Habitual Miso (Fermented Soybean Paste) Consumption Is Associated with a Low Prevalence of Sarcopenia in Patients with Type 2 Diabetes: A Cross-Sectional Study

Insulin resistance is a risk of sarcopenia, and the presence of sarcopenia is high in patients with type 2 diabetes (T2DM). It has been reported that habitual miso soup consumption was associated with lower insulin resistance. However, the association between habitual miso consumption and the presence of sarcopenia in patients with T2DM, especially sex difference, was unclear. In this cross-sectional study, 192 men and 159 women with T2DM were included. Habitual miso consumption was defined as consuming miso soup regularly. Having both low skeletal muscle mass index (<28.64% for men, <24.12% for women) and low adjusted hand grip strength (<51.26% for men, <35.38% for women) was defined as sarcopenia. The proportions of sarcopenia were 8.7% in men and 22.6% in women. The proportions of habitual miso consumption were 88.0% in men and 83.6% in women. Among women, the presence of sarcopenia was lower in the group with habitual miso consumption (18.8% versus 42.3%, p = 0.018); however, there was no association between habitual miso consumption and the presence of sarcopenia in men. Habitual miso consumption was negatively associated with the presence of sarcopenia in women (adjusted odds ratio (OR), 0.20 (95% confidence interval (CI): 0.06–0.62), p = 0.005) but not in men. This study indicated that habitual miso consumption was associated with the presence of sarcopenia in women but not in men.


Introduction
The number of older patients with type 2 diabetes (T2DM) in Japan is increasing, since the aging of the adult population is increasing [1]. These patients often complicate with sarcopenia, which is characterized as loss of muscle mass, power, and function [2]. In the last decade, sarcopenia has been reported as a risk factor for fatty liver, cardiovascular diseases, and death [3][4][5]. In insulin-resistant conditions including T2DM, insulin-stimulated glucose disposal is severely impaired in the skeletal muscle [6]. Insulin resistance and oxidative stress are reported to be a cause of sarcopenia [7][8][9], and they are related to vascular modification [10], chronic inflammation [11], and lipid inflation in muscles [12,13]. Moreover,

Study Participants
The KAMOGAWA-DM cohort study, a prospective cohort study, started in 2014 and is currently ongoing [30]. In this cross-sectional study, patients with data of questionnaires from January 2016 to December 2018 were included. Exclusion criteria were as follows: no data of multifrequency impedance body composition analyzer, no data of hand grip strength, incomplete questionnaires, and patients without T2DM. This study was approved by the local research ethics committee (No. RBMR-E-466-6) and was carried out in accordance with the Declaration of Helsinki. All patients gave written informed consent.

Data Collection
Duration of diabetes, family history of diabetes, smoking status, and exercise habit were asked by a standardized questionnaire. The patients were divided into smokers and nonsmokers. We defined regular exercisers as patients who regularly played any type of sport >1×/week. Further, venous blood was collected from the participants who had fasted overnight, and the levels of triglycerides, high-density lipoprotein cholesterol, creatinine (Cr), uric acid, and fasting plasma glucose were measured. Estimated glomerular filtration rate (eGFR) was calculated using the equation of the Japanese Society of Nephrology, that is, eGFR = 194 × Cr-1.094 × age-0.287 (mL/min/1.73 m 2 ) (×0.739, if woman) [31]. Hemoglobin A1c (HbA1c) levels were estimated using high-performance liquid chromatography and were expressed as National Glycohemoglobin Standardization Program (NGSP) units. After the participants had 5 min of rest, blood pressure was measured by a HEM-906 device (OMRON, Kyoto, Japan) in a quiet space, automatically. Additionally, the data on medications, including those for diabetes and hypertension (renin-angiotensin-aldosterone inhibitor) were obtained from the patients' medical records.

Definition of Sarcopenia
Using InBody 720 (InBody, Japan, Tokyo, Japan), a multifrequency impedance body composition analyzer, the data of body weight (kg), appendicular muscle mass (kg), and body fat mass (kg) were gathered [32]. Then, body mass index (BMI, kg/m 2 ), dividing body weight (kg) by the square of height (m) and ideal body weight (IBW), 22 multiplied by the square of the patient's height in meters squared [33], were calculated. Percent body fat mass (%), dividing body fat mass (kg) by body weight (kg) × 100 and skeletal muscle mass index (SMI, %), appendicular muscle mass divided by body weight × 100 [34] for muscle mass were also calculated. Hand grip strength (HGS) was measured twice with each hand on a handgrip dynamometer (Smedley; Takei Scientific Instruments, Niigata, Japan), and the highest value was recorded. Adjusted grip strength (AGS) was calculated as HGS value divided by body weight × 100 [35] for muscle strength. The cutoff points for low muscle mass and low muscle strength were 28.64% and 51.26% in men and 24.12% and 35.38% in women [36]. Having both low muscle mass and low muscle strength was defined as sarcopenia [36].

Data of Habitual Diet Intake, Including Habitual Miso Consumption
Using a brief-type self-administered diet history questionnaire (BDHQ), habitual food and nutrient intake during the preceding 1 month period were evaluated [37]. The details and validity of BDHQ have been presented previously [38]. Patient data on the intakes of energy, fat, carbohydrate, protein, including animal and vegetable proteins, fiber, miso soup consumption, and alcohol consumption were obtained by BDHQ. Energy intake (kcal/kg IBW/day), protein intake (g/kg IBW/day), animal protein intake (g/kg IBW/day), vegetable protein intake (g/kg IBW/day), fat intake (g/kg IBW/day), and carbohydrate intake (g/kg IBW/day) were calculated. The ratio of carbohydrate to fiber intake was defined as carbohydrate intake divided by fiber intake [39]. In addition, the data on the intake frequency of miso soup were also collected and habitual miso consumption was defined as consuming miso soup regularly.

Statistical Analysis
The data are shown as means (standard deviation (SD)) or frequencies of potential confounding variables. Because the characteristics and dietary intakes differed between men and women, the data was analyzed by sex.
Patients were divided into two groups according to habitual miso consumption. We performed a Shapiro-Wilk test to investigate the distribution of variables. The differences in the continuous variables and categorical variables were evaluated by Student's t-test or the Mann-Whitney U test and the chi-square test, respectively. The correlation was analyzed by Pearson's correlation coefficient.
Further, to examine the effects of habitual miso consumption on the prevalence of sarcopenia, a logistic regression analysis was performed. The independent variables were sex, age, insulin treatment, habit of exercise, habit of smoking, duration of diabetes, HbA1c, energy intake, protein intake, and alcohol consumption. In addition, we performed a logistic regression analysis to examine the effect of miso soup intake, as a continuous variable, on the prevalence of sarcopenia. The independent variables were sex, age, insulin treatment, habit of exercise, habit of smoking, duration of diabetes, HbA1c, energy intake, protein intake, and alcohol consumption. Because miso soup intake was a skewed variable, logarithmic transformation was done before performing a logistic regression analysis, which were performed to evaluate the association of sarcopenia with log (miso soup intake + 1).
We performed subanalyses to examine the effects of habitual miso soup consumption on the prevalence of sarcopenia adjusted by age, according to the presence or absence of habit of smoking.
We used EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) [40] for statistical analyses. Differences with p values < 0.05 were set as statistically significant.

Results
In the present study, 523 patients (276 men and 247 women) were included. Among them, 109 patients (50 men and 59 women) who did not undergo the bioelectrical impedance analysis (BIA) test, 24 patients with no date of BDHQ (15 men and 9 women), a patient who did not undergo the hand grip strength test, and 38 patients without T2DM (19 men and 19 women) were excluded from the study. Therefore, 351 patients (192 men and 159 women) were the study participants ( Figure 1).
We performed subanalyses to examine the effects of habitual miso soup consumption on the prevalence of sarcopenia adjusted by age, according to the presence or absence of habit of smoking.
We used EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) [40] for statistical analyses. Differences with p values < 0.05 were set as statistically significant.

Discussion
We firstly investigated the relationship between habitual miso consumption and the presence of sarcopenia in patients with T2DM in this study. We revealed that habitual miso consumption was associated with a low prevalence of sarcopenia in women but not in men. Additionally, habitual miso consumption was associated with lower percentage of body fat mass in women.
The possible explanation for the association between habitual miso consumption and the low prevalence of sarcopenia is as follows. A previous study has shown that miso consumption suppresses fat accumulation in fat mass and showed anti-obesity effects [24]. Furthermore, there is a close association between increased visceral fat and muscle atrophy [41,42]. Secretion of proinflammatory cytokines such as interleukin-6 and tumor necrosis factor-α from obesity-induced hypertrophic fat cells causes muscle atrophy [43]. The consumption of miso soup every day is related to lower insulin resistance in women [21]. In fact, the percent body fat mass was lower in women with habitual miso consumption than in those without it, in this study. Furthermore, miso is a product made of fermented soybeans. Soybeans are rich in soybean-specific proteins (glycinin and conglycinin), vitamin E, isoflavones (daidzein, daidzin, genistein, and glycitein), lipids rich in polyunsaturated fatty acids, lecithin, and saponin [17,44,45]. Miso also contains pyroglutamyl leucine [46], which is spontaneously generated from peptides with a glutaminyl residue at the amino terminal, during storage and processing [47]. Pyroglutamyl leucine was found to improve gut dysbiosis and colitis in mice [46,48,49] and, in fact, suppresses the excess proliferation of phylum Firmicutes, which is associated with obesity [49]. The main ingredient of miso is soybeans. Soybeans contain high amounts of omega-3 and omega-6 fatty acids [50]. There is a negative correlation between omega-3 fatty acids intake and the presence of sarcopenia in elderly patients with T2DM [51]. In addition, both omega-3 and omega-6 intake is known to be important for muscle maintenance [52]. Moreover, soybean proteins and isoflavones are known to reduce accumulation of visceral fat mass in animal models [53,54]. Isoflavones have a structure similar to that of estradiol and have a high binding ability to the primary estrogen receptor in the vascular wall [55]. Therefore, isoflavones in miso may inhibit the accumulation of visceral fat and the loss of muscle mass and strength in women.
Another possible mechanism is the association between habitual miso consumption and diet quality. In this study, women without habitual miso consumption had lower energy intakes than those with habitual miso consumption. Moreover, compared to those with habitual miso consumption, the diets of women without habitual miso consumption showed higher carbohydrate-to-fiber ratios, which has a reported association with metabolic syndrome [39] and visceral fat accumulation [56]. Thus, women without habitual miso consumption may have had poor diet qualities, which may have contributed to obesity and sarcopenia. Adherence to a dietary pattern rich in foods characteristic of the Japanese diet is associated with a lower prevalence of sarcopenia [57]. Miso soup is well-known as a traditional Japanese food. Therefore, the consumption of miso soup contributes to the traditional Japanese diet, suggesting that women with habitual miso consumption have a lower prevalence of sarcopenia. On the other hand, habitual miso consumption was not associated with the presence of sarcopenia in men. However, it is unclear why there is no association between miso consumption and sarcopenia in men. It is probably because there was no association between habitual miso consumption and diet quality in men. In fact, a previous study revealed an association between habitual miso soup consumption and insulin resistance in women but not in men [21].
In women without a habit of smoking, habitual miso consumption was associated with the presence of sarcopenia, but in women with a habit of smoking, habitual miso consumption was not associated with the presence of sarcopenia. This is due to the high oxidative stress caused by smoking [58]. In fact, the habit of smoking is known to be a risk factor of sarcopenia [59].
However, our study had certain limitations. First, the data regarding the frequency of miso soup consumption were self-reported, and there were some concerns regarding the accuracy of the data. Second, because of a cross-sectional study, it was not possible to show the causal relationship. Third, the sample size of this study was relatively small. Fourth, women without habitual miso consumption might have poor quality of diet, thus, we cannot deny the possibility that diet quality contributed to this result. Finally, all study participants were Japanese patients only; hence, it is unclear whether the results can be generalized to other ethnic groups.
In conclusion, this study firstly revealed that habitual miso consumption was associated with a low presence of sarcopenia in women but not in men. Although further research is needed, miso soup is a part of the traditional Japanese diet, and it has been suggested that consuming miso soup may help prevent sarcopenia.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to restrictions eg privacy or ethical.