Hepatosteatosis

1. Hepatosteatosis Prof Samir Helmy Assaad-Khalil Unit of Diabetes & Metabolism Alexandria Faculty of Medicine 2007

2. Agenda • Definition • Common Causes of Steatosis • Prevalence Data • Ethnic and gender differences • Relationship between fatty liver disease & Met. Syndrome • Pathogenesis of fat accumulation in the liver • Screening tests / Diagnosis • Management

3. Definition • Hepatosteatosis is defined as fat deposition in the liver that exceeds 5% of the total weight of liver, or with > 5% of hepatocytes containing fat deposits under light microscopic examination • It occurs under several disease states.

4. Fatty disorders of the liver may be • Alcoholic (AFLD) • Non-alcoholic.(NAFLD) • NAFLD includes both: • Hepatic Steatosis • NASH l l l l

5. Causes of Nonalcoholic Fatty Liver Disease *HIV, human immunodeficiency virus.

6. Inborn Errors of Metabolism Associated With Steatosis • Abetalipoproteinemia • Familial hepatosteatosis • Galactosemia • Glycogen storage disease • Hereditary fructose intolerance • Homocystinuria • Systemic carnitine deficiency • Tyrosinemia • Resfum's disease • Schwachman's syndrome • Weber-Christian syndrome • Wilson's disease

7. Acquired Metabolic Disorders Associated With Steatosis • Diabetes mellitus • Obesity • Inflammatory bowel disease • Jejuno-ileal bypass • Kwashiorkor and marasmus • Serum lipid abnormalities • Starvation and cachexia • Severe anemia • Total parenteral nutrition

8. Drugs/Toxins Associated With Steatosis • Antibiotics • Azaserine • Bleomycin • Puromycin • Tetracycline • Other drugs • Amiodarone • Coumadin • Dichloroethylene • Ethionine • Ethyl bromide • Estrogens • Flectol H • Glucocorticoids • Hydrazine • Hypoglycin • Orotate • Perhexilenemaleate • Safrole • Metals • Antimony • Barium salts • Borates • Carbon disulfide • Chromates • Phosphorus • Rare earths of low atomic numbers • Thallium compounds • Uranium compounds • Cytotoxic/cytostatic drugs • l-Asparaginase • Azacytidine • Azauridine • Methotrexate

9. Prevalence Data: Risk Factors T2 DM 28-55% Obesity 60-95% Hyperlipidemia 20-92%

10. Caucasian African-American Latino – Hispanic Asian Other Distribution of NAFLD by Racial/Ethnic Group Estimated Alameda County Population (represented by KP Membership) NAFLD Study Population

11. Distribution of Gender in Persons with NAFLD Number of Patients

12. Disease Associations in Different Racial/ Ethnic Groups With NAFLD * Asian versus other groups combined * Asian versus other groups combined

13. Clinical features of MS in patients with NASH Yasemin H. et al., Annals of Hepatology 2006; 5(2): April-June: 109-114

14. Chronic hyperinsulinemia & carbohydrate ingestion • Chronic hyperinsulinemia & carbohydrate ingestion stimulate de novo lipogenesis by stimulating the activity of lipogenic enzymes such as the sterol regulatory element binding proteins (SREBP-1c). • The FFA stored in the liver can originate from hydrolysis of dietary chylomicrons or adiposity.

15. SREBPs suppress IRS-2-mediated insulin signalling in the liver • Insulin receptor substrate 2 (IRS-2) is the main mediator of insulin signaling in the liver, controlling insulin sensitivity. • Sterol regulatory element binding proteins (SREBPs) directly repress transcription of IRS-2 and inhibit hepatic insulin signaling. Ide T et al., Nat Cell Biol. 2004 Apr;6(4):351-7.

16. Pathogenesis “2 Hit” Paradigm • “Second hit” – Intrahepatic oxidative stress • Lipid peroxidation • TNF-alpha, cytokine cascade “First hit” – Excess fat accumulation

17. Oxidative Stress & Mitochondrial Changes • Chronic oxidative stress has been implicated with a) Formation of lipid hydroperoxides. b) Induction of certain microsomal enzymes e.g. cytochrome P450 2 El. • Mitochondrial changes: a) Mitochondrial damage with inhibition of mitochondrial electron transport chain activity. b) Release of mitochondrial free radicals. c) Depletion of mitochondrial glutathione pools.

18. Kupffer`s Cell Dysfunction • Hepatic steatosis also promotes: a) Kupffer’s cell dysfunction b) Impaired phagocytosis c) Chronic low grade endotoxemia. • Endotoxemia stimulate hepatic production of: a) TNF-  b) IL-6 and IL-8 c) Promote neutrophil chemotaxis & an inflammatory response.

19. Adiponectin • Decreased serum adiponectin concentrations correlates with hepatic fat content in patients with type 2 diabetes. • Hepatocyte growth factor (HGF) is not only an antiapoptotic and antifibrotic factor of liver, but it is also an adipokine.

20. Serum leptin & NASH studies in humans Leptin in the Field of Hepatic Fibrosis:A Pivotal or an Incidental Player? CVH, chronic viral hepatitis; NASH, nonalcoholic steatohepatitis; NAFLD,nonalcoholic fatty liver disease Sotirios K. et alDig DisSci DOI 10.1007/s10620-006-9126-0

21. Serum Leptin Levels Are Associated With Tamoxifen-Induced Hepatic Steatosis Serum leptin levels in patients with a normal liver or stable hepatic steatosis (group 1) and with increased hepatic steatosis (group 2) NazanGünel et al., Curr Med Res Opin 19(1):47-50, 2003.

22. Ghrelin in NAFLD • Ghrelin is reduced in NAFLD vs. controls • Insulin resistance is a major factor controlling ghrelin levels in subjects with and without NAFLD.

23. Tumor necrosis factor-α (TNF-α) gene • Tumor necrosis factor-α (TNF-α) gene expression is increased in adipose tissue in insulin resistant obese and type 2 diabetic patients. • In patients with NASH, TNF-α gene expression is increased in both hepatocytes and adipose tissue.

24. The microbial theory • Conversion of choline into methylamines by microbiata in strain 129S6 on a high-fat diet reduces the bioavailability of choline and mimics the effect of choline-deficient diets, causing NAFLD. • Fatty liver due to chronic choline-deficient diet exacerbates liver hepatitis, which is predominantly facilitated by T cells. • Delayed intestinal transit may contribute to intestinal bacterial overgrowth (IBO). Soza A et al., Dig Dis Sci. 2005 Jun;50(6):1136-40.

25. Rosiglitazone • Rosiglitazone decreases liver fat and increases insulin clearance. The decrease in liver fat by rosiglitazone is associated with an increase in serum adiponectin concentrations. • Despite this beneficial effect of TZDs, in certain individuals however, in addition to increasing fat mass, TZDs have the potential to exacerbate underlying hepatosteatosis.

26. Hepatic iron • Many patients with NASH have biochemical evidence of iron overload. • Yet, it has been suggested that the iron overload may be a result of hemachromatosis gene mutation which is ethnic specific and is probably not seen outside the population of Anglo-Celtic Caucasian descent. Uraz S et al., Dig Dis Sci. 2005 May;50(5):964-9.

27. Inflammatory liver steatosis caused by IL-12 and IL-18 • Acute fatty degeneration in the liver is caused by various agents, such as aspirin, valproic acid, and ibuprofen, that directly inhibit mitochondrial beta-oxidation of fatty acid and oxidative phosphorylation. • IL-12 and IL-18 may mediate inflammatory hepatosteatosis through impairment of the microcirculation, which leads to mitochondrial dysfunction in hepatocytes. Kaneda M et al., J Interferon Cytokine Res. 2003 Mar;23(3):155-62.

28. The Serum Levels of IL-1b, IL-6, IL-8 and TNF-a in Nonalcoholic Fatty Liver Serum cytokine values of NAFL and control groups. • IL-8 might play a more important role in the pathogenesis of liver steatosis than TNF-a, IL-1b and IL-6. Ülyas Tuncer et al., Turk J Med Sci 33 (2003) 381-386

29. Post-transplant NASH Re-transplantation Cirrhosis 12.5% NASH 33% Steatosis 60%

30. Steatosis in chronic hepatitis B : a result of metabolic causes attributable to the host rather than the effect of the viruses. Comparison of BMI of groups with & without steatosis(P<0.05 Comparison of cholesterol and triglyceride levels of groups with & without steatosis (P<0.05). Altlparmak E et al. Causes of steatosisWorld J Gastroenterol May 28, 2005 Volume 11 Number 20

31. NASH in patients with HCV • The presence of NASH in patients with HCV is strongly associated with: • Features of the metabolic syndrome • Is a risk factor for advanced fibrosis with bridging. • Risk factors for advanced fibrosis in patients with NASH & HCV are: • Weight. • Presence of diabetes. • Presence and degree of cytological ballooning.

32. Pathogenesis of NASH The liver metabolizes FFAs (partly mediated by lipase which is inhibited by insulin) Interruption of insulin signaling in hepatocytes causes fatty liver. Increase hepatic FA content causes hepatic IR Insulin resistance (IR) FFA When FFAs accumulate in the liver , they are oxidized by mitochondria . When FFAs accumulation exceeds oxidation ,the triglycerides and fat accumulate in the liver - Insulin signaling -  fat in liver - TNF TNF  plays an important role in IR evidenced by improvement of insulin sensitivity by genetic disruption of type 1 TNF receptors Bacterial endotoxins Steatosis Endotoxins from intestinal bacteria escape into the mesenteric blood & trigger a sustained hepatic inflammatory cytokine response (TNF). Lipid peroxidation TNF Cytokines NASH patients have higher concentration of total & FFA and total saturated & monounsaturated FAs, mainly due to the increase of: Hexadecanoic acid, Hexadecenoic acid & Octadecenoic acid. While absolute PUFA was not increased Chronic oxidative stress with a) Induction of certain microsomal enzymes (cytochrom P450 2 El) b) Lipid peroxidation. c) Mitochondrial damage with depletion of mitochondrial glutathion pools and release of mitochondrial free radicals which induce lipid peroxidation of hepatocyte membranes; initiate an inflammatory response with release of cytokines (TNF , IL-6, IL- 8) and stimulate fibrosis. Steatohepatitis Reduced antipyrine clearance (Cl-AP (reflects hepatic microsomal oxidative capacity) Iron TGF- Cytokines Hepatic iron may contribute to the pathogenesis of NASH by : 1- Induction of lipid peroxidation of organelle membranes resulting in membrane disruption. 2- Impaired mitochondrial oxidative metabolism. 3- Hepatocyte injury and death. 4- Lipocyte activation and stimulation of collagen type I gene activation & fibrosis. Cirrhosis

33. Clinical Features of NASH • Symptoms • Vague (fatigue, malaise, right upper quadrant discomfort) • Variable • Mostly absent • Signs • Hepatomegaly common • Splenomegaly in some • Portal HTN unusual • Laboratory values • Increased AST, ALT typical • +/2 increased alk. phos., GGT • Increased cholesterol, triglycerides common • Increased glucose common • Viral markers (2) • Autoantibodies (2) • Iron studies abnormal sometimes • Imaging • Fatty liver

34. Transaminases • Alanine transaminase (ALT) levels are higher than aspartate transaminase (AST) levels in most instances. • AST level may occasionally be higher than the ALT level, especially in the presence of cirrhosis. • The usual AST/ALT ratio is < 1 in patients with NASH and may be used to differentiate it from alcoholic liver disease (> 2 in the latter). • NASH can present with normal ALT values (in 20-25% of patients) despite the presence of a full histologic spectrum.

35. Symptoms of obstructive sleep apnea in patients with nonalcoholic fatty liverdisease • Approximately one-half of NAFLD patients, whether NAFL or NASH, have Syndrome of Sleep Apnea. Singh H et al., Dig Dis Sci. 2005 Dec;50(12):2338-43.

36. Demonstration of fat in the liver, staging & grading • The presence of fat in the liver can be demonstrated by various imaging modalities; however, no current noninvasive method can distinguish NASH from NAFLD. • Liver biopsy remains the gold standard for staging and grading

37. Histologic criteria • There are 2 histologic patterns of NASH: • Steatosis. • Steatohepatitis • The principle histologic features of NASH are: • The presence of macrovesicular fatty change in hepatocytes • Displacement of the nucleus to the edge of the cell. • Leucocytic infiltration in steatohepatitis.

38. Imaging • Transabdominal ultrasound is a sensitive, noninvasive method for detecting NAFLD. However, diagnostic criteria are highly operator-dependant and non-standardized • Spleen-minus-liver attenuation difference (DeltaS-LA) derived from CT shows a good correlation with the pathology of hepatosteatosis. Duman DG et al.,Dig Dis Sci. 2006 Feb;51(2):346-51.

39. Scintigraphy • Using scintigraphy, a strong, significant inverse correlation between the severity of steatosis, hepatic triglycerides content, and 99mTc-mebrofenin uptake rate was observed. • In the future, noninvasive "dynamic" breath tests may disclose specific alterations in metabolic pathways.

40. Proposed Histologic Spectrum NAFLD Fat Ballooning Degeneration Fibrosis +/-Mallory Bodies Fat Inflammation Ballooning Degeneration Fat + Inflammation Stage IV Stage III Stage II Stage I FAT Matteoni et al, Gastroenterol 1999 Examples of non-alcoholic fatty liver disease (NAFLD)

41. NASH as a Cause of End-Stage Liver Disease • Primary indication for OLT in 31/1,207 (2.6%) of patients evaluated at Mayo between 1993-98. • 16/546 (2.9%) underwent transplantation for end-stage NASH OLT= Organ Liver Transplant Charlton et al. Liver Transpl, 2001

42. Current Management & Future Therapy for NASH

43. NASH and effect of weight loss • Histological & laboratory improvement occurs with a 10% decrease in body weight. • However, in some patients, rapid weight loss may result in mild increase in inflammatory lesions (hepatitis), despite the regression of steatosis. • This may result from rapid mobilization of fatty acids or cytokines from adipose tissue, especially visceral fat.

44. Silymarin • Therapeutic approaches include silymarin, the seed extract of milk thistle, which is a mixture of flavonolignans • A hepatoprotective agent stimulating secretion of adiponectin. • This flavanoid has also antioxidant, antifibrotic, and membrane-stabilizing effects. Schuppan D et al., Hepatology 1999;30:1099-1104

45. Vitamin E & Betaine • Vitamin E, an antioxidant agent, reduces liver inflammation and necrosis. • Betaine, a naturally occurring choline metabolite, improves both biochemical parameters and liver histology.

46. Metformin • Metformin improves biochemical indices of hepatocellular injury and insulin resistance • Long-term metformin (500 mg 3 times /day for 4 months) resulted in: • Reduced transaminase levels, which returned to normal in 50% of actively-treated patients. • Improved insulin sensitivity • Decreased liver volume by 20%. • Mechanism of action of metformin in NASH: • Inhibits the hepatic TNF- activity . • Inhibits the expression of UCP-2 .

47. PPAR Agonists • Thiazolidinediones reduce hepatic fat stores in type 2 diabetes mellitus. • PPAR-alpha agonists exert antioxidative effects by inducing antioxidant enzymes, in addition to diminishing steatosis

48. Insulin therapy • Insulin therapy improves hepatic insulin sensitivity • It slightly but significantly reduces liver fat content, independent of serum adiponectin.

49. Patients receiving long-term total parenteral nutrition • Patients receiving long-term total parenteral nutrition may develop NAFL partially because of choline deficiency. • Besides, bacterial overgrowth is enhanced in the resting intestines. • Choline supplementation has been reported to improve or revert hepatic steatosis. Buchman AL et al., Gastroenterology 1992; 102:1363-1370 Buchman AL,et al. Hepatology 1995;22:1390-1403 Pappo I et al., J Clin Res 1991;51:106-112 Freud HR rt al., J Surg Res 1985;38: 356-363

50. Iron depletion • Hepatic iron overload is frequent in NAFLD, and iron depletion improves liver function tests and insulin resistance. • The addition of iron depletion therapy to standard diet/exercise reduces serum ALT levels in NASH independently from iron stores, thus representing a promising effective, safe, and low-cost therapy.