Introduction

Nonalcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide, affecting an estimated 25% of the adult population [1]. NAFLD is a disease covering a wide spectrum, ranging from nonalcoholic fatty liver (NAFL), which is usually a benign condition, to nonalcoholic steatohepatitis (NASH), which can sometimes lead to liver cirrhosis or hepatocellular carcinoma without significant alcohol consumption [2, 3]. The prevalence of NAFLD is increasing rapidly because of Western dietary patterns and a lack of exercise. NAFLD is also a well-known risk factor for type 2 diabetes mellitus, chronic kidney disease, and cardiovascular disease.

Although several factors, such as a habitually high-calorie diet, low levels of physical activity, elevated oxidative stress, and genetics can contribute to the pathogenesis of NAFLD, insulin resistance is one of the most pivotal mechanisms underpinning NAFLD progression. Insulin resistance is induced by liver dysfunction as well as skeletal muscle and body fat disorders which play a key role in glucose and lipid metabolism. Such perturbations are often closely related to the onset and progression of NAFLD. Sarcopenia, characterized by a decline in skeletal muscle and low muscle strength, affects clinical outcomes, including quality of life, infection rate, and survival in patients with cirrhosis [4,5,6]. In general, idiopathic sarcopenia is referred to as primary (or age-related) sarcopenia, whereas the etiologic basis of secondary sarcopenia relates to other diseases, such as chronic liver disease, renal disease, inflammatory disease, and malignant tumors. In European populations, approximately 0.5–1.0% of skeletal muscle mass is lost per year after the age of 30 years, with the rate of decline dramatically accelerating after age 65 [7]. In Japan and Western countries, secondary sarcopenia reportedly occurs in 40–70% of patients with cirrhosis [8, 9].

Abnormal body composition, which includes obesity-related body fat mass and reduced skeletal muscle mass, is estimated to be associated with the progression of NAFLD. Therapeutic strategies for NAFLD need to focus on better managing these components of body composition.

The aim of this study was to determine (1) the association between skeletal muscle mass and NAFLD, and (2) the effect of body composition on the liver function of patients with NAFLD in Japan.

Methods

Patients

From January 2014 to October 2017, 347 patients who were consecutively diagnosed with NAFLD on the basis of liver biopsy findings of steatosis in ≥ 5% of hepatocytes—in the absence of other liver diseases such as viral hepatitis, autoimmune hepatitis, and drug-induced liver disease—were screened for study inclusion. Patients who were consuming more than 20 g of alcohol per day or for whom there was evidence of decompensated liver cirrhosis or hepatocellular carcinoma were excluded from the study. Among the patients who underwent a liver biopsy, 128 were excluded because of a lack of body composition data. A total of 219 patients were enrolled in the study. A follow-up cohort comprised 139 patients who underwent serial body composition tests 12 months after baseline. All patients provided written informed consent at the time of liver biopsy, and the study was conducted 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), alanine aminotransferase (ALT), γ glutamyl transpeptidase (GGT), total cholesterol, triglycerides, fasting plasma glucose (FPG), and type IV collagen 7 s. Hemoglobin A1c (HbA1c) was assayed using high-performance liquid chromatography and expressed in National Glycohemoglobin Standardization Program (NGSP) units (%). Parameters were measured with standard clinical chemistry laboratory techniques. Body mass index (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.

Liver histology

All enrolled patients underwent an ultrasound-guided percutaneous liver biopsy. Paraffin-embedded liver sections were prepared for hematoxylin and eosin or Masson’s trichrome staining. Specimens were evaluated by 2 hepatic pathologists (Y.S. and N.M.) who were blinded to the clinical findings. An adequate liver biopsy sample was defined as a specimen > 1.5 cm in length and/or with more than 11 portal tracts. The criteria for a diagnosis of NASH was (1) any degree of steatosis in addition to centrilobular ballooning and/or Mallory-Denk bodies or (2) any degree of steatosis along with centrilobular pericellular/perisinusoidal fibrosis. Patients with liver biopsy specimens showing simple steatosis or steatosis with nonspecific inflammation were identified as the NAFL cohort. Specimens with steatosis thresholds of < 5, 5–33, > 33–66, and > 66% were scored as grades 0, 1, 2, and 3, respectively. Histological grade and stage were scored as described. Necroinflammatory grades of NASH were defined as grade 1 (mild), grade 2 (moderate), and grade 3 (severe) based on the extent of hepatocellular steatosis, ballooning, and inflammation (acinar and portal). The severity of hepatic fibrosis (stage) was scored as follows: stage 1, zone 3 perisinusoidal fibrosis; stage 2, zone 3 perisinusoidal fibrosis with portal fibrosis; stage 3, zone 3 perisinusoidal fibrosis and portal fibrosis with bridging fibrosis; and stage 4, cirrhosis [10,11,12].

Body composition

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 and basal metabolic rate (BMR) was calculated using the Cunningham formula of BMR = 21.6 × fat-free mass (kg) + 370. Skeletal muscle index (SMI) was calculated as skeletal muscle mass (kg)/[height (m)]2. Body fat mass index (BFMI) was calculated as body fat mass (kg)/[height (m)]2. As indicated in the sarcopenia diagnostic criteria of the Japan Society of Hepatology [13], we defined a low SMI as < 7.0 kg/m2 in men and < 5.7 kg/m2 in women.

Statistical analysis

Results are presented as numbers for qualitative data or as medians for quantitative data. The distribution of subject characteristics was assessed using the Chi-square test or Fisher’s exact probability test, as appropriate. Logistic analysis was used to determine independent predictive factors associated with reduction in ALT in follow-up cohort. The hazard ratio (HR) and 95% confidence interval (CI) were also calculated. We performed multivariate logistic regression analysis with gender, age, complicating hypertension, hyperlipidemia, T2DM, serum level of AST, ALT, GGT, platelet count, hepatic fibrosis stage, and NAS to clarify the predictive factors associated with lowering SMI. The multivariate analysis to identify the predictive factors associated with reduction in ALT were performed with age, complicating hypertension, hyperlipidemia, T2DM, serum level of GGT, platelet count, hepatic fibrosis stage, NAS, and change in SF ratio. Statistical comparisons were performed using SPSS Ver.25 software (SPSS Inc., Chicago, IL). All P values < 0.05 were calculated using a two-tailed test and were considered significant.

Results

Patient characteristics

Baseline clinical characteristics of patients, as well as laboratory and histological data, are shown in Table 1. Total of 90 patients (41.1%) received the oral medication for T2DM, 98 patients (44.8%) received the oral medication for dyslipidemia, and 11 patients (5.0%) received the Vitamin E. The details of medication at baseline are described in Supplement Table 1. In this study of 219 patients, a cohort of 155 (70.8%) patients was diagnosed with NASH. T2DM was diagnosed in 61.9% of patients in the NASH group compared with 23.4% of patients in the NAFL group (P < 0.001). Of the 155 patients with NASH, 15 (9.7%) were stage 0, 59 (38.1%) were stage 1, 48 (31.0%) were stage 2, 19 (12.3%) were stage 3, and 14 (9.0%) had cirrhosis (stage 4). AST, ALT, GGT, FGP, HbA1c, and type IV collagen 7 s concentrations in serum, as well as BFMI, were significantly higher in patients with NASH than in patients with NAFL. In contrast, the platelet count was significantly lower in patients with NASH than in patients with NAFL. SMI was not significantly different between patients with NASH and NAFL, whereas SF ratio was significantly higher in patients with NAFL than in patients with NASH.

Table 1 Demographic characteristics, as well as laboratory and histological data, for patients with nonalcoholic fatty liver disease†
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Prevalence of low SMI in patients with NAFLD

Patients were stratified on the basis of low and normal SMI (Table 2). SMI was low in 27 (12.3%) patients and normal in 192 (87.7%) patients. The low SMI group included more patients who were female, older, and had higher serum concentrations of AST and triglycerides than the normal SMI group. BMI, BFMI, SMI, total body water, and BMR were significantly lower in the group of patients with a lower SMI as opposed to a normal SMI.

Table 2 Patient characteristics according to skeletal muscle index†
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The results for SMI, which was evaluated based on gender, age, and stage of hepatic fibrosis, are shown in Fig. 1. SMI decreased gradually with age in men. In women, SMI was significantly different between patients < 60 versus ≥ 60 years (P = 0.028). SMI was not correlated with stage of hepatic fibrosis. The prevalence of a low SMI for stages was as follows: stage 0, 14.8%; stage 1, 10.1%; stage 2, 6.1%; stage 3, 21.1%; and stage 4, 21.4%.

Fig. 1
figure 1

Skeletal muscle mass index (SMI) evaluated according to gender, age, and stage of hepatic fibrosis

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Factors associated with a low SMI

The multivariate analyses with gender, age, complicating hypertension, hyperlipidemia, T2DM, serum level of AST, ALT, GGT, platelet count, hepatic fibrosis stage, and NAS were performed to identify the risk factors associated with a low SMI in patients with NAFLD and revealed that the adjusted hazard ratio (HR) for women was 2.949 (95% CI 1.101–9.979; P = 0.031) and for the elderly (≥ 70 years) the HR was 5.114 (95% CI 1.259–20.78, P = 0.023; Table 3). The prevalence of a low SMI was 16.8% for women, 29.5% for men and women ≥ 70 years, and 35.7% in women ≥ 70 years in the NAFLD cohort.

Table 3 Factors associated with lowering SMI in patients with NAFLD as identified by multivariate analyses
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Changes in liver function and body composition in the follow-up cohort

A total of 139 patients underwent body composition tests at baseline and 12 months postbaseline. We performed only nutrition and exercise therapy, and no patients were added oral medication in follow-up period. The serum concentrations of AST, ALT, GGT, and HbA1c significantly decreased by the 12-month follow-up. Not only BFMI but also SMI significantly decreased by the 12-month follow-up (Table 4). Changes in the liver function parameters AST and ALT were negatively correlated with the ratio of SMI:BFMI (SF ratio) (Supplement Figure 1). Patients with an increased SF ratio exhibited a larger reduction in ALT (− 21.5 IU/L) than patients without an increased SF ratio (− 1.0 IU/L, P < 0.001). Even in the BMI increased group, the patients with increased SF ratio tended to show greater reduction in ALT (− 20.0 IU/L), than the patients with decreased SF ratio (0.5 IU/L, P = 0.053) (Fig. 2).

Table 4 Demographic characteristics as well as laboratory and histological data for patients in the followed up cohort†
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Fig. 2
figure 2

The change in ALT according to change in BMI and SF ratio. Delta change from baseline to 12 months

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To estimate the risk factors associated with reduction in ALT, we examined the correlation of patients’ backgrounds with reduction in ALT. We performed multivariate logistic regression analysis with age, complicating hypertension, hyperlipidemia, T2DM, serum level of GGT, platelet count, hepatic fibrosis stage, NAFLD activity score (NAS), and change in SF ratio in each sex. The multivariate analysis revealed that an increased SF ratio (HR of 10.99, P = 0.021 in men, HR of 6.849, P = 0.015 in women) was an independent predictive factor for a reduction in ALT in each sex, and the pathological findings having no discernable effect (Table 5). To compare the impact to liver function, we also performed multivariate logistic regression analysis with change in BMI instead of SF ratio (Supplement Table 2). The HR of increased SF ratio was greater than decreased BMI (HR 4.215, P = 0.031) in women. The change in BMI was not detected as a predictive factor in men.

Table 5 Factors associated with amelioration of ALT in patients with NAFLD
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Discussion

We investigated the SMI of Japanese patients with NAFLD by monitoring body composition. The prevalence of a low SMI was 12.3% in this study. A previous study investigating the prevalence of sarcopenia showed that 17.9% of patients with NAFL and 35.0% of patients with NASH had sarcopenia [14]. Another study by Lee et al. reported that 12.2% of patients with NAFLD were diagnosed with sarcopenia [15]. These two studies determined that the severity of hepatic fibrosis was associated with sarcopenia. In the present study, there was no correlation between SMI and histological features. Even among patients who did not have hepatic fibrosis, 14.5% of them met the low SMI criteria. The reason for this discrepancy is unclear; however, the definitions for sarcopenia, NAFLD, and the size and ethnicity of the population may be contributing that. The existence of patients with a low SMI during an early stage of NAFLD suggests that monitoring of SMI may be prudent at an early stage especially they are elderly patients.

The etiology for the reduction in SMI was different between men and women. The SMI decrease occurred gradually with progressive aging in men, whereas there was a significant decrease after the age of 60 years in women. One plausible hypothesis for these disparate results is that menopause affects the body composition of women. Menopause precipitates a decline in estrogen concentrations, and an increase in visceral adiposity, as well as a decrease in bone density, muscle mass, and muscle strength [16, 17]. The effect of menopause on skeletal muscle mass can occur directly as a consequence of estrogen receptors on muscle cells, and indirectly via an increase in circulating inflammatory cytokines [18]. Several studies also report a vitamin D deficiency can be associated with sarcopenia, especially in elderly women [19,20,21].

In this retrospective study, we identified the importance of the SF ratio in the pathology of NAFLD. After 12 months of follow-up, serum concentrations of AST, ALT, and BFMI decreased significantly. It is also worth mentioning that SMI declined significantly by 12 months. The changes in liver function were significantly associated with changes in the SF ratio. Targeting visceral fat stores using medication and/or exercise therapy to reduce them is considered essential to therapeutic approaches aiming to manage NAFLD [22,23,24]. A reduction in visceral fat accumulation is associated with improvements in insulin resistance, hypertension, dyslipidemia, and systemic chronic low-grade inflammation, and can decrease the risk of atherosclerosis and cardiovascular disease [25,26,27]. Because East Asian populations, including the Japanese, generally have a higher percentage of body fat than Caucasians of the same age, gender, and BMI [28,29,30,31], body fat or BMI is often the target of therapy.

Loss of skeletal muscle mass, especially when it is associated with sarcopenia, can be an independent risk factor for infection, a lower quality of life, and may even be prognostic in patients with cirrhosis [4, 5, 32, 33]. The effect of a low SMI on the pathology of NAFLD in the absence of cirrhosis is still a matter of debate. Interestingly, sarcopenic obesity—defined as a high fat mass and a low muscle mass—has been receiving attention as a risk factor for the impairment of physical activity and worse clinical outcomes compared with sarcopenia or obesity alone [34, 35]. Shida et al. suggest that a reduction in skeletal muscle mass may synergistically increase visceral fat in overweight subjects with NAFLD [36]. Leptin and inflammatory cytokines such as IL-6 are thought to play a major role in this synergy. Leptin stimulates fat degradation in skeletal muscle and improves insulin sensitivity [37]. In patients with sarcopenic obesity, serum leptin levels are reportedly higher than in patients with sarcopenia or visceral fat accumulation only [38]. Leptin resistance leads to a reduction in muscle mass and stimulates fat accumulation in muscles via an AMPK pathway-mediated reduction in muscle fatty acid oxidation. Serum IL-6 was reported to be high in subjects with sarcopenic obesity [39, 40]. The report by Shida et al. indicates that changes in body composition were associated with a change in adipokines, myokines, and hepatokines [36], thus supporting our result that changes in the SF ratio were associated with reduced ALT. The reduction in ALT concentrations in patients with an increased SF ratio after 12 months was significantly greater (− 21 IU/L) than for those patients with a decreased SF ratio (− 1.5 IU/L; P < 0.001). In the BMI decreasing group, the diminution of ALT was significantly greater in patients with increased SF ratio than in patients with decreased SF ratio (p = 0.005, Fig. 2). On the other hand, even the BMI increased, the patients with increased SF ratio showed reduction in ALT (− 20.0 IU/L) as much as the patients with decreased BMI and increased SF ratio (Fig. 2). From the standpoint of exercise therapy, there was no significant difference between aerobic and resistance exercise on changes in BMI, serum ALT, and hepatic steatosis [41]. Resistance exercise was reported to improve NAFLD in conjunction with consuming less energy, and patients unable to tolerate or adequately participate in aerobics exercise may benefit from performing resistance exercise as part of their therapeutic regimen [41].

This study has several limitations. It is a retrospective study conducted 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. Because of the low prevalence of advanced fibrosis (> stage 3, 15.1%), there is a possibility that the lowering SMI that was seen in the elderly cases in this study is simply exhibits general phenomenon in aging. Next, we did not measure muscle strength in this study. Because sarcopenia is diagnosed according to the loss of skeletal muscle mass and muscle strength, it is possible to overlook the size and number of muscle fiber decreases. 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. The previous report that compared the bioelectrical impedance analysis with dual X-ray absorptiometry showed that the ability of estimate the amount of skeletal muscle mass was equal in both methods [42]. Furthermore, the bioelectrical impedance analysis is a simple and noninvasive method rather than dual X-ray absorptiometry method including computed tomography or magnetic resonance imaging. From the economical point of view, the bioelectrical impedance analysis has also priority to Dual X-ray absorptiometry method.

In conclusion, patients with NAFLD were potentially susceptible to a low SMI, even during the early stage of disease development, and the changes in the SF ratio were correlated with parameters for liver function. Our results suggest that it would be prudent to evaluate the body composition of patients with NAFLD and incorporate the monitoring of these indices to reduce the risk of sarcopenia and a worse prognosis.