1. Hypothesis: AN ECTOPIC SYNTHESIS OF THE MELANIN IN ADIPOSE MAY HELP TO COUNTERACT SECONDARY COMPLICATIONS OF OBESITY Manpreet Randhawa, Tom Huff, Julio C. Valencia, Zobair Younossi, Vikas Chandhoke, Vincent J. Hearing, Ancha Baranova

3. Why obesity is so dangerous ? whyfiles.org/276metabolic_syndrome/

4. METABOLIC SYNDROME Common complications : Cardiovascular diseases Diabetes type II Non-alcoholic fatty liver disease (NAFLD) Non-alcoholic steatohepatitis (NASH)

5. Prevalence of the metabolic syndrome increases with age National Health and Nutrition Examination Survey III, 1988-1994 (8814 US adults)

6. Males Females Prevalence of metabolic syndrome increases with obesity, blacks;, Hispanics;, whites.

7. U.S. INCIDENCE OF DIABETES (BOTH TYPES): CDC graph

8. Cost burden of obesity (U.S. only) Direct cost only; 17% of total direct cost of heart disease, independent of stroke Direct and indirect costs Direct cost only; 17% of total direct cost of hypertension

9. Health-Care Costs for Obesity Top Those Related to Smoking Obesity is associated with an average increase in hospital and outpatient spending of $395 a year There has definitely been a feeling that smoking, drinking and substance abuse are bigger problems than obesity. This isn't the case.

10. Males Females Prevalence of metabolic syndrome increases with obesity, blacks;, Hispanics;, whites. Some obese people stay healthy despite very high BMI (even within bariatric cohort) WHY? Glass half full or half empty?

11. Non-Alcoholic Liver Disease as a model for the study of the secondary complications of obesity From: Ariel E. Feldstein and Marsha H. Kay, ACG website The majority of individuals with NAFLD have no symptoms and a normal examination NAFLD affects up to 20 % of adults and nearly 5 % of children. NASH  2-5 % of adult Americans; up to 20 % of obese subjects. Can we treat NAFLD ? Not really. Just try to lose weight…. increased risk of hepatocellular carcinoma Liver transplantation  Fibrosis 

12. For these who thinks that fat in the liver is microscopic change

13. IMPORTANT QUESTION: WHY SOME PATIENTS WITH VERY HIGH BMI (>45) STAY FREE OF LIVER DISEASE? ARE SOME PROTECTIVE FACTORS OR PREDISPOSING FACTORS INVOLVED? ARE THESE FACTORS PRESENT IN THE LIVER OR IN THE FAT?

14. Results of the visceral adipose profiling (40K chip) Results of the Liver biopsy profiling (5K chip) Dataset was downsized > 2.0 in the liver (NASH vs OC) = pro-NASH genes in the liver < 0.5 in the liver (NASH vs OC) = hepatoprotective genes in the liver > 2.0 in the adipose (NASH vs OC) = pro-NASH genes in the fat < 0.5 in the adipose (NASH vs OC) = hepatoprotective genes in the fat Obese Patients with NASH vs Obese patients without liver pathology

15. TNF-α and IL6 regulated genes expressed in adipose play a prominent role in the development of NASH Genes that are regulated by TGF-β signal in adipose are important for the development of the primary phenotype of the morbid obesity

16. Major finding: Adipose of the NASH patients overexpresses a number of genes encoding secreted, mostly pro-inflammatory molecules. IL18interleukin 18 (interferon-gamma-inducing factor) CCL26chemokine (C-C motif) ligand 26 CCL20chemokine (C-C motif) ligand 20 CTSBcathepsin B B2Mbeta-2-microglobulin UMODuromodulin (uromucoid, Tamm-Horsfall glycoprotein) PROCprotein C (inactivator of coagul. factors Va and VIIIa) FCN1ficolin (collagen/fibrinogen domain containing) 1 CLUclusterin (complement lysis inhibitor, apolipoprotein J) F3coagulation factor III (thromboplastin, tissue factor)

17. Other cells embedded in adipose, including macrophages Visfatin, Vaspin, Apelin, PAI-1, MCP1, other pro-inflammatory molecules IL-6 IL-8 NAFLD and NASH develop under the influence of the adipokines synthetized in the visceral adipose

18. ANOTHER study: Profiling of adiponectin, resistin, visfatin, apelin, TNF-alpha, IL-6, IL-8, IL1, IL1R, sIL-6 in the serum of patients with and without NAFLD  100 samples collected and profiled;  Correlation analysis with seven clinical parameters is completed;

20. Visfatin TNF-α IL-8IL-6 C.D. Controls I (N=38) Controls II (N=12) NAFLD (N=45) SS (N=19) NASH (N=26) Mean (pg/ml) Controls I (N=38) Controls II (N=12) NAFLD (N=45) SS (N=19) NASH (N=26) 1 10 100 1000 A. Mean (pg/ml) 100 B. Mean (pg/ml) Controls I (N=38) Controls II (N=12) NAFLD (N=45) SS (N=19) NASH (N=26) 100 10 1 1 Controls I (N=38) Controls II (N=12) NAFLD (N=45) SS (N=19) NASH (N=26) Mean (pg/ml) 10 1000 100 TNF-α Controls I (N=38) Controls II (N=12) NAFLD (N=45) SS (N=19) NASH (N=26) Mean (pg/ml) 10 1000 B. 100 TNF-α, pg/ml Obese Healthy (N = 38) 1.91+/-0.25 Lean Healthy (N = 12) 2.3+/-0.39 < 0.0001 Lower than normal levels of TNF-α may prevent the onset of NAFLD in morbidly obese patients. *

21. General Theme: Visceral fat in some obese subjects remains inert; Something protects obese people with inert fat from metabolic syndrome

22. Expression profiling in human OBESITY Comparisons of the visceral fat samples; >50 patients sampled during bariatric surgeries; 9 lean peoples donating their kidney;

23. Fat Tissue Stratagene human Ref. RNA Isolate total RNA Reverse Transcribe to cDNA, label with Cy3 Reverse Transcribe to cDNA, label with Cy5 Mix, Hybridize and Scan Linearly Amplify RNA Fat Tissue Stratagene human Ref. RNA Isolate total RNA Reverse Transcribe to cDNA, label with Cy3 Reverse Transcribe to cDNA, label with Cy5 Mix, Hybridize and Scan Linearly Amplify RNA Fat Tissue Stratagene human Ref. RNA Isolate total RNA Reverse Transcribe to cDNA, label with Cy3 Reverse Transcribe to cDNA, label with Cy5 Mix, Hybridize and Scan Linearly Amplify RNA Fat Tissue Stratagene human Ref. RNA Isolate total RNA Reverse Transcribe to cDNA, label with Cy3 Reverse Transcribe to cDNA, label with Cy5 Mix, Hybridize and Scan Linearly Amplify RNA Fat Tissue Stratagene human Ref. RNA Isolate total RNA Reverse Transcribe to cDNA, label with Cy3 Reverse Transcribe to cDNA, label with Cy5 Mix, Hybridize and Scan Linearly Amplify RNA METHOD :

24. Four comparisons were performed: Morbidly obese (N=50) vs. Controls Diabetic obese vs. normoglycemic obese –No significant genes found Diabetic obese (N= 9) vs. Controls NormoGlycemic Obese (N=22) vs. Controls

25. DMO = Obese with diabetes; MO =Obese without diabetes; NGO = NormoGlycemic Obese All comparison were performed vs. Non-Obese Controls

26. A LIST OF MELANOGENESIS RELATED GENES DIFFERENTIALLY EXPRESSED IN OBESE ADIPOSE Genesfold differenceAccession number TYRP12.66AA668457 DCT2.17N27147 RAB27A 2.47 AI309109 RAB27B 2.6 R39044 MITF1.5N67822 SOX1.36AA976578 CHS11.22N74383 Tyrosinase itself was not present on the chip

27. Expression of TYR, TYRP1 and DCT in adipose tissue as revealed by Quantitative RealTime-PCR P < 0.007P< 0.024NS

28. Fontana-Masson stain of human adipose tissue demonstrates melanin pigment (black staining) mainly in the periphery of the adipocytes. Cryoslicing of visceral adipose Melanin detection Fontana-Masson kit