Supplemental Methods Analytical determinations ALT and aspartate aminotransferase (AST) levels were measured using the α-ketoglutarate reaction (Roche, Basel, Switzerland). Glucose, triglyceride, total and high density lipoprotein (HDL) cholesterol concentrations were determined by enzymatic methods (Roche, Basel, Switzerland). The erythrocyte sedimentation rate (ESR) was measured automatically by the stopped-flow technique in a capillary microphotometer (Alifax Test 1 System). Plasma fibrinogen concentration and platelet count were determined by routine laboratory tests using Sysmex CA-7000 Automated Coagulation Analyzer, and Sysmex SE9500 (DASIT SpA, Milan, Italy), respectively. High sensitivity CRP levels were measured by automated instrument (CardioPhase hscrp, Milan, Italy). Albumin concentration was determined with a Alb2 kit on a Cobas C6000 analyzer (Roche Diagnostics, Milan, Italy). All others metabolites were measured by standard methods Real-Time RT-PCR Liver samples were stored at-80 C and distrupted/homogenized using a TissueRuptor apparatus (Qiagen, Milan, Italy) immediately before proceeding to nucleic acid extraction. Total RNA was extracted from treated and control HepG2 cells and from frozen liver samples using AllPrep DNA/RNA Micro Kit (Qiagen, Milan, Italy), reverse transcribed and analyzed by quantitative Real-Time PCR technique using commercial Assays-on-Demand kits (all Applied Biosystems, Foster City, CA) according to the manufacturer s protocol and normalized to housekeeping 18S gene. All reactions were performed in duplicate. Relative gene expression was calculated by using the comparative CT method. Definitions Type 2 diabetes was diagnosed according to the American Diabetes Association (ADA) criteria (1). The metabolic syndrome was defined according to the criteria of the consensus statement released in 2009 (2). Using this definition, a subject has metabolic syndrome if he or she meets three or more of the following criteria: 1) waist circumference >102 cm in men and >88 cm in women), 2) triglyceride >150 mg/dl, 3) HDL <40 mg/dl in men and <50 mg/dl in women, 4) blood pressure >130/85 mmhg, and 5) fasting glucose >100 mg/dl. The diagnosis of NAFLD was based on chronically elevated Alanine aminotransferase (ALT) for at least 6 months, alcohol consumption of <20 g/day in the year before liver biopsy, and evaluated by a
questionnaire, steatosis (>5% of hepatocytes) at histology with or without necroinflammation and/or fibrosis, and absence of other causes of liver disease or mixed etiologies (excessive alcohol consumption, regular use of steatosis-inducing drugs, hepatitis C, hepatitis B, autoimmune liver disease, Wilson s disease, hemochromatosis, α1-antitrypsin deficiency). The NAFLD fibrosis score was calculated according to the following formula: -1.675 + 0.0373 x age (years) + 0.0943 x body mass index (BMI kg/m 2 ) +1.13 x impaired fasting glycemia or diabetes (yes=1, no= 0) + 0.99 x AST/ALT ratio -0.013 x platelet (x10 9 /l - 0.66 x albumin (g/dl) (3). Two cutoff points (>0.676 and <-1.455) were used to divide the subjects in three groups: low risk of fibrosis (< -1.455), intermediate risk of fibrosis (-1.455-0.676), and high risk of fibrosis (>0.676), as described by Angulo and colleagues (3). The Kleiner classification (4) was used to compute the NAFLD activity score (from 0 to 8, on a scale including separate scores for steatosis, lobular inflammation, and hepatocellular ballooning) and to stage fibrosis from 0 to 4 in subjects with biopsies. References 1. American Diabetes Association. 2011 Standards of medical care in diabetes-2011. Diabetes Care 34 Suppl 1:S11-61 2. Alberti KG, Eckel RH, Grundy SM, Zimmet PZ, Cleeman JI, Donato KA, Fruchart JC, James WP, Loria CM, Smith SC, Jr. 2009 Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity. Circulation 120:1640-1645 3. Angulo P, Hui JM, Marchesini G, Bugianesi E, George J, Farrell GC, Enders F, Saksena S, Burt AD, Bida JP, Lindor K, Sanderson SO, Lenzi M, Adams LA, Kench J, Therneau TM, Day CP 2007 The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD. Hepatology 45:846-854 4. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, Unalp-Arida A, Yeh M, McCullough AJ, Sanyal AJ 2005 Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41:1313-1321
Variables Supplementary Table 1 Anthropometric and biochemical characteristics of Cohort 1 Whole study subjects Low risk of fibrosis (< -1.455) Intermediate risk of fibrosis (-1.455-0.676) High risk of fibrosis (>0.676) P P** P*** Gender (Male/Female) 122/99 39/36 71/47 12/16 0.20 --- --- Age (yrs) 61.3±9.2 57.0±7.2 61.7±8.8 c 68.9±8.6 c <0.0001* <0.0001* <0.0001* BMI (kg/m 2 ) 31.2±5.8 30.1±4.9 31.6±5.1 c 35.6±8.1 c <0.0001 <0.0001 <0.0001 Waist circumference (cm) 105±13 101±12 106±11 c 113±17 c <0.0001 <0.0001 <0.0001 Fasting Glucose (mgl/dl) 119±48 98±23 125±47 c 153±71 c <0.0001 0.03 0.02 Total cholesterol (mg/dl) 197±42 206±41 196±42 180±43 0.03 0.14 0.26 HDL (mg/dl) 48±14 49±15 47±13 44±10 a 0.04 0.24 0.18 Triglycerides (mg/dl) 154±69 148±67 153±69 151±14 0.83 0.51 0.99 ALT (UI/l) 27±15 27±12 27±15 27±14 0.87 0.81 0.71 AST (UI/l) 25±14 22±7 23±10 31±18 b 0.007 0.008 0.008 AST/ALT ratio 0.99±0.56 0.85±0.25 0.95±0.29 1.59±1.27 c <0.0001 <0.0001 <0.0001 Platelet count (x10 9 /l) 237±64 275±61 228±55 c 174±44 c <0.0001 <0.0001 <0.0001 Fibrinogen (mg/dl) 332±76 311±62 338±81 a 363±72 a 0.01 0.05 0.05 hscrp (mg/l) 4.0±3.5 2.9±1.9 4.4±3.8 b 5.2±4.3 b 0.008 0.02 0.04 ESR (mm/h) 12±10 9±7 12±9 a 19±13 c 0.001 0.009 0.008 Albumin (g/dl) 4.43±0.31 4.52±0.27 4.44±0.30 4.12±0.28 c <0.0001 <0.0001 <0.0001 IGF-1 (ng/ml) 135±54 150±54 131±53 a 116±53 a 0.03 0.04 0.01 Metabolic syndrome - yes/no (% 162/59 (73.3%) 42/33 (56%) 94/24 (79.7%) 26/2 (92.9%) <0.0001 --- ---
NFG/IFG/T2DM (n) 91/40/90 55/8/12 33/25/60 3/7/18 <0.0001 --- --- Oral antidiabetic agents/insulin (n) 45/17 5/1 33/9 7/7 0.001 --- --- Therapy with statins yes/no (%68/153 (30.8%) 18/57 (24%) 37/81 (31.4%) 13/15 (46.4%) 0.09 --- --- Data are means ± SD. Categorical variables were compared by χ 2 test. P values refer to results after analyses with adjustment for age, and gender. The risk groups were established as described by Angulo and colleagues (3, Supplemental data ).*P values refer to results after analysis with adjustment for gender. **P values refer to results after analyses with adjustment for age, gender, metabolic syndrome, glucose lowering treatment and statin therapy. ***P values refer to results after analyses with adjustment for age, gender, type 2 diabetes, glucose lowering treatment and statin therapy. ESR = erythrocyte sedimentation rate; ALT =Alanine aminotransferase AST= aspartate aminotransferase NFG=normal fasting glucose; IFG= impaired fasting glucose; T2DM2= type 2 diabetes. a P<0.05 vs. Low risk of fibrosis group.; b P<0.01 vs. Low risk of fibrosis group.; c P<0.0001 vs. Low risk of fibrosis group.
Supplementary Table 2 Anthropometric and biochemical characteristics of Cohort 2 Variables Age (yrs) 48.8±11.48 BMI (kg/m 2 ) 29.36±3.9 Fasting Glucose (mgl/dl) 101.5±27 Total cholesterol (mg/dl) 218±50 HDL (mg/dl) 53±24 Triglycerides (mg/dl) 153±71 ALT (UI/l) 72±44 AST (UI/l) 48±40 GGT 89±107 Data are means ± SD. ALT =Alanine aminotransferase AST= aspartate aminotransferase; GGT=gamma glutamyl-transferase