Claire Z. Larter

Nonalcoholic fatty liver disease : From steatosis to cirrhosis

Nonalcoholic steatohepatitis (NASH), the lynchpin between steatosis and cirrhosis in the spectrum of nonalcoholic fatty liver disorders (NAFLD), was barely recognized in 1981. NAFLD is now present in 17% to 33% of Americans, has a worldwide distribution, and parallels the frequency of central adiposity, obesity, insulin resistance, metabolic syndrome and type 2 diabetes. NASH could be present in one third of NAFLD cases. Age, activity of steatohepatitis, and established fibrosis predispose to cirrhosis, which has a 7‐ to 10‐year liver‐related mortality of 12% to 25%. Many cases of cryptogenic cirrhosis are likely endstage NASH. While endstage NAFLD currently accounts for 4% to 10% of liver transplants, this may soon rise. Pathogenic concepts for NAFLD/NASH must account for the strong links with overnutrition and underactivity, insulin resistance, and genetic factors. Lipotoxicity, oxidative stress, cytokines, and other proinflammatory mediators may each play a role in transition of steatosis to NASH. The present “gold standard” management of NASH is modest weight reduction, particularly correction of central obesity achieved by combining dietary measures with increased physical activity. Whether achieved by “lifestyle adjustment” or anti‐obesity surgery, this improves insulin resistance and reverses steatosis, hepatocellular injury, inflammation, and fibrosis. The same potential for “unwinding” fibrotic NASH is indicated by studies of the peroxisome proliferation activator receptor (PPAR)‐γ agonist “glitazones,” but these agents may improve liver disease at the expense of worsening obesity. Future challenges are to approach NAFLD as a preventive public health initiative and to motivate affected persons to adopt a healthier lifestyle. (Hepatology 2006;43:S99–S112.)

Animal models of NASH: Getting both pathology and metabolic context right

Non‐alcoholic fatty liver disease (NAFLD) is the most common cause of referral to liver clinics, and its progressive form, non‐alcoholic steatohepatitis (NASH), can lead to cirrhosis and end‐stage liver disease. The main risk factors for NAFLD/NASH are the metabolic abnormalities commonly observed in metabolic syndrome: insulin resistance, visceral obesity, dyslipidemia and altered adipokine profile. At present, the causes of progression from NAFLD to NASH remain poorly defined, and research in this area has been limited by the availability of suitable animal models of this disease. In the past, the main models used to investigate the pathogenesis of steatohepatitis have either failed to reproduce the full spectrum of liver pathology that characterizes human NASH, or the liver pathology has developed in a metabolic context that is not representative of the human condition. In the last few years, a number of models have been described in which the full spectrum of liver pathology develops in an appropriate metabolic context. In general, the underlying cause of metabolic defects in these models is chronic caloric overconsumption, also known as overnutrition. Overnutrition has been achieved in a number of different ways, including forced feeding, administration of high‐fat diets, the use of genetically hyperphagic animals, or a combination of these approaches. The purpose of the present review is to critique the liver pathology and metabolic abnormalities present in currently available animal models of NASH, with particular focus on models described in approximately the last 5 years.

Hepatic Free Cholesterol Accumulates in Obese, Diabetic Mice and Causes Nonalcoholic Steatohepatitis

BACKGROUND & AIMS Type 2 diabetes and nonalcoholic steatohepatitis (NASH) are associated with insulin resistance and disordered cholesterol homeostasis. We investigated the basis for hepatic cholesterol accumulation with insulin resistance and its relevance to the pathogenesis of NASH. METHODS Alms1 mutant (foz/foz) and wild-type NOD.B10 mice were fed high-fat diets that contained varying percentages of cholesterol; hepatic lipid pools and pathways of cholesterol turnover were determined. Hepatocytes were exposed to insulin concentrations that circulate in diabetic foz/foz mice. RESULTS Hepatic cholesterol accumulation was attributed to up-regulation of low-density lipoprotein receptor via activation of sterol regulatory element binding protein 2 (SREBP-2), reduced biotransformation to bile acids, and suppression of canalicular pathways for cholesterol and bile acid excretion in bile. Exposing primary hepatocytes to concentrations of insulin that circulate in diabetic Alms1 mice replicated the increases in SREBP-2 and low-density lipoprotein receptor and suppression of bile salt export pump. Removing cholesterol from diet prevented hepatic accumulation of free cholesterol and NASH; increasing dietary cholesterol levels exacerbated hepatic accumulation of free cholesterol, hepatocyte injury or apoptosis, macrophage recruitment, and liver fibrosis. CONCLUSIONS In obese, diabetic mice, hyperinsulinemia alters nuclear transcriptional regulators of cholesterol homeostasis, leading to hepatic accumulation of free cholesterol; the resulting cytotoxicity mediates transition of steatosis to NASH.

A fresh look at NASH pathogenesis. Part 1: the metabolic movers.

The strong relationship between over‐nutrition, central obesity, insulin resistance/metabolic syndrome and non‐alcoholic fatty liver disease (NAFLD) suggest pathogenic interactions, but key questions remain. NAFLD starts with over‐nutrition, imbalance between energy input and output for which the roles of genetic predisposition and environmental factors (diet, physical activity) are being redefined. Regulation of energy balance operates at both central nervous system and peripheral sites, including adipose and liver. For example, the endocannabinoid system could potentially be modulated to provide effective pharmacotherapy of NAFLD. The more profound the metabolic abnormalities complicating over‐nutrition (glucose intolerance, hypoadiponectinemia, metabolic syndrome), the more likely is NAFLD to take on its progressive guise of non‐alcoholic steatohepatitis (NASH). Interactions between steatosis and insulin resistance, visceral adipose expansion and subcutaneous adipose failure (with insulin resistance, inflammation and hypoadiponectinemia) trigger amplifying mechanisms for liver disease. Thus, transition from simple steatosis to NASH could be explained by unmitigated hepatic lipid partitioning with failure of local adaptive mechanisms leading to lipotoxicity. In part one of this review, we discuss newer concepts of appetite and metabolic regulation, bodily lipid distribution, hepatic lipid turnover, insulin resistance and adipose failure affecting adiponectin secretion. We review evidence that NASH only occurs when over‐nutrition is complicated by insulin resistance and a highly disordered metabolic milieu, the same ‘metabolic movers’ that promote type 2 diabetes and atheromatous cardiovascular disease. The net effect is accumulation of lipid molecules in the liver. Which lipids and how they cause injury, inflammation and fibrosis will be discussed in part two.

Apoptosis in experimental NASH is associated with p53 activation and TRAIL receptor expression

Background and Aims:  We examined extrinsic and intrinsic (endogenous) mitochondrial apoptosis pathways in experimental non‐alcoholic steatohepatitis (NASH).

MCD-induced steatohepatitis is associated with hepatic adiponectin resistance and adipogenic transformation of hepatocytes

BACKGROUND/AIMS In these studies, we tested the hypothesis that increased lipid intake would exacerbate the severity of nutritional steatohepatitis. METHODS C57Bl/6J mice were fed methionine-and-choline deficient (MCD) diets containing 20% (high) or 5% (low) fat by weight for 3 weeks and compared to lipid-matched controls. RESULTS MCD feeding increased serum ALT levels and induced hepatic steatosis, lobular inflammation and ballooning degeneration of hepatocytes, irrespective of dietary fat content. Hepatic triglyceride accumulation was similar between high and low-fat MCD-fed mice, but lipoperoxide levels were approximately 3-fold higher in the high-fat MCD-fed animals. Serum adiponectin levels increased in MCD-fed mice, although to a lesser extent in high-fat fed animals. AMPK phosphorylation was correspondingly increased in muscle of MCD-fed mice, but hepatic AMPK phosphorylation decreased, and there was little evidence of PPAR alpha activation, suggesting impaired adiponectin action in the livers of MCD-fed animals. Hepatocyte PPAR gamma mRNA levels increased in MCD-fed mice, and were associated with increased aP2 expression, indicating adipogenic transformation of hepatocytes. CONCLUSIONS Increased dietary lipid intake did not alter steatohepatitis severity in MCD-fed mice despite increased lipoperoxide accumulation. Instead, steatohepatitis was associated with impaired hepatic adiponectin action, and adipogenic transformation of hepatocytes in both low and high-fat MCD-fed mice.

Lipotoxicity: Why do saturated fatty acids cause and monounsaturates protect against it?

Saturated fatty acids (SFA) (e.g. palmitate [16 : 0]) are almost universally toxic to cells in culture, whereas the monounsaturated fatty acids (MUFA) (e.g. oleate [18 : 1]) are either non-toxic or cytoprotective. The opposing effects of SFA and MUFA have been observed in multiple cell types including islet b-cells, endothelial cells, cardiomyocytes, breast cancer cell lines, and in hepatocyte cell lines as shown by Ricchi et al. in this issue of the Journal. Importantly, the addition of MUFA to cell cultures dosedependently inhibits SFA-induced cell death. Elevated glucose clearly increases the toxicity of palmitate in b-cells, a process called glucolipotoxicity. The role of elevated glucose on lipotoxicity in other cell types has been under-investigated. An understanding of the mechanisms by which SFA are cytotoxic and MUFA are cytoprotective may give us clues to novel therapeutic approaches for relevant conditions, whether by diet or pharmacotherapeutic means. In most circumstances (e.g. steatohepatitis complicating non-alcoholic fatty liver disease [NAFLD]) the aim will be to inhibit cytotoxicity and/or promote cytoprotection. In some situations, however, inhibition of MUFA-induced cytoprotective mechanisms may have a role (e.g. in cancer therapy). So, why do the differing fatty acid types behave so differently with respect to cell survival? Fatty acids and their metabolites have numerous biological functions. Not only are lipids the major form by which energy is stored, they are also involved in cell structure, and participate in intracellular, extracellular and whole animal (endocrine) signaling processes. It should be no surprise, therefore, that the metabolism and behavior of the various types of fatty acids differs greatly. Considering this, it is probable that the mechanisms and/or pathways involved in SFA-induced cytotoxicity will be multiple and differ from those of MUFA-mediated cytoprotection. Fatty acids may exert their effects directly, for example as ligands to cell surface receptors (e.g. G-protein coupled receptors [GPCR]) or to intracellular transcription factors (e.g. peroxisome proliferator-activated receptors [PPAR]). Alternatively, fatty acids may need to be metabolized intracellularly to have their effects. There is evidence for both these direct and indirect effects influencing cell viability. The mechanisms, however, remain to be clearly defined.

Roles of adipose restriction and metabolic factors in progression of steatosis to steatohepatitis in obese, diabetic mice

Background and Aims:  We previously reported that steatohepatitis develops in obese, hypercholesterolemic, diabetic foz/foz mice fed a high‐fat (HF) diet for 12 months. We now report earlier onset of steatohepatitis in relation to metabolic abnormalities, and clarify the roles of dietary fat and bodily lipid partitioning on steatosis severity, liver injury and inflammatory recruitment in this novel non‐alcoholic steatohepatitis (NASH) model.

Hepatic free fatty acids accumulate in experimental steatohepatitis : Role of adaptive pathways

BACKGROUND/AIMS We determined the effects of dietary lipid composition on steatohepatitis development with particular attention to the nature of lipid molecules that accumulate in the liver and pathways of hepatic triglyceride synthesis. METHODS Mice were fed methionine and choline deficient (MCD) diets supplemented with 20% fat as lard (saturated) or olive oil (monounsaturated), for 3 weeks. RESULTS Irrespective of dietary lipid composition, MCD-fed mice developed steatosis, ballooning degeneration and lobular inflammation. MCD-feeding increased hepatic free fatty acid (FFA) levels 2-3-fold, as well as total triglyceride levels. Hepatic FFA composition was characterized by increased ratio of monounsaturated: saturated FFA. There were reduced nuclear levels of the lipogenic transcription factor sterol regulatory element binding protein-1 in MCD-fed mice, but no consistent reduction in fatty acid synthesis genes (acetyl-CoA carboxylase and fatty acid synthase). Consistent with pathways of hepatic triglyceride synthesis, expression of diacylglycerol acyltransferase-1 and -2 was increased, as were delta-5- and delta-6- fatty acid desaturase mRNA levels. CONCLUSIONS In this nutritional model of steatohepatitis, accumulation of FFA occurs despite substantial suppression of lipogenesis and induction of triglyceride synthesis genes. Accumulation of FFA supports a lipotoxicity mechanism for liver injury in this form of fatty liver disease.

Activation of peroxisome proliferator-activated receptor alpha by dietary fish oil attenuates steatosis, but does not prevent experimental steatohepatitis because of hepatic lipoperoxide accumulation

Background and Aim:  Non‐alcoholic fatty liver disease is the result of an imbalance in hepatic lipid partitioning that favors fatty acid synthesis and storage over fatty acid oxidation and triglyceride secretion. The progressive, inflammatory disorder of steatohepatitis can be prevented or reversed by correcting this lipid imbalance by activating peroxisome proliferator‐activated receptor (PPAR) α, a transcription factor which regulates fatty acid oxidation. n‐3 polyunsaturated fatty acids (PUFA), such as those found in fish oil (FO), are naturally occurring PPARα ligands which also suppress lipid synthesis.