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Oliveira et al. Hepatoma Res 2020;6:xx I http://dx.doi.org/10.20517/2394-5079.2020.73 Page 3 of 13

Assessment of NAFLD
The liver histology was read by an experienced hepatopathologist at each site with multiple prior
publications related to NAFLD. The presence of NAFLD was defined by the presence of hepatic steatosis
(> 5%) confirmed by a liver biopsy in all instances; the nonalcoholic nature was established by clinical
assessment of alcohol consumption to be less than 2 units daily for women and 3 units daily for men
[11]
over the last 2 years . Steatohepatitis was defined by the presence of steatosis (> 5%) along with lobular
[11]
inflammation and cytological ballooning . The severity of individual histological features of NAFLD was
[12]
scored using the NIDDK NASH CRN criteria .
Assessment and stratification of body mass index
Lean, overweight and obese states were defined according to the World Health Organization and Modified
[13]
National Cholesterol Education Program, Adult Treatment Panel III guidelines . For subjects from India,
two separate analyses were performed. In the first, the same body mass index cutoffs used in the West were
used to define these states to directly relate the clinical-laboratory-histological findings across continents
matched for BMI. A second analysis using the thresholds used for the Asian subcontinent was used in order
to evaluate those who were lean versus obese using physiologically relevant parameters for the region [14,15] .

Assessment and stratification of IR
IR was derived from fasting blood glucose and insulin. The Homeostatic Model of Assessment for IR
[16]
(HOMA-IR) was calculated from the formula: [22.5 × fasting insulin (mU/mL) × glucose (mmol/L)] .

To further visualize the relationship of glucose and insulin, the plasma insulin was plotted as a function
of fasting plasma glucose. The resulting plot was divided in to four quadrants based on a fasting glucose
threshold of 100 mg/dL and fasting insulin of 12 μU/mL. A fasting glucose below 100 mg/dL represents
euglycemia and a fasting insulin level above 12 μU/mL has been associated with IR [17,18] .

Subjects with a glucose < 100 mg/dL and a fasting insulin < 12 μU/mL were considered insulin sensitive.
Those with a glucose < 100 mg/dL but insulin levels > 12 μU/mL were considered to have IR with relatively
intact beta cell function. Those with higher plasma glucose (> 100 mg/dL) and insulin > 12 μU/mL were
considered to have severe IR. When the fasting plasma glucose levels were higher than 100 mg/dL and the
corresponding insulin levels declined below 12 μU/mL subjects were considered to have advanced IR with
beta cell failure. Type 2 diabetes was defined by a fasting blood glucose > 126 mg/dL in patients who had a
[19]
hemoglobin A1c level of 6.5% or greater, an FPG level of 126 mg/dL or greater .

Plan of analysis
Data were analyzed using SPSS version 2. Descriptive statistics of subjects from each site was performed.
Data for lean versus overweight versus obese subjects were compared for each country individually.
Numerical data were compared using analysis of variance for normally distributed data. Quantitative
variables with asymmetric distribution were described as median and interquartile range and compared
between groups using the Kruskal-Wallis test. Next, subjects within each BMI strata from each country
were compared to other countries correcting for multiple comparisons using Tukey’s test. A similar analysis
of overweight or obese subjects was also performed. In another analysis, the cohort was stratified by the
HOMA scores of < 2, 2-4 and > 4 and a similar analysis performed. Finally, a multivariable regression
analysis was performed to evaluate the interactions between BMI, IR and other clinical parameters on liver
histology in lean subjects with NAFLD in each region. Significance was set at a P value of 0.05.

RESULTS
A total of 430 subjects with biopsy-proven NAFLD was enrolled (USA:Brasil:France:India 139:125:96:63)