Study has been conducted in tertiary level medical institute of northern India, King George’s Medical University, Lucknow, India. The study was a randomized trial with nested control of 24 weeks. The study was approved by Institutional Ethics committee (Ref. Code No. 66 ECM II-B/P4). Written informed consent was obtained from all the participants prior to enrollment. Patient attending medicine OPD or those admitted in indoor medical wards and having fatty liver on Abdominal Ultrasonography were enrolled over a period of one year (August 2013 to July 2014) after screening for the predesigned inclusion and exclusion criteria. Total of 98 subjects who consented for the study were subsequently enrolled. The subjects were re-evaluated with USG abdomen to calculate USG score for fatty liver by a competent radiologist who was blinded for the study. Subjects with history of significant alcohol consumption i.e., more than 20gm/day of alcohol consumption (as per AUDIT-C1 Questionnaire devised by WHO) were excluded. Subjects with acute liver failure (i.e., liver enzymes > 3 times the upper limit of normal) and chronic liver disease, cardiac failure, renal failure, clinical suspicion of autoimmune hepatitis, positive for hepatitis B and hepatitis C viral markers, macular oedema on fundoscopy, previously on insulin sensitizers and hypolipidemic drugs, pregnant females, prior history of drug reactions with any of the drugs under study, less than 18 years of age and subjects who did not consented for the study were excluded.

Study subjects were randomly allocated to all the three Groups and administered Metformin (Group I), Rosuvastatin (Group II) and Pioglitazone (Group III) with lifestyle modification and dietary intervention being common to all. All the three Groups were advised dietary intervention and lifestyle modification by a trained dietician as per American Association of the Study of Liver Disease guidelines. All the cases were advised low calorie, low fat along with high fibre diet.

Sonography was used for the diagnosis of fatty liver. A score has been devised by Gore R. M., for grading hepatic steatosis on the basis of sonographic findings [15]. This included four sonographic findings of diffuse fatty change in the liver, two for high score viz. Loss of definition of the diaphragm, liver echogenicity exceeding that of renal cortex, poor delineation of the intrahepatic architecture, attenuation of the ultrasound wave with each finding assigned a score of one. A score of two or more was used as working definition of fatty liver. All USG studies were done at the Department of Radiology using mid-range colour Doppler and ultrasonography, manufactured by TOSHIBA (model XAIRO-660A) with 3.5 MHz and 10 MHz transducer.

All study participants were interviewed and examined in detail during the study period to collect baseline socio-demographic details. The patients were followed at 4 weeks, 12 weeks and 24 weeks and checked for adherence to dietary intervention and lifestyle modification along with assessment of weight, BMI, compliance to drugs and development of any side effects. Also all the Groups were evaluated at 12 weeks and 24 weeks for liver function tests, fasting lipid profile, USG scores for NAFLD. The statistical analysis was done using SPSS (Statistical Package for Social Sciences) Version 15.0 Statistical Analysis Software. A p-value of <0.05 was considered significant for the study. At the end of the study weight, BMI, liver enzymes, and lipid profile at different Follow-up periods was compared using ANOVA test. InterGroup comparison of USG scores at different Follow-up periods was done using Kruskal Wallis test. Also, Wilcoxon Ranked test was then applied to assess the change in USG scores at different Follow-ups during the study.

InterGroup difference in weight and BMI was not found to be statistically significant at any of the Follow-up except of BMI at 12 weeks and 24 weeks which was found to be significantly higher in Group III than Group I and Group II (p<0.05). Group II showed an increasing trend of liver enzymes while Group I and Group III showed a decreasing trend of liver enzymes. InterGroup difference in SGOT levels were found to be statistically significant at 1^st visit (p<0.001) and at 24 weeks (p=0.012). Difference in SGPT levels were found to be significant only at the time of first visit (p<0.001) and was not found to be statistically significant at 12 weeks and at 24 weeks [Table/Fig-1].

In our study the significant change in baseline characteristics during Follow-up [Table/Fig-2] was observed. InterGroup difference in cholesterol levels was statistically insignificant at first week and 12 weeks (p=0.065) while at 24 weeks (p=0.162) differences was found to be statistically significant (p<0.001). InterGroup difference in triglyceride levels were found to be statistically significant at 1^st visit (p<0.001) and at 24 weeks (p<0.001) while difference was not found to be statistically significant at 12 weeks (p=0.560). InterGroup difference in VLDL levels was not statistically significant at 1^st visit (p=0.561) and at 12 weeks (p=0.432) while difference was found to be statistically significant at 24 weeks (p=0.008). InterGroup difference in LDL levels were not statistically significant at 1^st visit (p=0.736) while difference was found to be statistically significant at 12 weeks (p=0.005) and 24 weeks (p<0.001). InterGroup difference in HDL levels was statistically insignificant at subsequent Follow-up visits.

In Group I, II and III decline in cholesterol values from that at 1^st Visit was observed at all-time intervals and these changes were found to be statistically significant. In both the study Groups, decline in VLDL values from that at 1^st Visit was observed at all-time intervals and these changes were found to be statistically significant except change in Group II between Visit I and 12 weeks. Maximum decline was found between 1^st Visit and 24 weeks and minimum decline between Week 12 and Week 24. In both the above study Groups, an increase in HDL values from that at 1^st Visit was observed at all-time intervals and these changes were found to be statistically significant. Maximum increase was found between 1^st Visit and 24 weeks in both the Groups, while minimum increase between 1^st Visit and 12 Weeks.

[Table/Fig-3,4] shows baseline USG score in all Groups and changes occurred during Follow-up in all three Groups. In Group I, USG scores showed an increase in later half of the study though the increase was not found to be statistically significant. In Group II, USG score declined with time and this change was found to be statistically significant between all the time intervals except for change in USG grade between 1^st Visit and 12 weeks. In Group III, USG score declined with time and this change in USG grade was found to be statistically significant as compared to the USG scores at the start of the study.

In our study out of 98 subjects, 30% were females and 70% were males. Many recent studies have reported that male gender was a risk factor for fatty liver disease [16]. Maruti et al., conducted a study on 26,527 subjects undergoing medical checkups, and found that the prevalence of NAFLD was 31% in men and 16% in women [17]. Group I (Metformin) and Group II (Rosuvastatin) showed a decline in weight during the course of the study as was attributable to the dietary intervention and life style modification done in all the three Groups. But Group III (Pioglitazone) showed an initial decline in weight followed by a later increase in weight. The increase in weight was significant at 12 weeks and 24 weeks as compared to previous visits. This increase in weight was most probably due to Pioglitazone as it is known to cause weight gain via Peroxisome Proliferator Accelerated Receptor (PPAR) mediated sodium and water retention. BMI also followed trend similar to that of weight in all the three Groups. Studies also showed an increase in abdominal fat content in patients on Pioglitazone. Belfort et al., studied the effect of Pioglitazone (45 mg/day) in a Randomized Controlled Trial (RCT) in subjects with NASH having impaired glucose tolerance or T2DM [18]. Subjects in the study showed a significant weight gain (2.5 ± 0.5 kg) with Pioglitazone, but it also showed significant improvement in aminotransferase levels, steatosis, hepatocellular ballooning, and inflammation. Shadid and Jansen, in 2003 also reported gain in weight with the use of Pioglitazone [19].

We have also studied the effect of the individual drugs on the natural history of hepatic steatosis and for this we used the USG scoring as described before. Initially there was no statistically significant difference among the three Groups for baseline USG score. When followed up at 12 weeks and 24 weeks, Group II and Group III showed a declining trend in the mean USG scores for NAFLD at 12 weeks as well as 24 weeks. The favourable results in Group II and Group III suggest that apart from dietary modification and lifestyle intervention advised to both these Groups of the study, Rosuvastatin and Pioglitazone appears to have beneficial effects in reversing the steatosis in patients of NAFLD. USG scores for NAFLD showed an early stationary trend followed by an insignificant increase in USG scores during the later course of the study. This increase could be due to eight subjects in Group I developing a rapid loss of weight during the initial 12 weeks of the study period which in turn could have precipitated hepatic steatosis in these subjects. This was also corroborated by the increase in the USG scores in the same subjects during the latter part of the study.

Baseline liver enzymes were comparable when the study started. Group I and Group III showed decline in SGOT levels and SGPT levels whereas, in Group II the liver enzymes showed progressive increase from 1^st visit towards the end of the study. Though, there was an increase in liver enzymes in Group I, none of the subjects had shown a rise of more than three times of the upper limit of normal which would have been suggestive of inflammatory liver injury. Previous studies have shown that statins cause hepatocellular inflammation and were associated with derangement of liver enzymes [20]. Therefore, it is advisable to monitor for liver enzymes while treating patients of NAFLD with statins and to avoid them in patients with liver enzymes of more than three times of the baseline. The improvement in lipid profile was significant in all the three Groups at both the Follow-up visits (at 12 weeks and 24 weeks). This was much better in Group II as compared to Group I and Group III. As compared to Group I and Group III the decline in serum cholesterol was greater in Group II. Triglycerides also showed a declining trend towards the end of the study period in all the three Groups. Serum VLDL showed a significant decline in all the three Groups during the course of the study. All the three Groups also showed an increase in serum HDL levels. This robust improvement across the fractions lipid profile parameters in all three Groups could be attributed to the well validated effects of change in dietary habits and lifestyle modifications. Our findings were supported by a previous study which reported that there was improved metabolic response in the form of decrease in serum triglyceride levels after 16 weeks of calorie-restricted low-glycaemic index diet and Metformin in subjects with impaired glucose tolerance [21]. A similar response was reported with Pioglitazone with respect to serum triglyceride levels [16]. It is previously reported that Rosuvastatin administration was associated with improvements in lipid profiles especially decrease in serum cholesterol levels [17]. The decline in serum VLDL as observed in our study is further supported by a previous study using Metformin and Pioglitazone [22,23]. The role of Rosuvastatin on serum HDL levels is still inconclusive as various studies have shown conflicting results. One of the study reported that Rosuvastatin administration was associated with improvement in serum HDL levels [24], while in other study Deguchi et al., 2014 observed a significant decline in serum HDL levels in dyslipidemic subjects with cerebral infarction when treated with rosuvastatin [25].

Thus it appears that Rosuvastatin and Pioglitazone could have better effects in reversing the changes of NAFLD as compared to Metformin when used as add on therapy over dietary intervention and lifestyle modification. As Pioglitazone being already incor-porated in the treatment guidelines by AASLD, Rosuvastatin as well may be useful as a pharmacotherapy for treating patients with NAFLD. Rosuvastatin use for prolonged periods may lead to increase in liver enzymes but acute decompensation is unlikely. Pioglitazone when used for prolonged periods may lead to weight gain and increase in BMI. Rapid weight loss in short period of time may lead to worsening of hepatic steatosis. Dietary and life style intervention has favourable effect on hepatic steatosis as observed by USG scores. A further aspect of clinical research from the above study is that the improvement in USG scores for NAFLD by Rosuvastatin used in the study is a molecule effect of Rosuvastatin per se or is the effect of class of drug.