1. Signal Pathway Proteomic Analysis of Human Adipose Tissue Using Phosphoprotein Arrays: Insights into the Pathophysiology of Nonalcoholic Fatty Liver Disease (NAFLD) and Type 2 Diabetes (DM) Valerie S. Calvert 1 ; Rochelle Collantes 2 ; Hazem Elariny 2 ; Ancha Baranova 1 ; Arian Afendy 2 ; Zachary Goodman 3 ; Lance A. Liotta 1 ; Zobair M. Younossi 2 ; Emanuel F. Petricoin 1 1 George Mason University, Manassas, VA; 2 Inova Fairfax Hospital, Fairfax, VA; 3 Armed Forces Institute of Pathology, Washington, DC

2. Obesity in the U.S. Nearly one-third of U.S. adults are obese (BMI > 30).* All adults (20+ years old): 61.3 million (30.5 %) Women (20+ years old): 34.7 million (33.4 %) Men (20+ years old): 26.6 million (27.5 %) BMI Categories: Underweight = <18.5 Normal weight = 18.5-24.9 Overweight = 25-29.9 Obesity = BMI of 30 or greater * Flegal KM, Carroll MD, Ogden CL, Johnson CL. Prevalence and trends in obesity among US adults, 1999-2000. Journal of the American Medical Association. 2002;288:1723-1727

3. Non-Alcoholic Fatty Liver Disease (NAFLD) Accumulation of Fat in Liver Cells Does not Damage the Liver Common in Patients who are Obese or have Diabetes Mellitus Treatment Depends on the Cause

4. Non-Alcoholic Steatohepatitis (NASH) Inflammation of the liver associated with accumulation of fat in the liver Inflammation causes damage to the liver Typically occurs in middle-aged, overweight, and often diabetic patients who do not drink alcohol NASH can result in the development of fibrosis in up to 40% of patients or cirrhosis in 5-10% of patients No specific treatment

5. Signaling Pathways Associated with Obesity Identify new therapeutic targets Characterize disease pathophysiology of organs and organ functions

6. Reverse Phase Protein Microarrays Measure phosphorylation levels of hundreds of signaling molecules at once from human biopsy material Immobilization of cell lysate Only requires one antibody Signal amplification generated by 3rd generation enzymatic reactions (e.g. tyramide amplification) Very sensitive assay

7. Problems with Laser Capture Microdissection of Adipose Tissue Samples Unable to cut 8 um sections Little cellularity in samples Too much time required to LCM enough material for reverse-phase microarrays Mostly homogeneous cell population A - adipose cell N - nuclei of adipose cells S - loose connective tissue septa

8. Hydrostatic Pressure Cycling Single-use, disposable pulse tube with two chambers Placed in an enclosed chamber of a Barocycler Subjected to rapid cycles of high & low pressure Optimal settings for adipocyte tissue samples: Pressure = 35,000 PSI Number of cycles = 10 High pressure = 20 sec Ambient pressure = 20 sec Pressure Biosciences, Inc. The Barocycler efficiently disrupts intracellular & extra- cellular materials. Proteins are completely released. The Barocycler NEP3229 (Bench Top Model)

9. Preparation of Adipocyte Samples Per manufacturer: Extraction efficiency of Barocycler best at 150 - 300 mg of sample Each pulse tube must contain 1.3 ml to 1.5 ml, sample + lysis buffer Test array - compare to lysate of known protein concentration Used 200 mg sample (0.8 cm 2) + 1.2 ml SDS lysis buffer Samples scored for hemolysis 1 to 4+ Samples were analyzed for phospho-tyrosine content Total Protein stain Anti-Phospho-Tyrosine

10. Reverse Phase Protein Microarrays Aushon 2470 Arrayer 350 um pins 158 samples printed in duplicate on 3 sets of 100 slides http://aushon.org/ A431 control Sample lysates 4 ug 2 ug 1 ug 500 ng 250 ng

11. Array Staining - Dako Autostainer 66 Antibodies: Focusing on growth factor, insulin signaling and apotosis Pathways

12. Image Analysis MicroVigene software (VigeneTech) Local background subtraction Average replicates Normalize total protein

13. Conclusions Human adipose tissue can be effectively analyzed for cell signaling using Reverse Phase Protein Microarrays. We have developed a lysing procedure for human adipose tissue, a specimen that in the past was difficult to analyze on a molecular level. Able to analyze multiple signaling endpoints from only a few hundred nanograms of material.