Seko et al 2019 Hepatology Research

bs_bs_banner Hepatology Research 2019 doi: 10.1111/hepr.13321 Original Article Clinical and pathological features of sarcopenia-related indices in patients with non-alcoholic fatty liver disease Yuya Seko, Naoki Mizuno, Shinya Okishio, Aya Takahashi, Seita Kataoka, Keiichiroh Okuda, Mitsuhiro Furuta, Masashi Takemura, Hiroyoshi Taketani, Atsushi Umemura, Taichiro Nishikawa, Kanji Yamaguchi, Michihisa Moriguchi and Yoshito Itoh Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto Background: Sarcopenia is diagnosed with the skeletal muscle index (SMI) or the sarcopenia index (SI). We previously reported that the ratio of skeletal muscle mass to body fat mass (SF ratio) was a novel index of sarcopenia in patients with nonalcoholic fatty liver disease (NAFLD). The aim of this retrospective study was to evaluate sarcopenia with these indices in patients with NAFLD. Methods: One hundred and fifty-six consecutive patients with hepatic fibrosis stage <2, the SI and the SF ratio were significantly greater than in patients with fibrosis stage ≥2. There was no difference in SMI between groups. In the cohort assessed at baseline and 12 months later, transaminase activity and SMI decreased significantly, and the SF ratio increased over time. A multivariate analysis revealed the presence of the PNPLA3 G allele and an increase in SF ratio (odds ratio, 7.406) as predictive factors of ALT reduction >30% from baseline. biopsy-proven NAFLD and alanine aminotransferase (ALT) >40 IU/L were enrolled. Liver function and body composition were evaluated in 121 patients after 12 months. We evaluated the relationship between histological findings, changes in liver function, and the SMI, SI, and SF ratio. Conclusions: Due to the high prevalence of obesity, we should consider both skeletal muscle mass and body fat mass in the diagnosis and treatment of NAFLD. The SF ratio could be a useful index in sarcopenic NAFLD. Results: Of the 156 patients enrolled, 13.5% and 26.3% were di- Key words: NAFLD, PNPLA3, sarcopenia, SF ratio, SI agnosed with sarcopenia with the SMI and SI. In patients with INTRODUCTION S ARCOPENIA IS NOW a common problem not only for elderly people, but also for people with diseases such as renal disease, inflammatory disease, malignant tumors and chronic liver disease, including non-alcoholic fatty liver disease (NAFLD). Sarcopenia, characterized by a loss of skeletal muscle mass, muscle strength, and function, affects clinical outcomes, including quality of life, infection rate, and survival in patients with cirrhosis.1–3 In European populations, approximately 0.5–1.0% of skeletal muscle mass is lost per year after the age of Correspondence: Dr Yuya Seko, Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan. Email: yuyaseko@koto.kpu-m.ac.jp Conflict of interest: The authors have no conflict of interest. Financial support: None declared. Received 19 December 2018; revision 17 January 2019; accepted 29 January 2019. © 2019 The Japan Society of Hepatology 30 years, and up to 60% of patients with end-stage liver disease had sarcopenia.4 Non-alcoholic fatty liver disease is currently the most common liver disease worldwide, affecting an estimated 25% of the adult population,5 and is the most common indication for liver transplantation.6 It is a disease covering a wide spectrum, ranging from nonalcoholic fatty liver (NAFL), which is usually a benign condition, to non-alcoholic steatohepatitis (NASH), which can sometimes lead to liver cirrhosis or hepatocellular carcinoma without significant alcohol consumption.7,8 The prevalence of NAFLD is increasing rapidly in Asian countries because of westernized dietary patterns and a lack of exercise. Recently, sarcopenia has become known as a novel risk factor for the development of NAFLD.9 A clear mechanism underlying the relationship between NAFLD and sarcopenia remains unknown, but insulin resistance (IR) and systemic inflammation could play a key role. Insulin resistance facilitates lipolysis and leads to free fatty acid (FFA) flux in liver. Excessive FFA also inhibits the growth hormone/insulin growth factor-1 axis, which 1 2 Y. Seko et al. protects muscle from age-related loss.10 Because skeletal muscle is the tissue responsible for glucose disposal, the existence of sarcopenia promotes IR.11 Chronic inflammation injures the liver and leads to fibrosis progression.12 In addition, cytokines such as tumor necrosis factor-α and transforming growth factor-β exacerbate protein catabolism and lead to sarcopenia.13 There are several definitions of sarcopenia and methods of diagnosis from some groups. The European Working Group on Sarcopenia in Older People and the Asian Working Group for Sarcopenia (AWGS) recommend the diagnosis of sarcopenia in elderly subjects based on gait speed, grip strength, and skeletal muscle index (SMI).14,15 The Japan Society of Hepatology (JSH) published diagnostic criteria for sarcopenia in liver disease based on grip strength and SMI in all ages.16 Bioelectrical impedance analysis is widely used to measure skeletal muscle mass because of its simplicity and lack of exposure to radiation. In addition to these definitions, some previous studies used original definitions to estimate sarcopenia. The Foundation for the National Institutes of Health (NIH) Sarcopenia Project defined the sarcopenia index (SI; appendicular skeletal mass [ASM] to body mass index [BMI] ratio) from nine sources of communitydwelling elderly cohorts. The Foundation defined a sarcopenia cut-off value of SI <0.789 in men and <0.512 in women.17 Koo et al. adopted a definition of sarcopenia of ASM to bodyweight ratio developed by the NIH Sarcopenia Project.18 We previously reported that the ratio of skeletal muscle mass to body fat mass (SF ratio) was associated with liver function independent of age and other metabolic features in Japanese patients with NAFLD.19 These three indices include both skeletal muscle mass and body fat mass. Thus, there are several definitions of sarcopenia, and which definition is used generally reflects the liver histology. It is still unclear which definition is suitable for therapeutic target of Japanese patients with NAFLD. The aims of this study were to compare the SMI, SI, and SF ratio on the association with liver histology and to determine the therapeutic target of these indices in clinical aspects of Japanese patients with NAFLD. METHODS Patients F ROM JANUARY 2014 to October 2017, 347 consecutive patients were diagnosed with NAFLD by liver biopsy findings of steatosis in ≥5% of hepatocytes and the exclusion of other liver diseases, including viral hepatitis, autoimmune hepatitis, and drug-induced liver disease at © 2019 The Japan Society of Hepatology Hepatology Research 2019 our institution. Patients consuming more than 20 g alcohol per day and those with evidence of decompensated liver cirrhosis or hepatocellular carcinoma were excluded from the study. Among patients who underwent a liver biopsy, 128 were excluded because of a lack of body composition data. Sixty-three patients with alanine aminotransferase (ALT) <40 IU/L were also excluded. A total of 156 patients were enrolled. A follow-up cohort comprised 121 patients who underwent serial body composition tests 12 months after baseline. No patients were given additional oral medications during the follow-up period. We instructed patients to decrease caloric intake by 500–1000 kcal/day and undertake moderate-intensity exercise. All patients provided written informed consent at the time of liver biopsy, and the study was carried out in accordance with the Declaration of Helsinki. This study protocol was approved by the institution’s human research committees. Laboratory and clinical parameters Venous blood samples were collected in the morning after a 12-h overnight fast. Laboratory assays included blood cell counts and measurements of serum aspartate aminotransferase (AST), ALT, γ-glutamyl transpeptidase (GGT), total cholesterol, triglycerides, and fasting plasma glucose. Parameters were measured with standard clinical chemistry laboratory techniques. The BMI was calculated as weight (kg)/(height [m])2. Type 2 diabetes mellitus (T2DM) was diagnosed according to the Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus or confirmed on the basis of patients being prescribed antihyperglycemic T2DM agents. Patients with serum cholesterol concentrations >220 mg/dL, triglyceride concentrations >160 mg/dL, or who were prescribed antidyslipidemia agents were considered dyslipidemic. Patients with systolic blood pressure >140 mmHg or diastolic blood pressure >90 mmHg, or who were prescribed antihypertensive drugs were considered to have hypertension. Liver histology All enrolled patients underwent a percutaneous liver biopsy under ultrasonic guidance. Liver specimens were embedded in paraffin and stained with hematoxylin–eosin or Masson’s trichrome. The specimens were evaluated by two hepatic pathologists (N.M. and Y.S.) who were blinded to the clinical findings. An adequate liver biopsy sample was defined as a specimen of length >1.5 cm and/or having more than 11 portal tracts. Nonalcoholic steatohepatitis was defined as steatosis with lobular inflammation and ballooning degeneration, with or without Mallory–Denk Hepatology Research 2019 bodies or fibrosis. Patients with liver biopsy specimens showing simple steatosis or steatosis with non-specific inflammation were identified as the NAFL cohort. Histological grade and stage were scored as described. Necroinflammatory grades of 1, 2, and 3 were defined as mild, moderate, and severe, respectively, for hepatocellular steatosis, ballooning, and inflammation (acinar and portal). The NAFLD activity score (NAS) was the sum of steatosis, lobular inflammation, and hepatocellular ballooning scores. The NAS ranged from 0 to 8. The severity of hepatic fibrosis (stage) was scored as follows: 1, zone 3 perisinusoidal fibrosis; 2, zone 3 perisinusoidal fibrosis with portal fibrosis; 3, zone 3 perisinusoidal fibrosis and portal fibrosis with bridging fibrosis; and 4, cirrhosis.20–22 Body composition and sarcopenia definition We analyzed the body composition of participants using the Inbody720 multifrequency impedance body composition analyzer (Inbody Japan, Tokyo, Japan). Body composition was measured in kilograms. The SMI was calculated as appendicular skeletal muscle mass (kg)/(height [m])2. As indicated in the sarcopenia diagnostic criteria of the Japan Society of Hepatology,16 we defined a low SMI as <7.0 kg/m2 in men and <5.7 kg/m2 in women. The SI was calculated as ASM (kg)/BMI (kg/m2). The NIH Sarcopenia Project23 defined sarcopenia using the cut-off points of SI <0.789 in men and <0.521 in women. The SF ratio was calculated as ASM (kg)/body fat mass (kg). Alanine aminotransferase response We defined an ALT response as a decrease of 30% or more from baseline, in accordance with a proposal by the NASH Clinical Research Network.24 This definition was found to have the highest area under the receiver operating characteristic curve (AUROC) for predicting an improvement in the NAS and liver fibrosis. DNA preparation and single nucleotide polymorphism (SNP) genotyping Genomic DNA was extracted from blood samples using DNeasy Blood & Tissue kit (Qiagen, Tokyo, Japan). The patatin-like phospholipase 3 (PNPLA3) SNP rs738409 was genotyped in each sample using TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA, USA) with commercially available predesigned SNP-specific primers for polymerase chain reaction amplification and extension reactions according to the manufacturer’s protocol. The precise protocol was carried out in a manner similar to our previous study.25 Validity of SF ratio in sarcopenic NAFLD 3 Statistical analysis Results are presented as numbers for qualitative data or as medians for quantitative data. The distribution of subject characteristics was assessed with the χ 2-test or Fisher’s exact probability test, as appropriate. To determine the cut-off point of the SF ratio to define sarcopenia, we calculated the sensitivity (Se) and specificity (Sp), and then constructed receiver operating characteristic (ROC) curves by plotting the Se against (1 – Sp) at each value. The diagnostic performance of the SF ratio was assessed by analysis of ROC curves. The most commonly used index of accuracy was the AUROC, with values close to 1.0 indicating high diagnostic accuracy. Statistical comparisons were undertaken with SPSS version 25 software (SPSS, Chicago, IL, USA). All P-values <0.05 were calculated with a two-tailed test and were considered significant. RESULTS Patient characteristics T ABLE 1 SHOWS THE patients’ clinical characteristics, as well as laboratory and histological data at baseline. A total of 64 patients (41.0%) received oral medication for T2DM, 61 patients (39.1%) received oral medication for dyslipidemia, and 10 patients (6.4%) received vitamin E. The details of medication at baseline are described in Table S1. This cohort included 82 (52.6%) female patients, and the median age was 57.5 years. A total of 112 cases (71.8%) were obese (BMI >25 kg/m2), and 117 cases (75.0%) were diagnosed with NASH. Forty-four (28.2%) of the 156 patients were Brunt stage 0, 54 (34.6%) were stage 1, 37 (23.7%) were stage 2, 11 (7.1%) were stage 3, and 10 (6.4%) had cirrhosis (stage 4). In this cohort, the PNPLA3 rs738409 genotype frequencies were: CC, 18 (16.4%) patients; CG, 51 (46.4%) patients; and GG, 41 (37.3%) patients. Prevalence of sarcopenia in patients with NAFLD We evaluated the relationship between histological features and the three indices. The median SI and the SF ratio in the non-significant fibrosis group (hepatic fibrosis stage <2) were significantly greater than those in the significant fibrosis group (hepatic fibrosis stage ≥2) (P = 0.008 and P = 0.047, respectively). The SI and the SF ratio in patients with NAS <6 were also significantly greater than those in patients with NAS ≥6 (P = 0.019 and P = 0.035, respectively) (Fig. 1). In this cohort, the SMI did not show a significant difference, regardless of fibrosis stage and NAS (Fig. 1). With the SMI, the prevalence of sarcopenia was 21 (13.5%) patients. However, with the SI, the prevalence © 2019 The Japan Society of Hepatology 4 Y. Seko et al. Hepatology Research 2019 Table 1 Characteristics of patients with non-alcoholic fatty liver disease: Total cohort and follow-up cohort Variable Female gender Age, years PNPLA3, CC/CG/GG NASH Type 2 diabetes mellitus Hypertension Albumin, g/dL AST, IU/L ALT, IU/L GGT, IU/L Platelet count, ×103/μL Total cholesterol, mg/dL Triglycerides, mg/dL LDL-C, mg/dL HDL-C, mg/dL FPG, mg/dL HbA1c, % NAS, 3/4/5/6Fibrosis stage, 0/1/2/3/4 BMI, kg/m2 SMI, kg/m2 SI SF ratio Total cohort n = 156 Follow-up cohort n = 121 82 (52.6) 57.5 (17–84) 18/51/41 117 (75.0) 79 (50.6) 69 (57.0) 56 (17–79) 15/38/34 96 (79.3) 62 (51.2) 66 (42.3) 4.4 (3.2–5.2) 51 (22–192) 66 (40–237) 63 (15–716) 215 (90–412) 54 (44.6) 4.4 (3.2–5.2) 52 (23–192) 67 (40–202) 69 (15–533) 217 (90–412) 201 (123–355) 205 (123–355) 132 (43–923) 123 (52–254) 52 (22–105) 106 (82–325) 6.3 (5.0–11.0) 35/49/31/41 44/54/37/11/10 122 (49–923) 126 (52–254) 53 (22–105) 106 (82–325) 6.3 (5.0–11.0) 22/39/24/36 30/41/31/10/9 26.9 (19.2–44.2) 7.36 (4.86–10.43) 0.70 (0.43–1.11) 0.77 (0.36–3.76) 27.2 (19.4–44.2) 7.37 (4.89–10.43) 0.68 (0.43–1.11) 0.74 (0.36–2.08) Results are presented as a number (n) or n (%) for qualitative data or as median (range) for quantitative data. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; FPG, fasting plasma glucose; GGT, γ-glutamyl transpeptidase; HbA1c, hemoglobin A1c; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; NAS, non-alcoholic fatty liver disease activity score; SF, skeletal muscle mass to body fat mass; SI, sarcopenia index; SMI, skeletal muscle mass index. was 41 (26.3%) patients. Only seven patients were diagnosed with sarcopenia by both indices. The concordance rate of sarcopenia diagnosis in each index was 0.058. Next, we determined the cut-off value of the SF ratio to define sarcopenia. Figure 2 shows the ROC curves for the SF ratio to differentiate sarcopenia based on the SI. The cut-off values of the SF ratio were 0.90 in men and 0.56 in women. The SF ratio had an AUROC of 0.880 in men and 0.852 in women. At the cut-off value, the sensitivity and specificity were 0.830/0.810 in men and 0.839/0.700 in women. © 2019 The Japan Society of Hepatology Changes in liver function and body composition in the follow-up cohort A total of 121 patients underwent body composition tests at baseline and 12 months postbaseline. We carried out only nutrition and exercise therapy, and no patients were given oral medication in the follow-up period. The serum concentrations of AST, ALT, and GGT decreased significantly by the 12-month follow-up. In terms of body composition, not only BMI, but also SMI, decreased significantly by the 12-month follow-up. The SF ratio increased significantly, from 0.74 to 0.76, but the SI did not change significantly (P = 0.290) (Table 2). Factors associated with ALT response A total of 69 patients (57.0%) were ALT responders in this study. Univariate analysis identified seven parameters that correlated with ALT response: sex (female, P = 0.065), PNPLA3 (G allele, P = 0.046), platelet count (≤200 × 103/μL, P = 0.024), fibrosis stage (≥2, P = 0.044), NAS (≥6, P = 0.072), SF ratio (increase, P < 0.001), and SI (increase, P = 0.003). We undertook multivariate logistic regression analysis with these factors. The adjusted odds ratio (OR) of presence of the PNPLA3 G allele was 5.181 (95% confidence interval [CI], 1.134–23.81; P = 0.034), and that of increased SF ratio was 7.406 (95% CI, 1.796– 30.54; P = 0.006) for ALT response (Table 3). The prevalence of ALT responders increased gradually with changes in the SF ratio and the SI. The ALT response rate of patients with a decreased SF ratio was 31.8%, and that in patients with an increased SF ratio was 70.1%. Likewise, the prevalence of ALT responders with PNPLA3 CG/GG was 56.9%, which was greater than that in patients with PNPLA3 CC (26.7%) (Fig. 3). DISCUSSION W E INVESTIGATED THE features of several sarcopenia-related indices in this study. The diagnosis of sarcopenia was quite different between the SMI and the SI. According to the SMI, which is adopted in the AWGS and JSH criteria, the prevalence of sarcopenia was 13.5%. In contrast, 26.3% of patients were diagnosed with sarcopenia by the SI. The difference was not only the proportion of sarcopenia. The concordance rate of sarcopenia between the two indices was only 0.058. From the view of liver histology, the SI and the SF ratio were associated with both fibrosis stage and the NAS, but the SMI did not show a significant relationship. Several factors, including age, obesity, and T2DM, are known to be associated with liver fibrosis. In Japanese non-viral liver disease patients, the prevalence of NAFLD and significant liver fibrosis was Hepatology Research 2019 Validity of SF ratio in sarcopenic NAFLD 5 Figure 1 Sarcopenia-related indices according to fibrosis stage and non-alcoholic fatty liver disease activity score (NAS) in Japanese patients. SF, skeletal muscle mass to body fat mass; SMI, skeletal muscle index. Figure 2 Receiver operating characteristic curves of the skeletal muscle mass to body fat mass (SF) ratio for sarcopenia in Japanese men (a) and women (b) with non-alcoholic fatty liver disease. reported to be associated with hemoglobin A1c (HbA1c) level.26 Previously, Lee et al. reported that sarcopenia diagnosed by the SI was associated with significant liver fibrosis independent of obesity and IR.9 In that study, they also concluded that the existence of sarcopenia was associated with a twofold increased risk of fibrosis. Several studies reported a relationship between sarcopenia and hepatic fibrosis;9,18,27 to the best of our knowledge, all of them diagnosed sarcopenia with indices including both skeletal muscle mass and body fat mass, such as ASM/BMI © 2019 The Japan Society of Hepatology 6 Y. Seko et al. Hepatology Research 2019 Table 2 Characteristics of patients with non-alcoholic fatty liver disease (NAFLD) in the follow-up cohort at baseline and 12 months Patients with NAFLD Variable Age, years Albumin, g/dL AST, IU/L ALT, IU/L GGT, IU/L Platelet count, ×103/μL Total cholesterol, mg/dL Triglycerides, mg/dL LDL-C, mg/dL HDL-C, mg/dL FPG, mg/dL BMI, kg/m2 SMI, kg/m2 SI SF ratio Baseline n = 121 12 months n = 121 P-value† 56 (17–79) 4.4 (3.2–5.2) 52 (23–192) 67 (40–202) 69 (15–533) 217 (90–412) 205 (123–355) 122 (49–923) 126 (52–254) 53 (22–105) 106 (82–325) 27.2 (19.4–44.2) 7.37 (4.89–10.43) 0.68 (0.43–1.11) 0.74 (0.36–2.08) – 4.4 (3.0–7.7) 34 (14–139) 44 (12–262) 42 (14–360) 220 (93–419) 196 (55–280) 136 (40–809) 124 (54–217) 50 (28–94) 110 (84–251) 26.4 (19.2–46.2) 7.22 (4.92–9.67) 0.69 (0.45–1.09) 0.76 (0.38–2.41) – 0.968 <0.001 <0.001 <0.001 0.866 0.640 0.538 0.595 0.136 0.052 <0.001 <0.001 0.290 0.003 Results are the median (range) for quantitative data. †Bold font for P-values indicates <0.05. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; FPG, fasting plasma glucose; GGT, γ-glutamyl transpeptidase; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; SF, skeletal muscle mass to body fat mass; SI, sarcopenia index; SMI, skeletal muscle mass index. Table 3 Factors associated with amelioration of alanine aminotransferase >30% in patients with non-alcoholic fatty liver disease (NAFLD) Multivariate analysis Variable Sex Category 1: male 2: female PNPLA3 1: CC 2: CG/GG Platelet count, 1: >200 ×103/μL 2: ≤200 Fibrosis stage 1: 0, 1 2: 2, 3, 4 NAS 1: <6 2: ≥6 Change in SI 1: decrease 2: increase Change in SF ratio 1: decrease 2: increase OR (95% CI)† P-value‡ 1.772 (0.572–5.489) 0.321 5.181 (1.134–23.81) 0.034 3.714 (1.042–13.23) 0.043 1.396 (0.401–4.860) 0.600 1.283 (0.367–4.484) 0.696 1.354 (0.362–5.066) 0.652 7.406 (1.796–30.54) 0.006 †Estimated using Cox proportional hazards regression analysis. ‡Bold font for P-values indicates <0.05. CI, confidence interval; NAS, NAFLD activity score; OR, odds ratio; SF, skeletal muscle mass to body fat mass; SI, sarcopenia index. or ASM/body weight. Our results were consistent with these previous data. Differences might be based on the method used to diagnose sarcopenia. The AWGS used © 2019 The Japan Society of Hepatology two standard deviations below the mean muscle mass of a young reference group as the cut-off value determination.15 In contrast, the Foundation for the National Institutes of Health (FNIH) determined the cut-off value of SI that best discriminated weakness (grip strength <26 kg in men, <16 kg in women) in elderly subjects.17 Moreover, 67.6% of the study population of FNIH included overweight/obese patients. To evaluate sarcopenia in patients with NAFLD precisely, we should choose an index that includes not only skeletal muscle mass, but also body composition. Because the SF ratio was constructed with consideration of body composition, including skeletal muscle mass and body fat mass, we adopted the SI to set a cut-off value of the SF ratio to diagnose sarcopenia. We defined the cutoff values of the SF ratio as <0.90 in men and <0.56 in women. The AUROC of the SF ratio for sarcopenia was 0.880 in men and 0.852 in women. Our results showed that the SF ratio can predict sarcopenia in NAFLD with sufficient accuracy. Further analyses that verify the utility of the SF ratio in a longitudinal study are needed. Next, we investigated the relationship between sarcopenia-related indices and changes in liver function. In a previous study, muscle function reduction and muscle volume loss in patients with liver cirrhosis showed a significant relationship with decreasing serum albumin levels.28 We also previously reported that Hepatology Research 2019 Validity of SF ratio in sarcopenic NAFLD 7 Figure 3 Prevalence of alanine aminotransferase (ALT) responders according to change in (a) skeletal muscle mass to body fat mass (SF) ratio, (b) sarcopenia index (SI), and (c) PNPLA3 genotype in Japanese patients with non-alcoholic fatty liver disease. Change from baseline to 12 months. Japanese patients with NASH whose serum ALT level did not decrease by at least 30% from baseline have a higher risk of disease progression, including both liver fibrosis and NAS.29 In that study, the inclusion criteria were ALT >40 U/L at baseline; that was why we evaluated patients with ALT >40 U/L at baseline in this study. In this study, changes in the SI and the SF ratio showed a significant correlation with ALT response. However, a change in the SMI was not correlated with a change in ALT. Multivariate analysis revealed that presence of the PNPLA3 G allele and an increase in the SF ratio were predictive factors for ALT response. The PNPLA3 gene was already identified as the strongest genetic determinant of liver fat concentration and serum level of ALT by genomewide association studies in European and Japanese populations.30,31 Additionally, the presence of the PNPLA3 G allele was associated with a greater reduction in intrahepatic triglycerides in patients with NAFLD who were undergoing lifestyle modification.32 Marzuillo et al. reported that PNPLA3 GG homozygotes had greater reductions in serum ALT level and waist-to-hip ratio in a study of 129 obese children participating in a weight loss program.33 They suggested that the interaction of visceral adipocytes and PNPLA3 might account for this difference in liver response. With regard to NAFLD patients with T2DM, patients with the PNPLA3 G allele showed a more positive correlation between change in HbA1c and ALT response to therapy with alogliptin.34 Individuals who have the PNPLA3 G allele are possibly more sensitive to the beneficial effect of lifestyle modification and appropriate treatment. Patients with an increased SF ratio had an OR of 7.406 for ALT response. It is already well known that sarcopenia can be an independent risk factor for infection, lower quality of life, and might even be prognostic in patients with cirrhosis.35,36 The longitudinal effect of sarcopenia on the pathology of early-stage NAFLD is still a matter of debate. To reduce visceral fat is considered an essential target of therapeutic strategies in NAFLD.37–39 A reduction in visceral fat leads to improvements in insulin resistance, hypertension, dyslipidemia, and chronic inflammation. Recently, sarcopenic obesity has received attention as a novel risk factor for NAFLD. Subjects with high fat mass and low muscle mass had worse clinical outcomes compared with subjects with sarcopenia or obesity alone.40,41 Actually, a reduction in skeletal muscle mass could © 2019 The Japan Society of Hepatology 8 Y. Seko et al. synergistically increase visceral fat in overweight subjects with NAFLD.42 Although the mechanism underlying this synergy is still unclear, leptin, which stimulates fat degradation in skeletal muscle and improves insulin sensitivity,43 and inflammatory cytokines such as interleukin-6 are thought to play a major role in this synergy.44,45 Shida et al. indicated that changes in body composition were associated with a change in adipokines, myokines, and hepatokines,42 thus supporting our result that changes in the SF ratio were associated with ALT response. A reduction in body fat and an increase in skeletal muscle could have the greatest impact on liver histology. However, it is too difficult a task to achieve in an actual clinical situation. It is acceptable to maintain skeletal muscle and reduce body fat as much as possible. In this study, we defined the cut-off value of SF ratio to diagnose sarcopenia. Furthermore, the SF ratio has superiority to other indices as a marker to reflect pathological change in the longitudinal evaluation of patients with NAFLD. Further studies that investigate whether patients with sarcopenia diagnosed with the SF ratio have worse mobility, quality of life, and prognosis are needed. This study has several limitations. It is a retrospective study undertaken at a single center, and the number of patients studied was not large. A multicenter, prospective study enrolling a larger number of patients will be required to draw firm conclusions. Next, we did not measure muscle strength in this study. Because sarcopenia is characterized by the loss of skeletal muscle mass and muscle strength, it is possible to overlook the size and number of muscle fibers. However, the SI was defined as discriminating patients with low grip strength. Thus, we consider that our results are reliable. A major strength of this study is that all enrolled patients were diagnosed by liver biopsy. A second strength relates to the use of bioelectrical impedance analysis to evaluate body composition. A previous report that compared bioelectrical impedance analysis with dual X-ray absorptiometry showed that the ability to estimate the amount of skeletal muscle mass was the same with both methods.46 Furthermore, bioelectrical impedance analysis is a simple and non-invasive method compared with dual X-ray absorptiometry, including computed tomography or magnetic resonance imaging. In conclusion, the SI and the SF ratio reflect disease severity and correlate well with changes in liver function in patients with NAFLD. Japan is a rapidly aging society, and the population of individuals with obesity is also increasing. 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Nutrients 2016; 29(8): 189. © 2019 The Japan Society of Hepatology Hepatology Research 2019 SUPPORTING INFORMATION A DDITIONAL SUPPORTING INFORMATION may be found online in the Supporting Information section at the end of the article. Table S1 Details of medications at baseline in this study.