver steatosis due to reduced expression of the genes involved in lipid uptake, storage, and de novo lipogenesis (
      • Schweiger M.
      • Romauch M.
      • Schreiber R.
      • Grabner G.F.
      • Hütter S.
      • Kotzbeck P.
      • et al.
      Pharmacological inhibition of adipose triglyceride lipase corrects high-fat diet-induced insulin resistance and hepatosteatosis in mice.
      ). In addition, the effect of Atglistatin on adipose tissue improved glucose homeostasis and insulin resistance (
      • Schweiger M.
      • Romauch M.
      • Schreiber R.
      • Grabner G.F.
      • Hütter S.
      • Kotzbeck P.
      • et al.
      Pharmacological inhibition of adipose triglyceride lipase corrects high-fat diet-induced insulin resistance and hepatosteatosis in mice.
      ).
      The second lipase involved in the release of FAs from the LDs is the hormone sensitive lipase (HSL), that hydrolyzes diacylglycerol. HSL is modulated by protein kinase A, which phosphorylates perilipin 1 allowing access to the LD core (
      • Wang H.
      • Hu L.
      • Dalen K.
      • Dorward H.
      • Marcinkiewicz A.
      • Russell D.
      • et al.
      Activation of hormone-sensitive lipase requires two steps, protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins.
      ). Insulin mediates the deactivation of HSL by inhibiting protein kinase A and, therefore, preventing phosphorylation of perilipin 1 (
      • Wang H.
      • Hu L.
      • Dalen K.
      • Dorward H.
      • Marcinkiewicz A.
      • Russell D.
      • et al.
      Activation of hormone-sensitive lipase requires two steps, protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins.
      ). Mice with adipose tissue HSL knockout developed liver steatosis, liver inflammation and insulin resistance; by contrast, mice with liver specific HSL knockout did not show alteration in hepatic fat content (
      • Xia B.
      • Cai G.H.
      • Yang H.
      • Wang S.P.
      • Mitchell G.A.
      • Wu J.W.
      Adipose tissue deficiency of hormone-sensitive lipase causes fatty liver in mice.
      ).
      The third lipase involved in the release of FAs from the LDs is the monoacylglycerols lipase (MGL) that hydrolyzes monoacylglycerols to form FAs (
      • Zechner R.
      • Zimmermann R.
      • Eichmann T.O.
      • Kohlwein S.D.
      • Haemmerle G.
      • Lass A.
      • et al.
      FAT SIGNALS--lipases and lipolysis in lipid metabolism and signaling.
      ,
      • Tardelli M.
      • Bruschi F.V.
      • Trauner M.
      The Role of Metabolic Lipases in the Pathogenesis and Management of Liver Disease.
      ). MGL has a pivotal role in hydrolyzing 2-arachidonoylglycerol into arachidonic acid, which is a precursor of prostaglandin synthesis and a main driver of inflammation. This production of arachidonic acid causes hepatic injury via endocannabinoid and eicosanoid signaling (
      • Cao Z.
      • Mulvihill M.M.
      • Mukhopadhyay P.
      • Xu H.
      • Erdélyi K.
      • Hao E.
      • et al.
      Monoacylglycerol lipase controls endocannabinoid and eicosanoid signaling and hepatic injury in mice.
      ).
      Autophagy is an intracellular pathway responsible for the degradation of lipids, damaged organelles, protein aggregates, membranes, mitochondria, and peroxisomes. Autophagy is an important cellular process for maintaining cytoplasmic homeostasis (
      • Ravikumar B.
      • Sarkar S.
      • Davies J.E.
      • Futter M.
      • Garcia-Arencibia M.
      • Green-Thompson Z.W.
      • et al.
      Regulation of mammalian autophagy in physiology and pathophysiology.
      ,
      • Weidberg H.
      • Shvets E.
      • Elazar Z.
      Lipophagy: selective catabolism designed for lipids.
      ). There are three forms of autophagy depending on the type of cargo delivered to the lysosome and the type of delivery methods: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Macroautophagy is a non-selective process that starts with the isolation and nucleation of the membrane (autophagosome) from various intracellular organelles such as ER, mitochondria, ER-mitochondria contact site and ER-Golgi site (
      • Lamb C.A.
      • Yoshimori T.
      • Tooze S.A.
      The autophagosome: origins unknown, biogenesis complex.
      ). After nucleation and expansion of the isolation membrane, the autophagosome surrounds the target, fuses with a lysosome to create an autolysosome, and degrades the sequestrated cargo (cytoplasmic components, LDs, or organelles). This process expunges materials for cellular biosynthetic processes and energy production (
      • Lamb C.A.
      • Yoshimori T.
      • Tooze S.A.
      The autophagosome: origins unknown, biogenesis complex.
      ). In microautophagy, by contrast, the lysosomal membrane infolds to form a cargo-containing vesicle (
      • Li W.W.
      • Li J.
      • Bao J.K.
      Microautophagy: lesser-known self-eating.
      ). The vesicle fuses and buds into the lysosome lumen to be degraded by lysosomal hydrolases. Lastly, CMA is a selective form of autophagy. It involves the selective degradation of a substrate containing a KFERQ-like pentapeptide motif recognized by the heat shock cognate protein (Hsc70), a cytosolic chaperone protein. This allows the uptake of the cargo into the lysosome through the interaction with the lysosome associated membrane protein type 2A (LAMP-2A) (
      • Schneider J.L.
      • Suh Y.
      • Cuervo A.M.
      Deficient chaperone-mediated autophagy in liver leads to metabolic dysregulation.
      ) (
      • Kaushik S.
      • Cuervo A.M.
      Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis.
      ). As PLIN2 contains the pentapeptide motif for the recognition and binding of Hsc70, CMA degrades PLIN2 found on the LD surface and facilitates the action of the cytosolic ATGL and the autophagy of LDs (
      • Tasset I.
      • Cuervo A.M.
      Role of chaperone-mediated autophagy in metabolism.
      ).

      Autophagosome structure

      The major site of autophagosome formation is the ER. The formation of the autophagosome involves three stages: initiation, nucleation, and elongation. The autophagosome membrane initiation starts with the phosphorylation of the ULK1 protein (unc-51 like autophagy activating kinase 1). Activated ULK1 forms a complex consisting of the ULK2, ATG13, the FAK family kinase interacting protein (FIP200) and ATG101 (
      • Jung C.H.
      • Jun C.B.
      • Ro S.H.
      • Kim Y.M.
      • Otto N.M.
      • Cao J.
      • et al.
      ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery.
      ,
      • Hara T.
      • Takamura A.
      • Kishi C.
      • Iemura S.
      • Natsume T.
      • Guan J.L.
      • et al.
      FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells.
      ). Another important signaling component in autophagy initiation is the PI3K-III (phosphatidylinositol 3-kinase, class III) complex which is composed of vacuolar protein sorting 34 and 15 (vps34 and vps15), Beclin 1 and ATG14. ULK1 phosphorylates AMBRA1 (activating molecule in Beclin 1-related autophagy 1), a scaffold protein that activates the Beclin1/ATG14/VPS34 complex and forms the PI3K-III complex (
      • Menon M.B.
      • Dhamija S.
      Beclin 1 Phosphorylation - at the Center of Autophagy Regulation.
      ). PI3K-III complex generates PI3Ps (phosphatidylinositol 3-phosphate) that recruits effector proteins such as WIPIs (WD-repeat domain phosphoinositide-interacting) proteins, DFCP1 (double-FYVE containing protein 1) and additional ATG proteins (
      • Menon M.B.
      • Dhamija S.
      Beclin 1 Phosphorylation - at the Center of Autophagy Regulation.
      ).
      Initiation drives the nucleation of the omegasome membrane which involves two ubiquitination-like proteins, ATG12 and ATG8 (LC3). In the first ubiquitin-like activity, ATG12 conjugates to ATG5 resulting in the formation of the ATG12-ATG5-ATG16L1 complex that binds to the pre-phagophore and phagophore (
      • Mizushima N.
      • Yoshimori T.
      • Ohsumi Y.
      Role of the Apg12 conjugation system in mammalian autophagy.
      ). LC3 is the second ubiquitin-like protein that functions as an E3-like ligase and that is involved in the elongation of the edges of the phagophore. The cytoplasmic form of LC3 is cleaved by ATG4 to form LC3-I. WIPI proteins specifically bind to PI3P at the nascent autophagosome membrane. WIPI/PI3P complex facilitate the recruitment of ATG12-5/ATG16 complex that conjugates LC3-I to phosphatidylethanolamine (PE) resulting in LC3 lipidation (LC3-II). LC3-II is involved in cargo recognition and hemi-fusion with the lysosome. LC3-II remains on the phagophore and autophagosome membranes even after Apg12–Apg5 dissociates. Thus, LC3-II levels correlate with autophagosome numbers. LC3-II interacts with p62 which functions as a selective cargo receptor for autophagy. P62 contains several domains for cargo recognition (including ubiquitin-associated domain) and cargo delivery to the autophagosome (through the LC3-interacting region). Through non-covalent interaction with ubiquitinate proteins, P62 recruits the cargo targeted for enclosure within the autophagosome.

      Regulation of autophagy

      Autophagy is regulated by the nutrient-sensing mechanistic target of rapamycin kinase (mTOR). In the fed state, mTOR is activated and plays a key anabolic role in promoting cell growth and proliferation by phosphorylating and inactivating ULK1 and ATG13 that in turn block autophagosome formation (
      • Shanware N.P.
      • Bray K.
      • Abraham R.T.
      The PI3K, metabolic, and autophagy networks: interactive partners in cellular health and disease.
      ). During starvation, AMP-activated protein kinase (AMPK) activates and inhibits mTORC1, which in turn activates the ULK1 proteins necessary for autophagosome formation (
      • Chan E.Y.
      mTORC1 phosphorylates the ULK1-mAtg13-FIP200 autophagy regulatory complex.
      ). This catabolic role of mTORC1 favors degradation of cytosolic organelles as well as lipids (lipophagy), proteins and carbohydrates for the production of energy allowing the cells to adapt to the new homeostatic status (
      • Allaire M.
      • Rautou P.E.
      • Codogno P.
      • Lotersztajn S.
      Autophagy in liver diseases: Time for translation?.
      ). During chronic overnutrition and the early stages of steatosis, there is a decrease in autophagy by approximately 70% (
      • Tasset I.
      • Cuervo A.M.
      Role of chaperone-mediated autophagy in metabolism.
      ,
      • Yang L.
      • Li P.
      • Fu S.
      • Calay E.S.
      • Hotamisligil G.S.
      Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance.
      ).
      Reduced autophagy causes an accumulation of small LDs. There is evidence that LD morphology influences LD catabolism as ATGL-driven lipolysis operates preferentially on larger LDs, whereas autophagy targets small LDs (with diameter <1μm) (
      • Schott M.B.
      • Weller S.G.
      • Schulze R.J.
      • Krueger E.W.
      • Drizyte-Miller K.
      • Casey C.A.
      • et al.
      Lipid droplet size directs lipolysis and lipophagy catabolism in hepatocytes.
      ). As described above, in the presence of ER stress there is an increase in Ca2+ accumulation in the cytosol. Thus, this environment inhibits LD autophagy by downregulating its fusion between autophagosome and lysosome. This causes an accumulation of ubiquitinated proteins and LDs that in turn increase ER stress and ROS production (
      • Park H.W.
      • Park H.
      • Semple I.A.
      • Jang I.
      • Ro S.H.
      • Kim M.
      • et al.
      Pharmacological correction of obesity-induced autophagy arrest using calcium channel blockers.
      ,
      • Arruda A.P.
      • Hotamisligil G.S.
      Calcium Homeostasis and Organelle Function in the Pathogenesis of Obesity and Diabetes.
      ).

      5. Lipid droplets and hepatocyte ballooning

      The progression from simple steatosis to NASH is characterized by hepatocellular ballooning. As described in the literature, ballooned hepatocytes feature dilated ER, cytoskeletal injury with Mallory-Denk bodies formation, parenchymal lesions, and substantial accumulation of LDs (Figure 4a-b) (
      • Argo C.K.
      • Ikura Y.
      • Lackner C.
      • Caldwell S.H.
      The fat droplet in hepatocellular ballooning and implications for scoring nonalcoholic steatohepatitis therapeutic response.
      ,
      • Fujii H.
      • Ikura Y.
      • Arimoto J.
      • Sugioka K.
      • Iezzoni J.C.
      • Park S.H.
      • et al.
      Expression of perilipin and adipophilin in nonalcoholic fatty liver disease; relevance to oxidative injury and hepatocyte ballooning.
      ,
      • Caldwell S.
      • Ikura Y.
      • Dias D.
      • Isomoto K.
      • Yabu A.
      • Moskaluk C.
      • et al.
      Hepatocellular ballooning in NASH.
      ).
      Figure thumbnail gr4
      Figure 4a and b. Diagram showing the association between hepatocyte ballooning, hypoxia, and hepatic stellate cells activation. Created with BioRender.com. In the endoplasmic reticulum (ER) membrane bilayer, specific enzymes esterify free fatty acids (FFAs) generated by extracellular and intracellular sources, synthesizing them into triglycerides (TG). TGs are then packed into nascent lipid droplets (LDs). At this stage, there are 3 possible pathways for LDs. 1) Degradation – depending on their size, LDs undergo lipolysis and/or autophagy and, as a result, release FFAs for β-oxidation. 2) Growth – LDs collide with other LDs, creating a contact site with the PLIN1/P3Kγ/CIDE complex and, ultimately creating a single, larger LD. Notably, a reduced degradation rate of LDs and/or excessive growth of LDs cause a compression of the space of Disse. This rheological change causes a release of hypoxia factors. 3) Embedding – LDs remain embedded within the ER membrane bilayer, producing ER stress and releasing reactive oxygen species. Hypoxia and ER stress cause the activation of hepatic stellates cells, with consequent collagen deposition and fibrosis induction. Created with BioRender.com

      Hepatocellular ballooning and lipotoxicity

      Ballooned hepatocytes feature a dysregulated lipid metabolism, with release of FAs and free cholesterol from the core of LDs that contribute to lipotoxicity and hepatic inflammation (
      • Marra F.
      • Svegliati-Baroni G.
      Lipotoxicity and the gut-liver axis in NASH pathogenesis.
      ).
      Regarding FAs, they are released from the core of LDs by neural lipase, resulting in the activation of the c-Jun N-terminal kinase signal pathways (JNK pathways) which cause cellular stress, inflammation, apoptosis and mitochondrial dysfunction (
      • Malhi H.
      • Bronk S.F.
      • Werneburg N.W.
      • Gores G.J.
      Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis.
      ,
      • Gan L.T.
      • Van Rooyen D.M.
      • Koina M.E.
      • McCuskey R.S.
      • Teoh N.C.
      • Farrell G.C.
      Hepatocyte free cholesterol lipotoxicity results from JNK1-mediated mitochondrial injury and is HMGB1 and TLR4-dependent.
      ). Moreover, the JNK pathways phosphorylate PPARγ that, in turn, inhibits FA β-oxidation with consequent exacerbation of lipotoxicity and hepatic inflammation. In addition, the release of FAs from the core of LDs causes the accumulation of DAG which are responsible for the development of insulin resistance through the activation of protein kinase (
      • Kolczynska K.
      • Loza-Valdes A.
      • Hawro I.
      • Sumara G.
      Diacylglycerol-evoked activation of PKC and PKD isoforms in regulation of glucose and lipid metabolism: a review.
      ). In the presence of insulin resistance there is further stimulation of de novo lipogenesis and an increase of FA flux from the adipose tissue to the liver (
      • Kim J.Y.
      • Nasr A.
      • Tfayli H.
      • Bacha F.
      • Michaliszyn S.F.
      • Arslanian S.
      Increased Lipolysis, Diminished Adipose Tissue Insulin Sensitivity, and Impaired β-Cell Function Relative to Adipose Tissue Insulin Sensitivity in Obese Youth With Impaired Glucose Tolerance.
      ). Regarding free cholesterol, it is released from the core of LDs by the action of the neutral cholesterol ester hydrolase enzyme. This increases ROS production which also activates the JNK pathways (
      • Hager L.
      • Li L.
      • Pun H.
      • Liu L.
      • Hossain M.A.
      • Maguire G.F.
      • et al.
      Lecithin:cholesterol acyltransferase deficiency protects against cholesterol-induced hepatic endoplasmic reticulum stress in mice.
      ).

      Hepatocellular ballooning and rheological changes

      Ballooned hepatocytes, due to their altered structure, cause disruption of the space of Disse and initiate the metabolic pathways that lead to NASH and fibrosis (
      • Carr R.M.
      VCAM-1: closing the gap between lipotoxicity and endothelial dysfunction in nonalcoholic steatohepatitis.
      ,
      • Ijaz S.
      • Yang W.
      • Winslet M.C.
      • Seifalian A.M.
      Impairment of hepatic microcirculation in fatty liver.
      ,
      • Baffy G.
      Origins of Portal Hypertension in Nonalcoholic Fatty Liver Disease.
      ). The space of Disse is a subendothelial space that lies between the liver sinusoidal endothelial cells (LSECs) and hepatocytes. LSECs are highly specialized cells with a characteristic fenestration forming the interface between blood cells on one side and hepatocytes and hepatic stellate cells on the other side. LSECs receive blood flow from the portal vein (70%) and the hepatic artery (30%), and their fenestrations allow the blood to flow from the sinusoidal area to the space of Disse (

      DeLeve LD. Vascular Liver Disease and the Liver Sinusoidal Endothelial Cell. In: DeLeve LD, Garcia-Tsao G, editors. Vascular Liver Disease: Mechanisms and Management. New York, NY: Springer New York; 2011. p. 25-40.

      ). LSEC fenestration is regulated by the vascular endothelial growth factor (VEGF), an angiogenic protein produced by hepatocytes, LSECs and HSCs. The compression of the space of Disse by the ballooned hepatocytes due to LD accumulation dilated ER, cytoskeletal injury, causes sinusoidal capillary compression, sinusoidal space restriction, and distortion of the sinusoidal pattern (reducing sinusoidal space by as much as 50% compared with normal liver) (
      • Ijaz S.
      • Yang W.
      • Winslet M.C.
      • Seifalian A.M.
      Impairment of hepatic microcirculation in fatty liver.
      ,
      • Farrell G.C.
      • Teoh N.C.
      • McCuskey R.S.
      Hepatic microcirculation in fatty liver disease.
      ). It is this compression of the space of Disse that ultimately causes hypoxia by increasing intrahepatic resistance and portal venous pressure, while concurrently reducing oxygen supply (
      • Francque S.
      • Laleman W.
      • Verbeke L.
      • Van Steenkiste C.
      • Casteleyn C.
      • Kwanten W.
      • et al.
      Increased intrahepatic resistance in severe steatosis: endothelial dysfunction, vasoconstrictor overproduction and altered microvascular architecture.
      ,
      • Hirooka M.
      • Koizumi Y.
      • Miyake T.
      • Ochi H.
      • Tokumoto Y.
      • Tada F.
      • et al.
      Nonalcoholic fatty liver disease: portal hypertension due to outflow block in patients without cirrhosis.
      ).

      Hepatocellular ballooning and hypoxia

      Recent evidence lends support to hypotheses linking the compression of the space of Disse to hypoxia and ultimately the progression of NAFLD (
      • Suzuki T.
      • Shinjo S.
      • Arai T.
      • Kanai M.
      • Goda N.
      Hypoxia and fatty liver.
      ) to NASH. Experimental studies in steatotic animal models indicate that moderate steatosis reduces sinusoidal blood flow by approximately half, because of sinusoidal distortion engendered by ballooned hepatocytes (
      • Seifalian A.M.
      • Piasecki C.
      • Agarwal A.
      • Davidson B.R.
      The effect of graded steatosis on flow in the hepatic parenchymal microcirculation.
      ). These alterations are associated with increased intrahepatic resistance that gives rise to post-sinusoidal non-cirrhotic portal hypertension (
      • Sarin S.K.
      • Kapoor D.
      Non-cirrhotic portal fibrosis: current concepts and management.
      ). In patients with NAFLD, the hepatic venous pressure gradient is ≥5 mmHg greater than patients without NAFLD (which is 4 mmHg or less in a normal individual (
      • Lee S.S.
      • Hadengue A.
      • Moreau R.
      • Sayegh R.
      • Hillon P.
      • Lebrec D.
      Postprandial hemodynamic responses in patients with cirrhosis.
      )), indicating sinusoidal portal hypertension that is related to the steatosis grade and not to the presence of extensive fibrosis or cirrhosis (
      • Francque S.
      • Verrijken A.
      • Mertens I.
      • Hubens G.
      • Van Marck E.
      • Pelckmans P.
      • et al.
      Noncirrhotic human nonalcoholic fatty liver disease induces portal hypertension in relation to the histological degree of steatosis.
      ).
      Crucially, hypoxia leads to HSCs activation through multiple pathways. First, hypoxia causes HSCs activation through VEGF expression and type I collagen. Specifically, hypoxia damages hepatocytes and increases the production of VEGF which in turn stimulates angiogenesis to counteract the hypoxic condition. VEGF is a signal protein that stimulates the formation of blood vessels. In normal physiological conditions, VEGF maintains the undifferentiated state of the LSECs and HSCs. By contrast, in hypoxic conditions, there is an increase in VEGF expression and type I collagen which jointly activate HSCs (
      • Corpechot C.
      • Barbu V.
      • Wendum D.
      • Kinnman N.
      • Rey C.
      • Poupon R.
      • et al.
      Hypoxia-induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis.
      ). Notably, VEGF and type I collagen activation of HSCs are independent of TGF-β activation (
      • Corpechot C.
      • Barbu V.
      • Wendum D.
      • Kinnman N.
      • Rey C.
      • Poupon R.
      • et al.
      Hypoxia-induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis.
      ).
      Second, hypoxia indirectly activates HSCs through tumor necrosis factor (TNF) and TGF-β. Specifically, the endothelial disfunction caused by hypoxia is associated with a reduced nitric oxide bioavailability that in turn promotes the production of inflammatory cytokines including tumor necrosis factor (TNF) and TGF-β that trigger the activation of HSCs.
      Third, hypoxia activates HSCs through the non-coding RNA plasmacytoma variant translocation (PVT1) pathway. In the presence of hypoxia, HIF-1α and HIF-2α translocate to the nucleus and activate transcription of target genes (
      • Schödel J.
      • Ratcliffe P.J.
      Mechanisms of hypoxia signalling: new implications for nephrology.
      ,
      • Triantafyllou E.A.
      • Georgatsou E.
      • Mylonis I.
      • Simos G.
      • Paraskeva E.
      Expression of AGPAT2, an enzyme involved in the glycerophospholipid/triacylglycerol biosynthesis pathway, is directly regulated by HIF-1 and promotes survival and etoposide resistance of cancer cells under hypoxia.
      ). Hypoxia upregulates the expression of long non-coding RNA PVT1. PVT1 is an oncogene associated with a variety of cancers, and it is significantly elevated in HCC (
      • Schödel J.
      • Ratcliffe P.J.
      Mechanisms of hypoxia signalling: new implications for nephrology.
      ). Under hypoxic conditions, HSCs overexpress PVT1 which in turn up-regulate the expression of miR-152/ATG14 signaling pathway inducing autophagy. The activation of autophagy will reduce retinyl esters contained in the LDs thus leading to HSC activation (
      • Zheng J.
      • Yu F.
      • Dong P.
      • Wu L.
      • Zhang Y.
      • Hu Y.
      • et al.
      Long non-coding RNA PVT1 activates hepatic stellate cells through competitively binding microRNA-152.
      ,
      • Deng J.
      • Huang Q.
      • Wang Y.
      • Shen P.
      • Guan F.
      • Li J.
      • et al.
      Hypoxia-inducible factor-1alpha regulates autophagy to activate hepatic stellate cells.
      ,
      • Yu F.
      • Dong B.
      • Dong P.
      • He Y.
      • Zheng J.
      • Xu P.
      Hypoxia induces the activation of hepatic stellate cells through the PVT1-miR-152-ATG14 signaling pathway.
      ).
      As a result, HSCs transition from a state of lipid-storage quiescence to a myofibroblast phenotype with fibrogenic properties, driving liver fibrosis and promoting the production and deposition of extracellular matrix (
      • Wynn T.A.
      Fibrotic disease and the T(H)1/T(H)2 paradigm.
      ). This extracellular matrix remodeling substantively contributes to the progression of NAFLD from simple steatosis to liver fibrosis, cirrhosis and ultimately end-organ failure (
      • Rockey D.C.
      • Bell P.D.
      • Hill J.A.
      Fibrosis--A Common Pathway to Organ Injury and Failure.
      ).

      6. Conclusions

      NAFLD is a heterogeneous disease and its progression towards liver fibrosis and hepatocellular carcinoma is not clearly understood.
      In the past, NAFLD research has limited its focus to TG accumulation in the hepatocytes. Such an approach fails to appreciate that TGs are stored in LDs.
      This review has shown that the biology of LDs plays a material role in the progression of NAFLD towards NASH and fibrosis. First, lipid composition of LDs can impact their relationship with the ER bilayer as well as impact ER stress, leading to hepatic inflammation and possibly NASH development. Second, the LD proteome can vary markedly based on genetic predisposition and the metabolic state of the cell, with relevant consequences on NAFLD development. Notably, the LD proteome can downregulate LD degradation hindering autophagy and lipolysis processes, further worsening steatosis. Third, increased LD number and size in hepatocytes cause liver anatomical changes, engendering the compression of sinusoidal cells and the reduction of blood flow, ultimately leading to hypoxia and increased portal pressure.
      It is crucial that we acquire a more comprehensive understanding of the biology of LDs, in hepatocytes and other liver cells to fully comprehend the molecular mechanisms and heterogeneity of NAFLD. Therefore, studying the changes that occur in LDs due to genetic predisposition, lifestyle, and metabolic diseases (such as diabetes, metabolic syndrome and obesity), as well as the LD localization and composition within the hepatocytes will be important areas of research. Equally, the genetic, proteomic and lipidomic profiles of LDs should be the object of exhaustive enquiry.

      Financial support

      Dr Carr receives research salary support from Intercept and Merck and has consulted for Boehringer. Dr Carr received research funding from NIH 1R01AA026302-01 and P30 DK050306.
      Dr Scorletti receive research salary support from the AFDHAL/MCHUTCHISON LIFER Award.
      The authors received no financial support to produce this manuscript.

      Authors’ contributions

      All the authors performed the research, writing, and review of all of the drafts of this paper and approved the final version.

      Conflict of interest

      Dr Carr was guest speaker for Arbutus Biopharma and expert witness for AstraZeneca.

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