Survey of Dietary Habits and Physical Activity in Japanese Patients with Non-Obese Non-Alcoholic Fatty Liver Disease

The incidence of non-obese non-alcoholic fatty liver disease (NAFLD), characterized by the presence of a fatty liver in individuals with a normal body mass index, is on the rise globally. Effective management strategies, including lifestyle interventions such as diet and exercise therapy, are urgently needed to address this growing public health concern. The aim of this study was to investigate the association between non-obese NAFLD, dietary habits, and physical activity levels. By elucidating these relationships, this research may contribute to the development of evidence-based recommendations for the management of non-obese NAFLD. The study had a single-center retrospective cross-sectional design and compared clinical data and dietary and physical activity habits between patients with and without non-obese NAFLD. Logistic regression analysis was utilized to investigate the relationship between food intake frequency and the development of NAFLD. Among the 455 patients who visited the clinic during the study period, 169 were selected for analysis, including 74 with non-obese NAFLD and 95 without NAFLD. The non-obese NAFLD group showed a less-frequent consumption of fish and fish products as well as olive oil and canola/rapeseed oil, while they showed more frequent consumption of pastries and cake, snack foods and fried sweets, candy and caramels, salty foods, and pickles compared to the non-NAFLD group. Logistic regression analysis revealed that NAFLD was significantly associated with the consumption of fish, fish products, and pickles at least four times a week. The physical activity level was lower and the exercise frequency was lower in patients with non-obese NAFLD compared to those without NAFLD. The results of this study suggest that a low consumption of fish and fish products and high consumption of pickles may be associated with a higher risk of non-obese NAFLD. Moreover, dietary habits and physical activity status should be taken into consideration for the management of patients with non-obese NAFLD. It is important to develop effective management strategies, such as dietary and exercise interventions, to prevent and treat NAFLD in this patient population.


Introduction
Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease [1], representing the hepatic manifestation of metabolic syndrome and leading to hepatic steatosis. The etiology of NAFLD includes primary factors, such as insulin resistance, as a consequence of obesity or metabolic syndrome, and secondary factors, which include endocrine disorders, such as hypothyroidism and dyslipidemia, as well as drugrelated causes [2]. On the other hand, non-obese NAFLD, also referred to as lean NAFLD, is a condition characterized by the presence of a fatty liver in individuals with a body mass

Study Design and Subjects
This research had a cross-sectional design and was based on that used in a previous study [22]. The study participants were patients who were first referred to the outpatient clinic for lifestyle-related liver diseases at the University of Tsukuba Hospital after being found to be obese or to have hepatic dysfunction, dyslipidemia, glucose intolerance, or hypertension during medical or physical examination. Additional study participants were added in subsequent surveys. In this study, data from 455 patients who attended our clinic between January 2017 and December 2021 were analyzed. NAFLD was diagnosed via interview, ultrasonography, and ultrasound elastography. The following exclusion criteria were applied: alcohol intake > 30 g/day if male and 20 g/day if female [23]; other chronic liver disease; a diagnosis of NAFLD was not possible because no ultrasound examination was performed; refusal to participate; questionnaire not returned; or a BMI ≥ 25 ( Figure 1).

Study Design and Subjects
This research had a cross-sectional design and was based on that used in a previous study [22]. The study participants were patients who were first referred to the outpatient clinic for lifestyle-related liver diseases at the University of Tsukuba Hospital after being found to be obese or to have hepatic dysfunction, dyslipidemia, glucose intolerance, or hypertension during medical or physical examination. Additional study participants were added in subsequent surveys. In this study, data from 455 patients who attended our clinic between January 2017 and December 2021 were analyzed. NAFLD was diagnosed via interview, ultrasonography, and ultrasound elastography. The following exclusion criteria were applied: alcohol intake > 30 g/day if male and 20 g/day if female [23]; other chronic liver disease; a diagnosis of NAFLD was not possible because no ultrasound examination was performed; refusal to participate; questionnaire not returned; or a BMI ≥ 25 ( Figure  1).

Data Collection
The following clinical data used in this study were obtained from the medical records of the patients: anthropometric measurements (height, weight, BMI, body composition), muscle strength measurements (grip, knee extension strength), liver fat content and fibrosis (controlled attenuation parameters (CAPs), liver stiffness measurements (LSMs)), parameters related to liver function (albumin, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase, gamma-glutamyl transferase, cholinesterase),

Data Collection
The following clinical data used in this study were obtained from the medical records of the patients: anthropometric measurements (height, weight, BMI, body composition), muscle strength measurements (grip, knee extension strength), liver fat content and fibrosis (controlled attenuation parameters (CAPs), liver stiffness measurements (LSMs)), parameters related to liver function (albumin, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase, gamma-glutamyl transferase, cholinesterase), glucose metabolism (fasting blood glucose, insulin, homeostasis model assessment-insulin resistance (HOMA-IR), HbA1c), lipid metabolism (total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides), and blood pressure. The measurements were performed in the same way as described previously [24]. Obesity was defined as a BMI of ≥25, and sarcopenia-related indices, including the skeletal muscle index (limb skeletal muscle mass [kg]/height [m] 2 ) [25], sarcopenia index (limb skeletal muscle mass [kg]/BMI) [26], and skeletal muscle mass to visceral fat area (SV) ratio [27], were calculated. Surrogate markers, including insulin resistance via HOMA-IR [28], the FIB-4 index [29], and the NAFLD fibrosis score (NFS) [30], were calculated using the biochemical data. The severity of liver fibrosis was assessed using the FAST score [31], which incorporates the liver stiffness measurement (LSM), controlled attenuation parameter (CAP) to assess liver fat content, and aspartate aminotransferase (AST) to assess liver inflammation.
The data on dietary habits and physical activity were obtained using a questionnaire survey administered to the study participants. The survey was performed in the same way as described in a previous report [22]. The food items in the questionnaire were selected based on the five food categories commonly included in dietary assessments, including staple foods, main dishes, side dishes, milk and dairy products, and fruits [32], as well as foods that have been reported to have an impact on the development and progression of NAFLD in previous studies, to ensure comprehensive coverage of dietary intake. Details on physical activity level (PAL) and exercise (Ex) have also been provided in previous reports [22]. The questionnaire included a section on oral medications. The relationship between drugs, the clinical data, and comorbidities was carefully examined with reference to the patient's medical history.

Statistical Analysis
The statistical analysis was conducted with the use of the SPSS software package, version 26 (IBM Corp., Armonk, NY, USA). Categorical variables were expressed as frequency and percentage, and continuous variables were presented as mean ± standard deviation. All statistical tests were two-tailed, and statistical significance was set at the 5% level. The Mann-Whitney U test and the chi-squared test were used for between-group comparisons. Confounding by covariates measured between the non-obese NAFLD group and the non-NAFLD group was reduced through the use of the propensity score method [33]. A propensity score quantifying the probability of developing non-obese NAFLD was calculated using a logistic regression model. Covariates for inclusion in the propensity score model were selected based on their likelihood of being associated with non-obese NAFLD [3,11] and included age, sex, skeletal muscle mass, knee extension stretch, and PAL. A multivariate model was created with the propensity score as a predictor variable, and binomial logistic regression analysis was performed using a model with the presence or absence of non-obese NAFLD as the outcome and food intake frequency of ≥4 times/week as a factor. The results are reported as the odds ratio (OR) and corresponding 95% confidence interval (CI).

Ethics Approval and Consent to Participate
The study was approved by the Ethical Review Committee of the University of Tsukuba Hospital (approval number H26-181) and conducted in compliance with the principles of the 1975 Declaration of Helsinki. All study participants signed an informed consent document after receiving a detailed explanation of the purpose of the study.

Cohort Formation and Patient Demographic and Clinical Characteristics
Out of the 455 patients who visited the outpatient clinic from January 2017 to December 2021, a total of 169 patients were included in the analysis. The patients were divided into a non-obese NAFLD group (n = 74) and a non-NAFLD group (n = 95) ( Figure 1).

Association between Non-Obese NAFLD and Food Intake Frequency
To determine whether food intake frequency was associated with non-obese NAFLD, logistic regression analysis was performed with the presence of NAFLD as the outcome, a food intake frequency of ≥4 times/week as the risk factor, and propensity variables calculated using the propensity score method as the covariates (Table 3). There was a significant association between non-obese NAFLD and the consumption of fish and fish products (OR 0.261, 95% CI 0.114-0.594; p = 0.001) and pickles (OR 2.880, 95% CI 1.264-6.558; p = 0.012) ≥4 times/week. The results did not change when the analysis was conducted using a model that included the prevalence of diabetes and dyslipidemia in the propensity score. Outcome: NAFLD. Factors: fish and fish products, pastries and cake, candy and caramels, and pickles ≥ 4 times/week. Adjusted for propensity score that included age, sex, skeletal muscle mass, knee extension stretch, and PAL. NAFLD: non-alcoholic fatty liver disease, OR: odds ratio, CI: confidence interval.

Discussion
As far as we are aware, this is the first study using a questionnaire to examine the association between dietary habits and physical activity in Japanese patients with nonobese NAFLD.
The results of the questionnaire revealed that patients with non-obese NAFLD consumed fish and fish products and olive oil or canola/rapeseed oil less frequently and consumed pastries and cake, snack foods and fried sweets, candy and caramels, salty foods, and pickles more frequently. Dietary risk factors for non-obese NAFLD have been reported to include an excessive intake of cholesterol and fructose [7]. In this study, there was no significant between-group difference in the frequency of intake of cholesterol-rich chicken eggs [34]; however, the frequency of intake of pastries and cake and of snack foods and fried sweets [34], which contain high levels of saturated fatty acids [35] that indirectly increase serum cholesterol, was higher in the patients with non-obese NAFLD. The background of excessive fructose intake has been attributed to a high consumption of soft drinks and juices containing corn syrup fructose [36]. In our study, the between-group difference in the frequency of soft drink consumption was not statistically significant, although there was a trend toward a higher frequency of consumption in patients with non-obese NAFLD. However, the frequency of intake of candy and caramels [34] with a higher amount of corn syrup fructose per gram was higher in patients with non-obese NAFLD. These findings are consistent with those of previous studies [7].
Our findings suggest that there are several previously unrecognized dietary risk factors for non-obese NAFLD. In this study, the consumption of fish and fish products and olive oil or canola/rapeseed oil was less frequent in the non-obese NAFLD group. Furthermore, the consumption of fish and fish products ≥ 4 times/week had a suppressive effect on the fatty liver. This finding suggests that patients with a non-obese NAFLD lack sufficient unsaturated fatty acids in their diet. A dietary pattern characterized by a high intake of olive oil and fish, known as the Mediterranean diet, has been reported to reduce liver fat [16]. Levy et al. [19] reported that ω3 polyunsaturated fatty acids reduce hepatic lipidosis, and the administration of ω3 polyunsaturated fatty acids has been shown to be effective in treating hepatic lipids in patients with NAFLD [37]. Olive oil and rapeseed/canola oil contain high levels of oleic acid [34], a monounsaturated fatty acid of the ω9 family that has the effect of reducing insulin resistance and lipidosis [38]. Olive oil has been demonstrated to reduce the accumulation of triglycerides in the liver in a rodent model of NAFLD [39], and once-daily rapeseed/canola oil consumption has been reported to be effective in the treatment of hepatic adiposity [40]. The Mediterranean diet may have a favorable effect on the fatty liver because of the unsaturated fatty acids contained in fish and olive oil.
In a previous study by Zhao et al. [41], it was found that a diet high in salt can have an impact on hypertriglyceridemia, oxidative stress, and inflammation, whereby all of which are known pathological factors in the development of NAFLD, and a higher prevalence of NAFLD has been reported in young and middle-aged adults who consume a high-sodium diet, according to a previous study [42]. In our study, patients with non-obese NAFLD had a higher frequency of consumption of salty foods and of pickles, which contain a high amount of salt. Furthermore, the consumption of pickles ≥ 4 times/week promoted the formation of the fatty liver. This finding suggests that a high-salt diet may contribute to the formation of NAFLD even in non-obese individuals.
Body composition analysis showed that an abnormal composition such as an increase in the internal fat cross-sectional area and a decreased tendency toward an SV ratio of sarcopenia body mass index was observed in those with non-obese NAFLD. It has been thought that visceral fat and muscle balance abnormalities are associated with the pathophysiology of non-obese NAFLD [11]. Our laboratory has reported that exercise intervention in patients with NAFLD has little effect on weight loss, but maintains lean body (skeletal muscle) mass and increases large muscle strength [43], suggesting that exercise improves the pathophysiology of NAFLD independently of weight loss. Given that the responses to our questionnaire showed that patients with non-obese NAFLD tended to have low PAL and Ex (Table 2), we believe that in addition to their daily PAL they should perform further voluntary exercise to maintain a good balance between visceral fat and muscle mass.
This study had several limitations. First, the Food Frequency Questionnaire was used as the dietary survey method. Dietary survey methods, regardless of which method is used, are reportedly subject to errors as a result of diurnal variation [44] and both underreporting and over-reporting by the respondents [45]. Second, because of the small number of patients in the non-obese NAFLD group, the propensity score method was used to eliminate the possibility of over-fitting when performing multivariate analysis; however, the study was retrospective in nature and the ability to control for differences was limited to variables for which data were available. Third, it should be noted that this was not designed as a prospective cohort trial and was conducted in a single hospital, making it likely to lead to spurious causality and bias. Therefore, in the near future, we are planning a prospective cohort study based on the results from this study.

Conclusions
Our questionnaire-based study revealed that a lower frequency of fish and fish product consumption, as well as a higher frequency of pickled food consumption, were associated with an increased risk of non-obese NAFLD. These findings suggest that dietary habits and physical activity should be taken into account in the routine clinical management of Japanese patients with non-obese NAFLD.

Institutional Review Board Statement:
This study was conducted in compliance with the principles of the 1975 Declaration of Helsinki and approved by the Ethical Review Committee of the University of Tsukuba Hospital (approval number H26-181).

Informed Consent Statement:
The purpose and significance of the study were fully explained to the participants, and written informed consent was obtained.

Data Availability Statement:
The datasets analyzed in this study are available from the corresponding author upon reasonable request. Further inquiries can be directed to the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.