Hepatic lipotoxicity and the pathogenesis of nonalcoholic steatohepatitis

“…the cause of steatosis, and not the fat accumulation by itself, produces cirrhosis”—Heribert Thaler, 19751 W ith nonalcoholic steatohepatitis (NASH), NASH cirrhosis, and NASH-related hepatocellular carcinoma becoming increasingly prevalent, the need for effective therapies has never been greater. Unfortunately, our understanding of what causes NASH at the molecular level remains mostly speculative, and thus our ability to design clinical trials that test rationally designed therapies is limited. More than a decade has passed since Christopher Day and Oliver James first proposed the oft-cited two-hit hypothesis of nonalcoholic fatty liver disease (NAFLD) to explain the pathogenesis of NASH.2, 3 According to this appealing hypothesis, the accumulation of lipid in the form of triglyceride is needed for the development of NASH and thus constitutes the first “hit” in this disease. The cause of injury in the setting of lipid-loaded hepatocytes was proposed to be oxidant stress leading to lipid peroxidation in the milieu of ample substrate. This second “hit” then triggers the necroinflammatory changes that we recognize histopathologically as NASH. Although this hypothesis has intuitive appeal, emerging data now suggests that in the liver, as in other organs, triglyceride accumulation in the form of lipid droplets truly is just an “innocent bystander” in the processes leading to cellular injury and inflammation,4, 5 an explanation presciently suggested by the eminent Austrian pathologist Heribert Thaler,1 considered by others,6 and acknowledged as a viable alternative by Day and James when they proposed the two-hit hypothesis.3 Convincing evidence for a central role of oxidant stress and lipid peroxidation in causing steatohepatitis has also not been established. A decade of research has confirmed that these processes occur,7 but studies have been unable to prove that oxidant stress or lipid peroxidation are necessary for the development of steatohepatitis in humans. As an alternative hypothesis, emerging evidence now points to metabolites of fatty acids as the real culprits in the hepatocellular injury in NASH, just as they are in other target organs of lipotoxicity (Fig. 1).8-12 Because triglyceride often accumulates as a parallel process during lipotoxic injury, it has been understandably difficult to separate the respective roles of triglyceride and fatty acid metabolites in causing injury and cell death. As with all good hypotheses, the nontriglyceride lipotoxicity hypothesis generates more questions than we can currently answer. Fortunately, it also provides new avenues for investigators to pursue in the search for effective treatments for this common disease. The lipotoxicity model of NASH. Emerging data indicate that metabolites of free fatty acids cause lipotoxic hepatocellular injury manifested as ER stress, inflammation, apoptosis, necrosis, and dysmorphic features such as ballooning and Mallory-Denk body formation. The generation of lipotoxic metabolites of fatty acids typically occurs in parallel with the accumulation of triglyceride droplets (steatosis), resulting in a phenotype recognized as NASH where steatosis and features of cellular injury are present together. A feature of this model that distinguishes it from earlier models of the pathogenesis of NASH is that the accumulation of triglyceride is not needed for the development of NASH and, in fact, it may be protective. A caveat is that steatosis does not “progress” to NASH. Metabolic abnormalities predisposing to lipotoxic injury include an increased supply or impaired disposal of free fatty acids. Insulin resistance plays a central role in these processes by allowing an excessive flow of fatty acids from adipose tissue and also impairing peripheral glucose disposal. Fatty acid disposal in the liver occurs through oxidative pathways and through the formation of triglyceride which is then stored temporarily as lipid droplets or secreted as VLDL. Supply-side salutary processes include reduction of carbohydrate precursors for de novo lipogenesis and prevention of inappropriate lipolysis by improving adipocyte insulin responsiveness. Favorable processes on the disposal side include increasing oxidative pathways (with the caveat that the intracellular antioxidant defenses must be adequate to handle the ROS produced) and increasing formation of triglyceride. Red arrows identify processes that contribute to the flux of fatty acids through hepatocytes, thus promoting lipotoxic injury; green arrows identify processes that eliminate fatty acids and thus reduce lipotoxic injury. Not shown are the mechanistic aspects of injury, repair, and fibrogenesis that ultimately determine whether lipotoxic liver injury leads to cirrhosis. ER, endoplasmic reticulum; IR, insulin resistance; P450, cytochrome P450 mixed function oxidases; ROS, reactive oxygen species; SER, smooth endoplasmic reticulum; VLCFA, very long chain fatty acids; VLDL, very low density lipoprotein. CoA, coenzyme A; CYP, cytochrome P450; DAG, diacylglycerol; DGAT2, diacylglycerol acyltransferase 2; DNL, de novo lipogenesis; ER, endoplasmic reticulum; FABP, fatty acid binding proteins; JNK, c-Jun N-terminal kinase; LPC, lysophosphatidyl choline; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen species; SCD-1, stearoyl-CoA desaturase-1; SREBP-1c, sterol regulatory element binding protein 1c; VLDL, very low density lipoprotein. Lipotoxicity is a term coined by Unger 15 years ago to describe the toxic effects of excessive free fatty acids on pancreatic beta cell survival.13 The term has never been formally defined but has been used variably to describe cellular injury and death caused by free fatty acids, their metabolites, and even triglyceride, although triglyceride is generally found to be relatively inert.8, 14 Because a wide variety of metabolites can be derived from fatty acids having varying chain lengths and varying degrees and locations of desaturation, not to mention cis versus trans configuration of double bonds, the number of permutations of fatty acid metabolites is enormous, making the identification of lipotoxic species challenging.15 Identifying important fatty acid derivatives remains an area of intense lipidomic study to understand lipotoxic diseases of the heart, pancreatic islets, and vasculature, as well as the liver.10, 12, 16-21 Shown in Fig. 2 are a few of the many putative mediators of nontriglyceride lipotoxicity. We should anticipate that as lipidomic studies advance and new metabolites are discovered, this list will appear quite naïve in retrospect. Possible fatty acid–derived mediators of lipotoxicity. Currently available data suggest that fatty acid metabolites participate in lipotoxic liver injury recognized as NASH. The reintroduction of diacylglycerol from triglyceride may play an important role in regulating the exposure of cells to these intermediaries and is highly regulated by a family of proteins.41, 67, 125 The tissue specificity and regulation of these pathways has been reviewed.66, 69, 70, 72 ACSL, acyl-CoA synthase; AGPAT, acyl-glycerolphosphate acyltransferase; ATGL, adipose triglyceride lipase; CPT, choline phosphotransferase; DAGK, diacylglycerol kinase; DGATs, diacylglycerol acyltransferases; GPAT, glycerol monophosphate acyltransferase; HSL, hormone-sensitive lipase; LPAAT, lysophosphatidic acid acyltransferase; LPAP, lysophosphatidic acid phosphatase; LysoPLD, lysophospholipase D; MAGK, monoacylglycerol kinase; MGL, monoacylglycerol lipase; MOGAT, monoacylglycerol acyltransferase; PLA, phospholipase A; PLD, phospholipase D. The accumulation of triglyceride as lipid droplets was once thought to be the underlying cause of insulin resistance in muscle, liver, and other tissues, but data now indicate that accumulation of lipid droplets is a parallel phenomenon and not the cause of altered insulin signaling pathways.12, 14, 22-24 Similarly, the data implicating triglyceride stored within hepatocyte lipid droplets as a cause of liver injury in human studies and animal models mostly demonstrate an the than triglyceride accumulation is not have suggested that triglyceride flow by of the during hepatocyte although this has been triglyceride does to injury during and during The accumulation of lipid droplets has been with increased of endoplasmic stress, although the role of this cellular in NASH is diacylglycerol acyltransferase 2 the triglyceride have increased triglyceride accumulation and of endoplasmic a of endoplasmic these not in the pathways of c-Jun N-terminal and that are typically in insulin resistance and in the fatty triglyceride accumulation but not lipotoxic injury caused by fatty acid exposure in cell and in the that to the role of triglyceride versus fatty acid–derived triglyceride precursors have been relatively in nontriglyceride lipotoxic injury. has been of of oxidative disposal pathways that can reduce the of fatty acids and the of evidence has also lipid accumulation from injury. triglyceride very low density by the triglyceride protein caused impaired of triglyceride, leading to triglyceride not appear to cause liver A has from studies that the formation of lipid droplets may be a that lipotoxicity from other fatty acid–derived 12, studies of lipotoxicity this in and a of triglyceride formation has been in pancreatic and of triglyceride as fat droplets is a the fatty acids in triglyceride must be at and the cell is unable to handle through other the stored triglyceride as a of lipotoxic fatty acids are highly to As a free fatty acids are with to and intracellular fatty acids are to fatty acid binding within cells that have for fatty acid hepatocytes, and of fatty acid in does not the flux through the but in with NASH, the flux of fatty acids through the liver is Because fatty acids are to the are mostly a function of and not of is through the have confirmed that the intracellular fatty acid is in the even with increased in the as with that of fatty acid in hepatocytes are to be with the increased of fatty acids to and that process is the other of in cells and of liver steatosis in the liver and liver cell that the role of this in lipotoxicity. as the intracellular for of fatty acids to that as and the the the more can be A study of in the of with NASH to with fatty liver but the of which remains Because fatty acids can be to many it has been difficult to prove whether free fatty acids or their metabolites are for the cellular injury in fatty acids can as for leading to a of study that or of function of can steatohepatitis in Fatty acids are also of leading to inappropriate of and causing of As for peroxisome proliferator-activated and fatty acids also effects that play and roles in of and as an and lipotoxicity by promoting the disposal of fatty acids through oxidative and data that fatty acids cause lipotoxic injury, other studies have shown that the formation of A is a in the development of fatty lipotoxicity. in liver cells and pancreatic have shown that acyl-CoA with lipotoxicity caused by fatty other metabolites of fatty acids need to be considered as more causes of lipotoxic liver injury. are fatty acid metabolites that thought to play a role in lipotoxic cellular and are increased in fatty liver lipotoxicity has been shown in pancreatic but studies that in other cell not fatty injury. the of with the not lipotoxicity in cell and not to be in lipotoxic cell death in cells their these will be to human liver disease remains to be but studies are now to other lipid species as more species are of protein a of for cellular signaling the of species is regulated to the of in and as for the formation of triglyceride and in species have been as a to lipidomic of liver from with with or steatohepatitis found although than was found in the increased may increased of fatty acids through the liver, but may not be to the cellular injury of studies of the role of in promoting steatohepatitis are needed to their role in lipotoxic liver injury. or is a of cell and lipid and it is a necessary of for formation and of fatty acid from the on the glycerol generates lysophosphatidyl choline a that has been in cellular injury as well as study found that be an important of lipotoxicity in this in liver from a of with found to be increased and in to disease A role of in lipotoxic liver injury was then human liver cell hepatocytes in and in the lipotoxic injury to on an with of through species can be through the of phospholipase on and of phospholipase shown to lipotoxicity in cell Although this important study it provides evidence for the of of fatty lipotoxicity. A wide variety of other metabolites of free fatty acids, and play a role in the lipotoxicity caused by excessive exposure to free fatty acids. lysophosphatidic acid species are that through a family of and acid species or and they also with a number of intracellular signaling regulating fat are a process of lipotoxic metabolites from free fatty acids is or that this by increasing the flux of fatty acids through the liver, through increased supply or impaired disposal of fatty acids, lipotoxicity. of the pathways that lipotoxic from fatty acids or of pathways that of also be to lipotoxicity. Because the understanding of lipotoxicity is just emerging and the mediators at the molecular level are currently data that of the role of such pathways in lipotoxicity. on that lipotoxic liver injury have been in cell and animal but evidence in human disease is limited. The of studies are The of fatty acid exposure and triglyceride accumulation and disposal have been for more than The of free fatty acids are adipocyte triglyceride lipolysis with of free fatty acids the or hepatocyte de novo lipogenesis the formation of new fatty acids from and acids as (Fig. tissue lipolysis is the of fatty acids to the liver, in with NASH, may contribute to of the fatty to the free fatty acid include of chain fatty acids from the and of and for of the triglyceride in are not a of fat for the This is important when treatments for NASH, to reduce triglyceride not have a on the The role of the supply side of fatty acid to the liver, through excessive peripheral lipolysis or excessive in the development of NASH has been well of triglyceride stored in adipose tissue with the of fatty acids the is regulated by the of and and is more than regulation of the hormone-sensitive as was once thought The process is by lipid and of adipocyte monophosphate Insulin is a of the of fatty acids from adipose tissue triglyceride and, as insulin resistance at the level of adipose tissue is for inappropriate lipolysis that leads to lipotoxic fatty acids are from adipose they can be in the liver to as triglyceride to the this has been proposed to play a role in it Although the has been that fatty acids by adipocyte are and to other tissues, data now also suggest that oxidative pathways within also be an important a role in and although this to be acids can also with adipose tissue to by adipose through the to monophosphate acids to and The of peripheral lipolysis on the liver can be by fatty acid is well recognized that adipocyte such as occurs during can more fatty acids to the liver than the liver can studies the of on liver but studies have also shown that the of such peripheral lipolysis on the liver can be by oxidative by of from the effects of excessive fatty acid but at the of effects of fatty acids on other will steatosis, but with impaired are to liver and the that has a in fat fatty acid or in that even to and are that need in clinical with to and the common that all The de novo of fatty acids is a of carbohydrate to the liver from and impaired peripheral glucose disposal. that include excessive carbohydrate in the form of may be common causes of the supply side of the fatty acid through the to fatty acid from the for this and thus provides an of as the for in have confirmed the of in and a study in has now with more fatty acids triglyceride, a of fatty acids must to fatty acids the of the glycerol is with an fatty The stearoyl-CoA effects this and is to the formation of triglyceride in the of adequate fatty acids. studies have shown that of triglyceride but at the of increased Similarly, excessive with a in but in caused liver injury but triglyceride accumulation in studies to the evidence that triglyceride formation is a than a cause of injury. studies shown that is often by in oxidative that as long as alternative pathways of fatty acid disposal are lipotoxic injury can be novo lipogenesis can also be by in sterol regulatory element binding protein the for the of for of in animal models has been as a to lipotoxic liver is by acids through the a for as a of inappropriate lipogenesis and of such as and with intracellular of fatty acids from the triglyceride by is a relatively to the fatty acid flux through the recognized and quite by is the role of in fatty acids from triglyceride within hepatocyte lipid this process play a role in the of hepatocyte lipid lipolysis is also by and of these as a of the generation of fatty acids has been understanding the effects of intracellular is by the in the effects of or are the liver, adipose or with acid in and in the that at the level of the liver play a role in this increased supply of fatty acids not be a disposal pathways are of disposal is to fat to and through As an important antioxidant must be to handle reactive oxygen species such as and that are in as are from to oxygen as fatty acids are Not the liver is with is present in in hepatocytes and hepatocyte and lipid and is a for the to eliminate reactive during oxidative of has not been shown to in that this is of the of ROS to during increased oxidative of fatty and studies have shown that this can be resulting in cell death and fatty acids to the liver are not needed as a of and are from to the formation of triglyceride. in the fatty acids are used as a of through to and fatty acids to oxidative pathways versus triglyceride formation is pancreatic glucose and to fatty acid the liver, is but peripheral lipolysis in adipose tissue to fatty acids to the liver, then this the disposal pathways at a and may to lipotoxicity. the to in NAFLD, a central role of in lipotoxic liver whether or is often with the steatohepatitis that these is to altered in the liver or is the of such as muscle, adipose or other peripheral has not been established. for have been shown to impaired function and are to The that or to and in is a second of oxidative fatty acid that be when are The role of fatty acid in NASH was difficult to understand in the of the the of ROS was a central role in causing NASH. of the in to cause NASH, and was found to be in which oxygen is to to oxygen to are well with to handle and ROS that are by such as pathways as a for human NASH has with mixed a available in has been shown to be in human studies of other have not been in clinical of in the or the role of in to in fatty acid and the of shown in human The oxidative smooth ER cytochrome P450 a of free fatty acids. This is of fatty acids such as chain and fatty acids with of or of double of fatty acids by leads to acids that are by of has been in NASH. Although thought to be a of ROS has not found this to be the of the oxidative and are regulated by the thus as a for a of species and, as a with the this to fatty acid the is also a for species but the formation and or of fatty acids as triglyceride. The accumulation of triglyceride as lipid droplets in NASH is by a of and that this predisposing to lipotoxic injury and in insulin signaling in cell and in animal human data has been difficult to of VLDL, the of and is not with liver that may be necessary for liver disease to in this This was also suggested to be the in the which and fatty liver steatohepatitis when studies have insulin resistance in the pathogenesis of As our understanding of insulin resistance has questions have which are important for the development of NASH. our of a common has been that NASH is a disease of the liver, insulin resistance in the liver must be of insulin to cause or lipotoxic liver injury in Because insulin pathways in the liver, even that insulin resistance be an that excessive de novo lipogenesis from data demonstrate that insulin resistance at the level of adipose tissue is the for the development of lipotoxic have shown that adipose tissue insulin resistance may be a target of of in adipose tissue may be a of adipose tissue insulin and of the or in have been shown to reduce inappropriate lipolysis and the in animal Because insulin resistance is to NASH, insulin may be an important in therapies for NASH. insulin can be the of glucose as the or the Insulin more with to NASH and the role of adipose insulin resistance be an of adipose insulin to insulin adipose insulin can be the of insulin free fatty acid to the a that be in studies of NASH and in the of with NASH. accumulation in the liver was once thought to insulin signaling and be a cause of insulin resistance in the liver and data have shown that triglyceride does not insulin signaling just as it does not cause lipotoxic injury; it is a of the increased supply of fatty acids or their impaired disposal leading to lipotoxic cell stress and in the insulin signaling that in A of the understanding of the of lipotoxic hepatocellular injury and the resulting to injury that leads to the phenotype of NASH has been 14 stress but role in cell injury and death has not been it plays a role in lipotoxic injury to be the of data for ROS as a to liver injury in all with a of oxidant stress may be important in insulin and studies have shown that excessive can contribute to insulin cell death to be central to the pathogenesis of lipotoxic injury in the as it is in other and of and may be of this is in understanding the of the liver to lipotoxic injury in animal models of NASH with the of the signaling as a leading from injury to The lipotoxicity model of NASH an it does not a number of other to the phenotype of liver injury recognized as NASH. impaired of excessive accumulation of in cell and with impaired flow or and such as or and are to be important to varying degrees in but are not for in this This the that what is currently recognized as NASH on a liver a of with a common The hypothesis that NASH is caused by nontriglyceride lipotoxicity has important lipotoxicity occurs of triglyceride then are have lipotoxic liver injury but not have triglyceride This triglyceride is impaired and the flux of fatty acids to the liver is not by oxidative disposal. lipotoxic liver injury present with liver and a inflammation, Mallory-Denk and but ballooning be present in such is Although a common is that cells are studies suggest that what is as ballooning on accumulation of very triglyceride droplets that are from during for A study liver but changes in liver fat of may be an of such A second caveat is that steatosis does not “progress” to NASH. Although studies this the of steatosis to steatohepatitis has in our of the of intuitive of of in the setting of steatosis or steatohepatitis found on a when steatosis was on an earlier relatively be by the of lipotoxic injury not by our relatively of cells or Mallory-Denk or lipotoxic injury in to such as in The in that a the that human NASH be more on this than is currently This hypothesis is by data in and in the study of with liver caused by triglyceride accumulation does not appear to cause liver injury, liver fat can be a of excessive fatty acid from peripheral adipose tissue and thus as a of peripheral insulin has been to the of treatments that peripheral by their effects on is when this the of steatosis not with the of steatohepatitis in changes in steatosis with in of injury in a clinical an to the formation of triglyceride, this fat accumulation in the liver, to lipotoxic injury. an of putative of is needed in steatosis as a for flux of fatty acids through the liver and of the lipotoxicity. the has with of an for the liver abnormalities in with flux of fatty acids through the liver and lipotoxic liver The term is but is a disease as not this it is to the steatosis is not to the pathogenesis of injury, then the need for a is more lipotoxic liver injury is not in form or but is and it from the that triglyceride can cause lipotoxic injury. it is a lipotoxic liver injury should with the caveat that triglyceride is generally and not a to lipotoxicity. and be considered by investigators in the in to a new not for lipotoxic liver injury but also for steatosis or injury NAFLD, a of A body of evidence now to the causes of NASH. thought to be a disease caused by triglyceride accumulation in hepatocytes with oxidant stress and lipid peroxidation causing and new data from animal studies and a number of human studies now evidence that triglyceride accumulation does not cause insulin resistance or cellular injury in the The lipotoxic liver injury hypothesis for the pathogenesis of NASH suggests that we need to our on the of fatty acids to the liver or in the This can be by improving insulin at the level of adipose tissue to inappropriate peripheral lipolysis and by de novo lipogenesis in the are the for de novo and carbohydrate through changes and increasing glucose through important of and prevention of lipotoxic liver injury, a disease NASH. to for this to the many investigators in the of lipotoxicity to the not