1. Faculty Disclosure Fernando Zapata, MD Dr. Zapata has listed no financial interest/arrangement that would be considered a conflict of interest.

2. NAFLD Epidemic Fernando Zapata Assistant Professor Pediatric Gastroenterology Division Children’s Hospital and Medical Center- Omaha 2

3. Prevalence Data 3 USA Canada Japan France UK Italy Spain Portugal South America Israel

4. 4 Adults: Prevalence of Overweight: 54% (USA) Prevalence of Obesity: 22% population > age 20 Steatosis 30.1 million obese adults NASH 8.6 million Angulo, NEJM 2002 Children: Prevalence of Overweight: 22% Non-Hispanic Blacks (girls) Mexican-American (boys) Prevalence of Obesity: 11% NHANES III

5. Prevalence Data 5 Type II DM 28-55% Hyperlipidemia 20-92% Obesity 60-95%

6. 6 Who Gets This???  Obesity  Type II Diabetes  Dyslipidemia Classic TRIAD Class I Obesity BMI 30-34.9 g/m2 Class II Obesity BMI 35-39.9mg/m2 Class III Obesity BMI > 40mg/m2 WHO Definitions:

7. Worldwide Demographic Data: Adults 7 LudwigUSA.1980205465 50 90 67 Itoh Japan1987165275 5 100 63 Diehl USA1988395281 55 71 20 Lee USA1989495378 51 69 --- PowellAustralia1990424983 36 95 81 PintoPortugal1996324975 34 47 28 BaconUSA19991325353 33 70 72 AnguloUSA19991445167 28 60 27 Author Country Year N Age Female NIDDM Obesity Lipids Expressed as percentages

8. 8 Metabolic Syndrome “Syndrome of Insulin Resistance” NIDDMTG HDLHypertension Visceral Obesity Steatosis NASH

9. HISTOPATHOLOGY 9

10. Non-Alcoholic Fatty Liver Disease (NAFLD) A new consequence of the obesity epidemic.

11. Non-Alcoholic Fatty Liver Disease (NAFLD) Fatty liver (Steatosis) Steatohepatitis - inflammation - inflammation - fibrosis - fibrosis Cirrhosis Normal liver

12. NAFLD 1.Most common of all liver disorders. 2. Most frequent cause of chronic liver disease. 3. Affects 3-20% of general population. 4. Present in up to 75% of individuals with obesity, type 2 diabetes. obesity, type 2 diabetes. 5. Present in 3% of children and >50% of obese children. obese children. Fatty Liver Disease: NASH and Related Disorders Blackwell Publishing, 2005

13. The Brief History of NAFLD Fatty Liver Disease: NASH and Related Disorders Blackwell Publishing, 2005 1979 ~8 papers published 1998 First NIH conference 1999 First Clinical Trials 2002 ~60 papers published 2004 Release of first book on NAFLD/NASH 2005 ~354 papers published

14. Non-Alcoholic Fatty Liver Disease (NAFLD) Fatty liver (Steatosis) Steatohepatitis - inflammation - inflammation - fibrosis - fibrosis Cirrhosis ? ?

15. Two-hit Hypothesis Fatty Liver 1 st Hit Damaged Liver 2 nd Hit Oxidative Stress Toxins InflammatoryMolecules Susceptibility Donnelly et al. J. Clin. Invest. 113: 1343, 2005 Day and James. Gastroenterol. 114: 842, 1998 DietFFABurnedVLDL-TG

16. Fatty Liver 1 st Hit Damaged Liver 2 nd Hit Susceptible Oxidative Stress Toxins InflammatoryMolecules Type  Saturated vs. vs. Unsaturated Unsaturated Addition to the Two-hit Hypothesis

17. Subject Characteristics Males = 24 Females = 27 Age (mean) 37 ± 5 36 ± 4 BMI (mean ± SD) kg/m² 38.6 ± 5.1 37.3 ± 5.9 Alcohol consumption ≤ 5 glasses/wk Impaired Glucose Tolerance or Type II Diabetes 56 Steatosis83%89% Collaboration with UT Southwestern

18. Fatty Liver 1 st Hit Damaged Liver 2 nd Hit Susceptible Oxidative Stress Toxins InflammatoryMolecules Type  Saturated vs. vs. Unsaturated Unsaturated Hypothesis

19. Relationship between saturated fatty acids and liver injury in morbidly obese subjects Amount of saturated lipids in liver Liver Injury Normal

20. Dietary models of hepatic steatosis Endocrinology 147:943, 2006

21. Increased susceptibility to lipopolysaccharide in steatosis characterized by increased saturated fatty acids Endocrinology 147: 943, 2006 Liver Injury Steatosis - + + + - + + + Sat FA - + - + - + - +

22. Two-hit Hypothesis + 1 Fatty Liver 1 st Hit Damaged Liver 2 nd Hit Oxidative Stress Toxins InflammatoryMolecules Susceptible Saturated Fats Damage

23. Non-Alcoholic Fatty Liver Disease (NAFLD) Fatty Liver NASH Fibrosis Cirrhosis Hepatocyte apoptosis increased in patients with non-alcoholic steatohepatitis (NASH). Canbay et al. Hepatol. 39: 273, 2004

25. Saturated fatty acids induce apoptosis in liver cells Am. J. Physiol. (In Press) 6 Hours 16 Hours

26. Ratio of saturated to unsaturated fatty acids determines degree of apoptosis in liver cells 16 Hours Am. J. Physiol. (In Press)

27. Two-hit Hypothesis + 1 Fatty Liver 1 st Hit Damaged Liver 2 nd Hit Oxidative Stress Toxins InflammatoryMolecules Susceptible Saturated Fats Unsaturated Fats Apoptosis

28. Liver Damage 2 nd Hit Liver Damage Sat FA 2 nd Hit Apoptosis Hepatocyte Mass Fatty Liver

29. Fatty Liver + Saturated Lipids Liver Damage Apoptosis Metabolism of and signals generated by saturated fatty acids Fibrosis ? ? ? ?

30. 30 Pathogenesis “2 Hit” Paradigm “Second hit” – Intrahepatic oxidative stress Lipid peroxidation Lipid peroxidation TNF-alpha, cytokine cascade TNF-alpha, cytokine cascade “First hit” – Excess fat accumulation

31. Worldwide Demographic Data : Children and Adolescent NAFLD 31 Author Country year n age range M/F Obesity IDDM Lipids Moran USA 1983 3 13 (10-15) 2/1 3 1 2/3 Baldridge USA 1995 14 13 (10-18) 10/4 14 0 5/10 Rashid Canada 2000 36 12 (4-16) 21/15 30 4 18 Manton Australia 2000 17 11 (9-15) 11/6 16 3 5/2 Schwimmer USA 2003 43 30/14

32. Histologic Features in Childhood and Adolescent NAFLD 32 Moran TN 1983 3 n/a 2/3 2 /3 0 Baldridge Boston 1995 14 24/31 14/14 14 /14 1 Rashid Canada 2000 36 24/31 21/24 17/24 1 Manton Australia 2000 17 10/11 8 9 Schwimmer USA 2003 27 27/43 1 Author Country Yr n US NASH Fibrosis Cirrhosis

33. Natural History Data for NAFLD 33 Author Study # Pt’s Follow-up % Improve No NASH Fibrosis Cirrhosis (years) Teli 1978-95 n=40 7-16 46% 7.5% 30% 3% 0% Teli et al. Hepatology 1995

34. Natural History Data for NASH 34 Lee 1968-82 n=49 1.2 - 6.9 12/39 0% 58% 25% 16.6% (1989) Powell 1960-89 n=42 1- 9 12/41 8.3% 41.6% 33% 16.6% (1990) Bacon 1990-93 n=33 4 -7 2/33 0% 50% 0% 39% (1994) AuthorStudy# Pt’s Follow-up N Improved No Change Fibrosis Cirrhosis (years)

35. Proposed Histologic Spectrum NAFLD 35 Fat + Inflammation Fat Inflammation Ballooning Degeneration Fat Ballooning Degeneration Fibrosis +/-Mallory Bodies FAT Stage I Stage II Stage III Stage IV Matteoni et al, Gastroenterol 1999

36. Comparison of Outcomes for Individual Histological Types of NAFLD 36 Type IType II Type IIIType IV Outcome(n=49)(n=10)(n=19)(n=54) Cirrhosis2 (4%)0 (0%)4 (21%)14 (26%) Death16 (33%)3 (30%)5 (26%)24 (44%) Liver-related1/49 (2%)0 (0%)1/19 (5%)*7/54 (13%)* Matteoni et al. Gastroenterol 1999

37. Clinical Outcomes Based on the Presence or Absence of Necroinflammation 37 Type I + II Type III + IV p-value Cirrhosis 3.4% 24.7% (p<0.0001) 5-yr Survival 75.6% 70.9% (p=0.12) Liver-Related 5.6%30.8% (p=0.06) Deaths Matteoni et al. Gastroenterol 1999 11% liver – related mortality for those with hepatocellular necrosis (Type III+IV)

38. Cryptogenic Cirrhosis 38  Derived from Greek “ kryptos” “genesis ”  3 rd most common indication for transplant  7-14% of transplant recipients - Actuarial 1 and 5 year survival: 72% and 58%

39. Prevalence of Risk Factors for NAFLD among Patients with Cryptogenic CirrhosisCaldwell(1999)Virginia70%47%53%21% 100% Caucasian Poonawala(2000)Maryland47%55%47%N/AN/A Ong(2001)Virginia60%25%34%N/A94%Caucasian 39 Study Population Female Obesity NIDDM Lipids Ethnicity Author Female Obese DM Lipids

40. 40 NASHCryptogenic 100% 60% 15% NASHCryptogenic Post-OLT Steatosis Post-OLT Steatohepatitis Charlton et al. 2002Ong et. al. 2001

41. NASH as a Cause of End-Stage Liver Disease 41  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 Charlton et al. Liver Transpl, 2001

42. Post-transplant Allograft Steatosis 42 Grade II Steatosis NASHHCVC2H5Cholestatic 4 months 4 months60%(N=15)8%(N=51)12%(N=40)4%(N=67) 12 months 60%(n=15)15% (n= 54) 15%(n=40)5%(n=54) Histology Primary Liver Disease Charlton et al. Liver Transpl, 2001

43. Post-transplant Allograft Fibrosis 43 Grade II Fibrosis NASHHCVC2H5Cholestatic 4 months 4 months20%(N=15)29%(N=51)18%(N=40)19%(N=67) 12 months 12 months33%(n=15)46% (n= 54) 13%(n=40)15%(n=62) Histology Primary Liver Disease

44. Post-transplant NASH 44 Steatosis NASH Cirrhosis Re-transplantation 33% 12.5% 60%

45. Post-transplant Recurrence of NASH 45

46. Steatosis Increases The Rate of Fibrosis in HCV 46 None1-30%>30% Hepatocytes with fat 8 yrs Per stage 7 yrs Per stage 4 yrs per stage Change in HAI Score/yrs of infection P<0.01 P<0.05 0.12.3.2.1 0 0.14.23 Genotype 1b, 10-14 yrs HCV infection Adinolfi et al, Hepatol 2001

47. Role of Familial Factors and Ethnicity (Cont’d) 47  Retrospective review of 90 patients age 14-70 with NASH seen at liver clinics at University of Tennessee or Medical University of South Carolina  NASH seen in 9 families, either in siblings or subsequent generations  28% patients had cirrhosis, almost ½ with complications of portal hypertension  Familial clustering was common 18%  Insulin resistance found in 85% tested. “ Ninety Patients with Nonalcoholic Steatohepatitis: Insulin Resistance, Familial Tendency, and Severity Willner et al, Am J Gastroenterol, 2001

48. Distribution of NAFLD by Racial/Ethnic Group 48 NAFLD Study Population Estimated Alameda County Population (represented by KP Membership) Caucasian African-American Latino – Hispanic Asian Other

49. Distribution of Serum Aminotransferase Levels in Persons With NAFLD 49 Mean Value U/L

50. Disease Associations in Different Racial /Ethnic Groups With NAFLD CaucasianLatino- Hispanic African- American AsianP- value BMI, mean (kg / m 2 )34.034.037.826.7P<0.001* NIDDM (%)47.945.650.025P=0.03* Lipids (%)49.243.457.157.1NS 50 * Asian versus other groups combined

51. Distribution of Gender in Persons with NAFLD 51 Number of Patients

52. Steatosis or NASH? 52

53. Liver Histopathology in Morbidly Obese Patients Undergoing Roux-en-Y Bypass 53 Normal Liver Histology 40% 40% 22% Steatosis 19% 19% 56% NASH 31% 28% 22% Bridging fibrosis/cirrhosis 2.9% 3.5% 0% Total Normal LFT’s Elevated LFT’s n=68 n-57 n=11 UCSF Bariatric Database NAFLD 36/68 (53%)

54. 54

55. Potential Pharmacologic Treatment Options for NALFD 55 Insulin Sensitizing Agents  Troglitazone/Rosiglitazone  Metformin Lipid-Lowering Agents  Clofibrate  Gemfibrizole Future Potential Treatments  Antifibrotics  Probiotics  Silymarin, SAMe  Vitamin E  Lecithin  Vitamin C   -Carotene  Selenium  Vitamin B Complex Anti-Oxidants Membrane-Stabilizing  Ursodeoxycholic Acid  Betaine ( SAMe)

56. Drugs Evaluated for NAFLD 56 Laurin,1999 URSO 24 0pen Label 12 mos Yes Yes Guma,1997 URSO 24 RCT 6 mos Yes none Ceriani,1998 URSO+diet 31 Open Label 6 mos Yes none Lauren, 1996 Clofibrate 16 Open Label 12 mos No none Basaranoglu,1999 Gemfibrozil 46 RCT 1 mo Yes none Levine, 2000 Vitamin E 11 Open Label 12 mos Yes none Abdelmalek, 2000 Betaine 24 Open Label 12 mos Yes Yes Gulbahar, 2000 NAC 11 Open Label 12 mos Yes none Marchesini, 2001 Metformin 20 Open Label 4mos Yes none Neuschwander- Tetri, 2001 Rosiglitazone 12 mos Yes Yes Promrat, 2004Pioglitazone 18 Open Label 12 mos Yes Yes Lindor, 2004 Urso 166 RCT 24mos Yes Yes Author Treatment # Pt’s Design Duration LFT’s Histology

57. 57