Patients and methods
Sixty patients diagnosed as NAFLD attending Liver clinic were included in the study. Diagnosis of NAFLD was based on the presence of fatty liver on ultrasonography and an elevated serum alanine aminotransferase (ALT) > 1.5 times the upper limit of normal for a minimum period of 3 mo. Patients with other causes of chronic hepatitis including viral hepatitis, autoimmune hepatitis, cholestatic liver disease, hemochromotosis, Wilson’s disease and alcoholic liver disease (alcohol use > 20 g/d) were excluded from study.
This study was carried out in accordance with the principles of the Helsinki declaration and was formally approved by institutional ethical committee. All patients gave a written, informed consent before participation in the study.
Clinical and anthropometric data
Detailed history including use of drugs, particularly oral contraceptives, corticosteroids and antituberculosis, antidiabetics, insulin sensitizers was obtained and clinical examination to look for any evidence of chronic liver disease was done at initial screening. Bodyweight was measured using self-zeroing weight scale with light clothing without shoes to the nearest half-kilogram. Height was measured to the nearest 2 mm with patient standing on bare feet closely apposed to each other and against the wall with patient looking straight. Body mass index (BMI) was calculated as follows: body weight (kg/m2
]. Waist circumference (WC) in centimeters was measured at a level midway between the lower rib margin and iliac crest and hip circumference at the widest portion of buttocks. Waist-hip ratio (WHR) was calculated by dividing waist circumference by hip circumference. Increased WHR was defined as ≥ 0.90 in men and ≥ 0.85 in women[19
Metabolic syndrome was defined according to National Cholesterol Education Program (NCEP) adult treatment panel III (ATP III) guidelines as the presence of 3 or more of the following 5 risk factors: (1) Waist circumference > 102 cm (men) and > 88 cm (women); (2) Fasting triglycerides ≥ 150 mg/dL; (3) High density lipoprotein cholesterol < 40 mg/dL (men) and < 50 mg/dL (women); (4) blood pressure ≥ 130/≥ 85 mmHg; (5) fasting glucose > 110 mg/dL[20
]. The cut off for normal waist circumference in adult Indians has been found to be lower than Caucasians; hence we used a cut off of > 90 cm (men) and > 80 cm (women) in our study[21
Overnight fasting blood sample were obtained for measurement of plasma glucose and serum lipids. Plasma glucose 2 h after 50 gm of glucose load was also done in all patients. Plasma glucose was measured with an automated analyzer using glucose oxidase and peroxidase method. Fasting lipid profile for total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, very low-density lipoprotein and triglycerides was obtained (RA-XT random access clinical chemistry analyzer, Bayer Diagnostics, Tarrytown, NY, USA). Serum iron studies, albumin, aspartate aminotransferase (AST) and ALT were done on all patients at baseline.
To exclude hepatitis B and hepatitis C, HBsAg (Hepanostika, Biomerieux bv, Boxtel, NL, US) and anti-hepatitis C virus (UBI, United Biochemicals Inc, Houppauge, NY, US) and to exclude autoimmune hepatitis, anti nuclear antibody, anti smooth muscle antibody, anti mitochondrial antibody and anti liver kidney microsomal antibody were done in all patients. Patients less than 40 years of age also underwent slit lamp examination to rule out Wilson’s disease.
Fasting samples of serum obtained after centrifugation were stored at -70 °C until assayed. Fasting insulin levels (mU/L) were measured using radioimmunoassay (Diagnostic Products Corporation, Los Angeles, CA). The hinsulin resistance was calculated on the basis of fasting values of plasma glucose and insulin according to homeostasis model assessment (HOMA) of insulin resistance (HOMA-IR) model formula,
HOMA-IR = fasting insulin (mU/L) × fasting glucose (mmol/L) ÷ 22.5[22
As previously recommended, insulin resistance was arbitrarily considered altered when it was > 2[23
Ultrasonography of liver was performed in all participants in fasting state by trained radiologist blinded for laboratory and anthropometric data. Brightness and posterior attenuation were considered indices of the extent of fatty infiltration. The diagnosis of fatty liver was made based on findings of echogenicity: graded as: grade 0: normal echogenicity; grade 1: slight, diffuse increase in fine echoes in liver parenchyma with normal visualization of diaphragm and intrahepatic vessel borders; grade 2: moderate, diffuse increase in fine echoes with slightly impaired visualization of intrahepatic vessels and diaphragm; and grade 3: marked increase in fine echoes with poor or non-visualization of the intrahepatic vessel borders, diaphragm, and posterior right lobe of the liver[24
In patients who gave consent, liver biopsy was done as indoor procedure using Menghini’s needle. All biopsies were reported by one pathologist blinded for clinical data using the Brunt’s scoring system for non-alcoholic steatohepatitis[25
]. Steatosis was graded according to percentage of cells with fatty droplets (grade I, 0%-33%; grade II, 34%-66%; grade III > 66%). Necroinflammation was graded 0-3 (0, absent; 1, occasional ballooning and no or minimal inflammation; 2, ballooning of hepatocytes with mild to moderate portal inflammation; 3, intra-acinar inflammation and portal inflammation). Fibrosis was graded as 0-4: (0, no fibrosis; 1, perisinusoidal/ pericellular fibrosis; 2, periportal fibrosis; 3, bridging fibrosis; 4, cirrhosis.
All patients were counseled to an aerobic exercise regimen. Moderately energy-restricted diet was advised by a qualified dietician to those with high BMI (> 25 kg/m2).
Exercise program and diet regimen
All patients were given training by professionally qualified physical instructor from “Health Zone” Lucknow about exercise programme at beginning and at regular interval. During workshop two physical trainers assessed and individualized the exercise program for each patient, and supervised their performance during workshops and were trained to achieve target heart rate. The exercise program included brisk walking; jogging or rhythmic aerobic exercises set to beat music, for a minimum period of 45 min/d, for at least 5 d/wk. They were counseled to achieve approximately 70% of their maximal heart rate for minimum period of 20 min. Maximum heart rate was estimated from the formula 220 - age (years) with a standard deviation of 10-12 beats/min[26
]. All patients were asked to maintain records about exercise programme and MHR in the provided Performa to assess their compliance. Those patients who exercised on less than 4 d/wk (16 d/mo) were considered as exercise non-compliant.
All patients received standardized nutritional counseling by registered Dietician, who supervised them regularly. Moderate energy restricted diet containing 60% carbohydrate, 20% fat, 20% protein and 200 mg cholesterol (National cholesterol education program step I diet, (25 kcal/kg ideal body weight) was advised to patients with high BMI[20
]. Ideal body weights were calculated in kg using the formula: Ht (cm) - 100 × 0.9[27
All patients were followed up monthly by measuring anthropometric data and laboratory assessment of serum albumin, ALT and AST levels. Fasting insulin levels were taken at baseline and after 6 mo of exercise program. Care was taken so that none of them took any other pharmacological intervention except for coexisting condition like hypertension.
Statistical analysis was performed using SPSS 10.0.1 software (SPSS Inc., Chicago, IL, US). All data was expressed as mean ± SD. Baseline parameters were compared between groups using Mann-Whitney U test and for comparing the variables before and after therapy, Wilcoxon signed ranks test was used. The degree of association between the decline in insulin resistance, ALT and liver histology; before and after exercise was done using Spearman’s correlation coefficient (rs). A significance level of P < 0.05 was considered as statistical significance.
Seventy-five patients were initially screened for study of which 60 agreed to participate. Forty five of 60 complied with the exercise program (compliant group) while 15 did not (non-compliant group). Majority of our patients had non-specific minor symptoms like dyspepsia, right upper quadrant heaviness or had been referred for incidental detection of fatty liver on ultrasonography(USG) and elevated transaminases on routine blood tests.
Majority of study population were male (70%, 42/70) with mean age being 40.0 ± 8.9 years. While 70% (42/60) patients were overweight, 95% (57/60) had central obesity (WC ≥ 0.90 in men and ≥ 0.80 in women). Two of our patient had essential hypertension and none of our patients were diabetic. All of our patients were nonalcoholic and had negative viral markers. On USG examination 19 (32%) patients had grade 1, 30 (50%) had grade 2 and 11 (18%) had grade 3 fatty liver.
Thirty-two (53%) patients gave consent for liver biopsy and findings were given in Table . All liver biopsies showed fatty infiltration predominantly macro vesicular. Grade 1 fatty infiltration was present in 12 patients, while grade 2 in 15 and grade 3 in 5. Ballooning degeneration was present in 8 (25%) and glycogenated nuclei in 11 (34.3%). Majority of the biopsies had mild to moderate necroinflammatory activity (grade 1 in 10, grade 2 in 17). Only 5 biopsies showed severe (grade 3) necroinflammatory activity. None of the biopsies showed cirrhotic changes, while grade 1 fibrosis was seen in 14 patients, grade 2 in 4 and grade 3 in 3 patients. Eleven patients had no fibrosis on liver biopsy.
Histological characteristics of nonalcoholic steatohepatitis on liver biopsy
Insulin resistance and metabolic syndrome
Mean insulin resistance level in our patients was 7.1 ± 5.1. Fifty-six (93%) patients had HOMA-IR > 2. The details of the presence of the components of metabolic syndrome according World Health Organization (WHO) criteria and according to modified Indian criteria are given in Table . 17% (10) patients had metabolic syndrome according to WHO criteria, but when the modified Indian criterion for WC was used 37% (22) fulfilled the criteria. Only one patient in our study population did not have any component of metabolic syndrome when Indian criteria were used. The most common feature observed was high WC (modified) in 57 (95%) followed by low HDL in 38 (63%).
Components of metabolic syndrome seen in our patients n = 60 (%) according to National Cholesterol Education Program adult treatment panel III and waist modified by Indian criteria
Baseline comparison of compliant and non-compliant groups
Exercise compliant and non-compliant groups had no significant difference in baseline characteristics as shown in Table . Demographic profile, anthropometric parameters and baseline biochemical results were not different in two groups.
Baseline demographic, anthropometric and biochemical characteristics of patients
Effect of exercise on BMI, WC, aminotransferases
Comparison of anthropometric measurements before after 6 mo of intervention is given in Table . Compliant group showed significant decrease in BMI [26.7 ± 3.3 kg/m2
vs 25.0 ± 3.3 kg/m2 (P < 0.001)] and WC [95.7 ± 8.9 cm to 90.8 ± 7.3 cm (P < 0. 001)]. ALT also showed significant improvement from 84.8 ± 43.5 U/L to 41.3± 18.2 U/L, (P < 0.001) respectively.
Sub-group analysis of the compliant group - exercise alone and both exercise and diet modification and comparison with non-compliant group
Among compliant group, there was 2.9 kg mean weight loss in patients who were advised both exercise and dietary restriction, while 0.3 kg decrease in weight was seen in patients who were advised only exercise (normal BMI group). Noncompliant group had increase of 0.4kg weight at the end of 6 mo. Patients who were advised exercise only showed no significant change in BMI [23.0 ± 1.4 kg/m2
vs 21.7 ± 1.6 kg/m2; P = not significant (NS)], but they had significant improvement in waist circumference (88.0 ± 7.0 cm vs 85.4 ± 7.2 cm, P = 0.001). Patients who were advised exercise and diet restriction (high BMI) showed significant improvement in BMI, waist circumference (28.5 ± 2.4 kg/m2
vs 26.6 ± 2.7 kg/m2, P < 0.001 and 99.5 ± 7.2 cm vs 93.6 ± 5.5 cm, P < 0.001, respectively).
Effect of exercise on insulin resistance and correlation with ALT
Insulin resistance showed a significant decline at the end of exercise program in the compliant group. Insulin resistance decreased significantly in both combined group and exercise alone group (6.5 ± 4.8 to 1.4 ± 1.1 and 6.2 ± 4.6 to 1.2 ± 0.8 respectively) (Figure ). In the non-compliant group there was no significant change in insulin resistance level before and after exercise (Table ). Using Spearman’s correlation, the decline in ALT correlated with decline in insulin resistance levels in the compliant group (P = 0.01, rs = 0.903), but there was no correlation in noncompliant group (P = NS), rs = 0.321), (Figure ).
Diagrammatic representation of homeostasis model assessment-estimated insulin resistance levels of all the patients in the 3 groups before and after exercise. HOMA-IR: Homeostasis model assessment-estimated insulin resistance.
Correlation of decline in homeostasis model assessment-estimated insulin resistance levels to serum alanine aminotransferase in the compliant and non-compliant group using Spearman’s test. NS: Not significant.
At the end of follow-up, 24 compliant patients had normal ALT levels while in none of the noncompliant patients ALT normalized.
Effect of exercise on liver histology and correlation with insulin resistance and anthropometry
Eight patients had repeat liver biopsy in compliant group, of which six showed improvement in steatosis, necroinflammatroy score with no change in fibrosis score and two had no change of the NASH score (Table and Figures and ). Total NASH score in eight patients decreased from 5.3 ± 1.5 to 3.3 ± 1.5 (P = 0.02).
Anthropometric, biochemical and histological characteristics of patients with paired liver biopsy (n = 8)
Change in nonalcoholic steatohepatitis score pre and post intervention (n = 8).
Liver histology pre (A) and post (B) intervention in compliant group.
Using spearman’s correlation decline in HOMA-IR correlated with decline in NASH score in these 8 patient with repeat histology (P = 0.03, rs = 0.73). Improvement in NASH score also correlated with decline in WC (P = 0.04, rs = 0.65), BMI (P = 0.05, rs = 0.62) and ALT (P = 0.05, rs = 0.54) (Table .)
Correlation between improvement in nonalcoholic steatohepatitis score and anthropometry, biochemical characteristics