Non-alcoholic steatohepatitis Wen-Ying, Chen 2006, 10 Chapter 11

Definition of nonalcoholic fatty liver disease Nonalcoholic Fatty Liver Disease (NAFLD) or Non-alcoholic steatohepatitis (NASH) is fatty inflammation of the liver when this is not due to excessive alcohol use. It is a major cause of cryptogenic cirrhosis of the liver. Nonalcoholic Fatty Liver Disease (NAFLD) or Non-alcoholic steatohepatitis (NASH) is fatty inflammation of the liver when this is not due to excessive alcohol use. It is a major cause of cryptogenic cirrhosis of the liver. It differs from the simple accumulation of fat in the liver (fatty liver, or hepatic steatosis) in that the inflammation of NASH causes damage to the liver cells while simple fatty liver probably does not. It differs from the simple accumulation of fat in the liver (fatty liver, or hepatic steatosis) in that the inflammation of NASH causes damage to the liver cells while simple fatty liver probably does not.

Chronic liver disease prevalence in the U.S. Nonalcoholic fatty liver disease 20 % Nonalcoholic fatty liver disease 20 % Nonalcoholic steatohepatitis 3 % Nonalcoholic steatohepatitis 3 % Chronic hepatitis C 2 % Chronic hepatitis C 2 % Alcoholic liver disease 0.7 % Alcoholic liver disease 0.7 % Chronic hepatitis B 0.4 % Chronic hepatitis B 0.4 % NASH is one of the commonest liver diseases in Western countries.

Introduction NASH is the most prevalent from of progressive liver disease and it is suggested that it affects between 10-39% of the global population, with an average incidence of 20%. NASH is the most prevalent from of progressive liver disease and it is suggested that it affects between 10-39% of the global population, with an average incidence of 20%. Diabetes, obesity and hypertriacylglycerolaemia are all well-known risk factors for NASH. Diabetes, obesity and hypertriacylglycerolaemia are all well-known risk factors for NASH.

Progression of NAFLD STEATOSISNASH Fibrosis (35 %) Cirrhosis (15 %) Liver Failure Hepatocellular Carcinoma (Caldwell SH. Hepatol. 2003; 37: 1202) 10 %

Aetiology Nutritional abnormalities Obesity 90% and lean people 6% Total parenteral nutrition; rapid weight loss Metabolic disorders DM; insulin resistance; lipodystrophy Hypertriglyceridaemia; abetalipoproteinaemia Hypolipoproteinaemia---- DrugsGlucocorticoidAspirin Synthetic oestrogen Tetracycline Surgery Jejuno-ileal bypass Gastopexy --- Occupational exposure Environmental toxins Conditions associated with non-alcoholic fatty liver disease

Diagnosis Clinical features Clinical features Biochemistry tests Biochemistry tests Imaging Imaging Histopathological features Histopathological features

Clinical features often asymptomatic----delays early diagnosis often asymptomatic----delays early diagnosis Discomfort in the upper right quadrant, fatigue---- Discomfort in the upper right quadrant, fatigue---- NASH is another feature of the metabolic syndrome NASH is another feature of the metabolic syndrome In one study of 66 patients: 98% insulin resistance and 39% diabetic In one study of 66 patients: 98% insulin resistance and 39% diabetic Obesity and insulin resistance are linked with NASH Obesity and insulin resistance are linked with NASH

Biochemistry tests Liver dysfunction in NASH Liver dysfunction in NASH serum alkaline phosphatase (ALP) and γ- glutamyltransferase (γ-GT) are increased serum alkaline phosphatase (ALP) and γ- glutamyltransferase (γ-GT) are increased Increased serum ferritin % Increased serum ferritin %

Imaging Ultrasound Ultrasound 87% sensitivity and 77% specificity 87% sensitivity and 77% specificity Computed tomography scanning Computed tomography scanning Magnetic resonance imaging Magnetic resonance imaging quantitative assessment of fatty infiltration is best achieved quantitative assessment of fatty infiltration is best achieved

Histopathological features Steatosis Steatosis Inflammatory cell infiltration Inflammatory cell infiltration Hepatocyte ballonning and necrosis Hepatocyte ballonning and necrosis Glycogen nuclei Glycogen nuclei Mallory’s hyaline Mallory’s hyaline Fibrosis Fibrosis liver biopsy is recommended for : 1. increased liver enzymes 2. metabolic syndrome: type 2 diabetes, hyperlipidaemia and obesity. liver biopsy is recommended for : 1. increased liver enzymes 2. metabolic syndrome: type 2 diabetes, hyperlipidaemia and obesity.

Pathogenesis Insulin resistance Insulin resistance Obesity Obesity Oxidative stress Oxidative stress Nuclear receptor regulation of gene expression Nuclear receptor regulation of gene expression

Pathogenesis insulin resistance Insulin resistance enhances TG lipolysis and inhibits esterification of free fatty acids (FFA) within adipose tissue. Hepatic TG synthesis is driven by the increased influx of FFA and favoured by insulin upregulated lipogenic transcription factors, such as PPARγ and SREBP-1c. TG export via VLDL may be inhibited by decreased synthesis of apo B.

Pathogenesis insulin resistance Rad (ras associated with DM),which interferes with essential cell functions (growth, differentiation, vesicular transport, and signal transduction). Rad (ras associated with DM),which interferes with essential cell functions (growth, differentiation, vesicular transport, and signal transduction). PC-1 (a membrane glycoprotein that has a role in I.R.), which reduces insulin-stimulated tyrosine kinase activity. PC-1 (a membrane glycoprotein that has a role in I.R.), which reduces insulin-stimulated tyrosine kinase activity. leptin, which induces dephosphorylation of insulin-receptor substrate-1 leptin, which induces dephosphorylation of insulin-receptor substrate-1 fatty acids, which inhibit insulin-stimulated peripheral glucose uptake. fatty acids, which inhibit insulin-stimulated peripheral glucose uptake. TNF- ,which down-regulates insulin-induced phosphorylation of insulin-receptor substrate-1 and reduces the expression of the insulin- dependent glucose-transport molecule Glut4. TNF- ,which down-regulates insulin-induced phosphorylation of insulin-receptor substrate-1 and reduces the expression of the insulin- dependent glucose-transport molecule Glut4. Adiponectin, which suppress TNF- . Adiponectin, which suppress TNF- . Several molecular targets have now been identified linked to the actions of insulin that contribute to the dyslipidaemia that may inform the pathogenesis of NASH.

Pathogenesis Obesity % of NASH patients are obese % of NASH patients are obese. Steatosis is a common observation in obesity and may be associated with inflammatory signs of non-specific hepatitis. Steatosis is a common observation in obesity and may be associated with inflammatory signs of non-specific hepatitis. The TNF-α gene is overexpressed in adipose tissue in the obese and in overweight or obese patients with type 2 diabetes, resulting in higher plasma TNF-α concentations that may contribute NASH. The TNF-α gene is overexpressed in adipose tissue in the obese and in overweight or obese patients with type 2 diabetes, resulting in higher plasma TNF-α concentations that may contribute NASH.

Pathogenesis Oxidative stress Mitochondria are the main cellular source of reactive oxygen species, which may trigger the inflammatory component of NASH. Mitochondria are the main cellular source of reactive oxygen species, which may trigger the inflammatory component of NASH. Numberous studies have emphasized the critical importance of oxidative stress as a crucial pathophysiological mechanism of NASH. Numberous studies have emphasized the critical importance of oxidative stress as a crucial pathophysiological mechanism of NASH.

Pathogenesis Oxidative stress Nitric oxide: iNOS may facilitate regeneration by inhibiting cell death in NASH. Nitric oxide: iNOS may facilitate regeneration by inhibiting cell death in NASH. In iNOS knockout mice: unscavenged superoxide production may cause toxic lipid peroxidation and ultimately microvesicular steatosis and NASH. In iNOS knockout mice: unscavenged superoxide production may cause toxic lipid peroxidation and ultimately microvesicular steatosis and NASH. Adiponectin: have hepatoprotective effects. Adiponectin: have hepatoprotective effects. 1). Increased lipid clearance through β-oxidation possibly mediated via activation of CPT-1. 1). Increased lipid clearance through β-oxidation possibly mediated via activation of CPT-1. 2). Direct anti-inflammatory effects through inhibition of TNF-α 2). Direct anti-inflammatory effects through inhibition of TNF-α 3). Reduce hepatomegaly, steatosis and serum ALT 3). Reduce hepatomegaly, steatosis and serum ALT Adiponectin may play a role in directly or indirectly regulating factors involved in the development of NASH.

Pathogenesis Oxidative stress Cytochrome P450 enzymes: CYP2E1 & CYP4A Cytochrome P450 enzymes: CYP2E1 & CYP4A are capable of generating free radicals to increase oxidative stress. are capable of generating free radicals to increase oxidative stress. CYP2E1: CYP2E1: 1). to be up-regulated persistently in type 2 diabetes, insulin resistance, central obesity and NASH; 1). to be up-regulated persistently in type 2 diabetes, insulin resistance, central obesity and NASH; 2). also is up-regulated by a high-fat/low-carbohydrate diet. 2). also is up-regulated by a high-fat/low-carbohydrate diet. 3). may play a role in initiating hepatic fibrosis. 3). may play a role in initiating hepatic fibrosis.

Insulin resistance Obesity/lipodystrophy Increased lipolysis/decreased Re-esterification PPAR γ activity Hepatocytes/stellate/ Kupffer cells Cyto P450 A P450E1 Glutathione Decreased: PPARα activity PPARδ activity Increased: SREBP 1a/1c SREBP2 activity Free radicals antioxidants NASH Oxidative stress & inflammation Kupffer cell NF-κB Leptin FFA Rad TNF- α PC-1 adiponectin NEFAs NO TNF- α ATP Inadequate oxidation of FFA & di/triacylglycerols Oxidative stress

Pathogenesis Nuclear receptor regulation of gene expression Peroxisome proliferator-activated receptors (PPAR) Peroxisome proliferator-activated receptors (PPAR) members a nuclaer receptor superfamily and key regulators of adipogenesis----involved in the pathogenesis of NASH. members a nuclaer receptor superfamily and key regulators of adipogenesis----involved in the pathogenesis of NASH.

Insulin resistance Obesity/lipodystrophy Increased lipolysis/decreased Re-esterification PPAR γ activity Hepatocytes/stellate/ Kupffer cells Cyto P450 A P450E1 Glutathione Decreased: PPARα activity PPARδ activity Increased: SREBP 1a/1c SREBP2 activity Free radicals antioxidants NASH Oxidative stress & inflammation Kupffer cell NF-κB Leptin FFA Rad TNF- α PC-1 adiponectin NEFAs NO TNF- α ATP Inadequate oxidation of FFA & di/triacylglycerols Oxidative stress

Pathogenesis Nuclear receptor regulation of gene expression Sterol regulatory element-binding proteins (SREBPs): important members of transcription factors that regulate hepatocyte cholesterol homeostasis. Sterol regulatory element-binding proteins (SREBPs): important members of transcription factors that regulate hepatocyte cholesterol homeostasis. In the liver, three SREBP isoforms, SREBP-1a, SREBP-1c and SREBP-2, regulate the production of lipid for export into the plasma as lipoproteins and into the bile as micelles. In the liver, three SREBP isoforms, SREBP-1a, SREBP-1c and SREBP-2, regulate the production of lipid for export into the plasma as lipoproteins and into the bile as micelles. Expression of SREBP-1c in the liver is regulated by LXR, which increases fatty acid synthesis. Expression of SREBP-1c in the liver is regulated by LXR, which increases fatty acid synthesis.

Pathogenesis Nuclear receptor regulation of gene expression Sterol regulatory element-binding proteins (SREBPs) Sterol regulatory element-binding proteins (SREBPs) 1). SREBP-1c levels are increased in the fatty livers of obese (ob/ob) mice with insulin resistance and hyperinsulinaemia caused by leptin deficiency, and SREBP-1c increases lipogenic gene expression, enhances fatty acid synthesis and accelerates triacyglycerol accumulation. 1). SREBP-1c levels are increased in the fatty livers of obese (ob/ob) mice with insulin resistance and hyperinsulinaemia caused by leptin deficiency, and SREBP-1c increases lipogenic gene expression, enhances fatty acid synthesis and accelerates triacyglycerol accumulation. 2). Overexpression of SREBP-1a in rats resulted in a 26-fold increase in fatty acid synthesis and a 5-fold increase in cholesterol synthesis. 2). Overexpression of SREBP-1a in rats resulted in a 26-fold increase in fatty acid synthesis and a 5-fold increase in cholesterol synthesis. 3). SREBP-2 overexpression in transgenic mice resulted in a 28-fold increase in cholesterol synthesis and a 4-fold increase in fatty acid synthesis. 3). SREBP-2 overexpression in transgenic mice resulted in a 28-fold increase in cholesterol synthesis and a 4-fold increase in fatty acid synthesis.

Insulin resistance Obesity/lipodystrophy Increased lipolysis/decreased Re-esterification PPAR γ activity Hepatocytes/stellate/ Kupffer cells Cyto P450 A P450E1 Glutathione Decreased: PPARα activity PPARδ activity Increased: SREBP 1a/1c SREBP2 activity Free radicals antioxidants NASH Oxidative stress & inflammation Kupffer cell NF-κB Leptin FFA Rad TNF- α PC-1 adiponectin NEFAs NO TNF- α ATP Inadequate oxidation of FFA & di/triacylglycerols

Treatment Peroxisome proliferator-activated receptor gamma agonists Peroxisome proliferator-activated receptor gamma agonists Biguanides Biguanides Peroxisome proliferator-activated receptor alpha agonists Peroxisome proliferator-activated receptor alpha agonists Antioxidants Antioxidants Weight loss Weight loss

Treatment PPAR γ agonists Rosiglitazone (24 weeks) in 30 NASH: improved insulin sensitivity, reduced liver fat content and improved ALT level. (may be due in part to the anti-inflammatory effects of PPARγ ligants. Rosiglitazone (24 weeks) in 30 NASH: improved insulin sensitivity, reduced liver fat content and improved ALT level. (may be due in part to the anti-inflammatory effects of PPARγ ligants. Troglitazone (400 mg for 6 months) in 10 NASH women: Troglitazone (400 mg for 6 months) in 10 NASH women: normal ALT level and mild histological improvement. normal ALT level and mild histological improvement. for 48 weeks: improved insulin sensitivity, reduced liver fat content and improved the biochemical and histological features of NASH. for 48 weeks: improved insulin sensitivity, reduced liver fat content and improved the biochemical and histological features of NASH. The main side-effects reported in these studies were weight gain and in total body fat.

Treatment Biguanides Metformin is the only biguanide FDA-approved for use in the United Stated. It regulates AMP-activated protein kinase in hepatocytes, which is a major cellular regulator of lipid and glucose metabolism. Metformin is the only biguanide FDA-approved for use in the United Stated. It regulates AMP-activated protein kinase in hepatocytes, which is a major cellular regulator of lipid and glucose metabolism. Improvement in liver biochemical test, insulin sensitivity and decreasing hepatic volume and body weight. Improvement in liver biochemical test, insulin sensitivity and decreasing hepatic volume and body weight. Thus, metformin may be beneficial in the treatment of NASH but some of the benefit may be a direct benefit of weight loss.

Treatment PPARα agonists a). Clofibrate (2g/day): no significant benefit in liver tests or hepatic histology b). Gemfibrozil (600 mg/day) in 46 NAFLD patients: a significant improvement in aminotransferase levles More evidence is needed from long-term studies with the newer PPARα agonists.

Treatment Antioxidants It is uncertain at present whether antioxidant therapy is beneficial in the treatment of NASH. It is uncertain at present whether antioxidant therapy is beneficial in the treatment of NASH. 1. Ursodeoxycholic acid (UDCA) (a epimer of chenodeoxycholic acid and non-hepatotoxic) a). 4 open-label pilot studies: have evaluated the therapeutic benefit of UDCA in patients with NAFLD. a). 4 open-label pilot studies: have evaluated the therapeutic benefit of UDCA in patients with NAFLD. b). 2 years randomized trials concluded that no significant differences in the degree of stestosis, inflammation or fibrosis occurred between the UDCA and placebo groups. b). 2 years randomized trials concluded that no significant differences in the degree of stestosis, inflammation or fibrosis occurred between the UDCA and placebo groups.

Treatment Antioxidants 2. Betaine is a normal component of the metabolism of methionine and increases the S-adenosylmethionine (SAM) level, which has been shown to protect against triacyglycerol deposition and liver injury in ethanol-fed rats. level, which has been shown to protect against triacyglycerol deposition and liver injury in ethanol-fed rats. a). In a recent study: betaine (20g/day for 12 months)---7 NAFLD patients decrease hepatic steatosis

Treatment Antioxidants 3. N-Acetylcysteine is an antioxidant that increases glutathione levels in hepatocytes. a). 11 NAFLD patients: N-acetylcysteine (1g/day for 3 m)----well tolerated and with a significant improvement in aminotransferase levles. 4. Vit E 5. α-tocopherol Thus, it is still uncertain whether there is benefit from treatment with antioxidants.

Treatment Weight loss Obesity is the condition most commonly associated with NASH and therefore weight loss is frequently advocated. Obesity is the condition most commonly associated with NASH and therefore weight loss is frequently advocated. However, the benefit of weight loss is difficult to evaluate because obese patients with NASH rarely achieve or maintain sustained reductions in body weight. However, the benefit of weight loss is difficult to evaluate because obese patients with NASH rarely achieve or maintain sustained reductions in body weight. Moreover, the effect of weight loss on NASH is not consistent. Moreover, the effect of weight loss on NASH is not consistent.

Treatment Weight loss For the future, weight loss combined with PPARα or perhaps PPARγ agonists will most likely be the treatments of choice but before this recommendation can be advocated more evidence of efficacy and safety is required. For the future, weight loss combined with PPARα or perhaps PPARγ agonists will most likely be the treatments of choice but before this recommendation can be advocated more evidence of efficacy and safety is required.