Circadian rhythm disruption is associated with an increased risk of sarcopenia: a nationwide population-based study in Korea.

Reduced sleep quality is associated with an increased risk of sarcopenia. However, the potential effects of disturbing the regular circadian rhythm, as occurs with shift work, on the risk of sarcopenia have not been established. Data from 9105 workers from the 2008-2011 Korean National Health and Nutrition Examination Survey were analyzed. Sarcopenia, measured by dual-energy X-ray absorptiometry, was defined as one standard deviation below the mean of the appendicular skeletal muscle/body mass index value of a young reference group. Compared to the group that had never experienced shift work, the odds ratio (OR) for sarcopenia with a 95% confidence interval (95% CI) for the shift work group was 1.7 (1.5-1.9); the association remained even after adjusting for confounding variables, including age, sex, total fat mass, insulin resistance profile, smoking, alcohol intake, diet, and physical activity. The results of the subgroup analysis indicated that the highest risk of sarcopenia was among workers engaging in shift work with an irregular schedule (OR 1.8, 95% CI 1.3-2.4). Disruption of circadian rhythm by shift work was associated with increased risk of sarcopenia. Intervention strategies are needed to prevent sarcopenia in shift workers.

Definition of sarcopenia. We defined sarcopenia in three different ways in accordance with the consensus report of the Asian Working Group for Sarcopenia, as below 1,23 : (1) weight-adjusted percentage muscle mass (ASM/ weight squared) below the lowest quintile computed for the study population; (2) height-adjusted percentage muscle mass (ASM/height squared) below the lowest quintile computed for the study population; (3) body mass index-adjusted percentage muscle mass (ASM/body mass index) below the lowest quintile computed for the study population in Body fat (kg) and appendicular skeletal muscle mass (ASM, kg) were measured through dual-energy X-ray absorptiometry (DISCOVERY-W fan-beam densitometer; Hologic, Inc., Bedford, MA) 22 . Among the total study population, subjects below the median value and subjects above the median value of the total study group were designated as the low and high total body fat mass groups, respectively. In the same manner, the low and high axial skeletal muscle mass groups were defined as subjects below the median and above the median axial skeletal muscle mass, respectively.
Definition of type of work and shift work. Type of work was categorized according to occupation: blue-collar workers (simple labor, agriculture, forestry, and fisheries), white-collar workers (professionals and office workers), and pink-collar workers (service workers) 22 .
Workers were divided into those who worked only in the daytime (regular daytime workers) and those with other work schedules (alternative shifts).
Other variables. Dietary patterns were assessed via single 24-h recall 19,22 . Individual energy and nutrient intakes were calculated using the seventh Food Composition Table prepared by the Korean National Rural Resources Development Institute 19,22 . Trained interviewers instructed respondents to recall and describe all foods and beverages consumed during the previous day 19 . Values indicating "excessive, " "appropriate, " and "lacking" in terms of nutrient status were those of the Dietary Reference Intakes for Koreans, revised in 2015. Exercise patterns were classified in accordance with exercise type and frequencies per week, such as walking exercise frequency and strength exercise frequency 19,22 . Smoking status was divided into nonsmoker and current smoker based on the response to the question "Do you currently smoke? 19,22 . " Alcohol drinking status was assigned based on the response to the question "Did you drink more than one glass of liquor during the last month? 22 . " Sleep duration (h/d) was self-reported as an answer to the question "How long do you usually sleep at night?" 22 . Household income was calculated as the sum of the monthly income of all family members and classified into groups including lowest (first quantile), middle-low (second quantile), middle-high (third quantile), and highest (4 th quantile) 22 . Education status was divided into four classes; education for 6 years or less as elementary school; 7-9 years as middle school; 10-12 years as high school; and 13 years or over as college or university. Homeostatic model assessment for insulin resistance (HOMA-IR) was defined as below; HOMA-IR = [Fasting blood insulin (µU/mL) × [Fasting blood glucose (mmol/L)]/22.5.
Statistical analysis. Continuous and categorical variables are expressed as the mean ± standard deviation (SD) and number with percentage (±SE), respectively. General characteristics were presented according to 1) the experience of shift work among the total population, and 2) the presence of sarcopenia among the shift work group. To compare 1) the shift work group vs the non-shift work group, and 2) members of the shift work group with sarcopenia vs members of the shift work group without sarcopenia, t-test and the chi-squared test were used. Odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between shift work experience and sarcopenic status were analyzed using multivariate logistic regression analyses with or without adjustment for confounding factors. Statistical analyses were performed using SPSS ver. 20 for Windows. All tests were two-sided, and P < 0.05 was taken to indicate statistical significance.

Results
characteristics of the study population according to shift work. A total of 9105 workers were included in our analyses. There were no significant differences in education level, income status, presence of a spouse, or medical history between non-shift workers and shift workers (Table 1). Hemoglobin A1C level (HbA1C), high-density lipoprotein (HDL), BMI, and axial skeletal muscle mass index were significantly different between the two groups, whereas fat mass index and waist circumference were not significantly different between the two groups ( Table 1).
Baseline health behaviors according to shift work type. Health behaviors, including diet, physical activity, smoking, alcohol ingestion, and sleep duration were examined according to the type of work schedule www.nature.com/scientificreports www.nature.com/scientificreports/ (Table 2). Energy excess, current smoking, current alcohol ingestion, frequency of strength exercise, and sleep duration of less than 5 hours per night were more prevalent in the shift worker group (Table 2). However, carbohydrate intake, protein intake, fat intake, and frequency of walking exercise were not significantly different between shift work and non-shift work groups (Table 2). www.nature.com/scientificreports www.nature.com/scientificreports/ characteristics of shift workers with and without sarcopenia. There were statistically significant differences in energy intake, smoking status, strength exercise frequency, BMI, total fat mass index, HbA1C, HDL, and homeostatic model assessment for insulin resistance (HOMA-IR) index between shift workers with and without sarcopenia (Table 3). odds ratio of sarcopenia according to shift work type in univariate and multivariate analyses. Sarcopenia was defined using different calculations, and all types of sarcopenia were associated with the type of shift work after adjustment for socioeconomic factors, health behaviors, fat mass, and laboratory indices (Table 4).

Discussion
In this nationally representative cohort study, we found that sarcopenia was more prevalent in shift workers than in those who had never experienced shift work. Shift workers had an increased risk of sarcopenia even after adjustment for potential confounding factors, including age, sex, low-protein diet, leisure time, physical inactivity, sleep duration, smoking, alcohol intake, insulin resistance, mental illness, and socioeconomic factors. Furthermore, among shift workers, irregularly scheduled shift work was more strongly associated with sarcopenia than regularly scheduled shift work. The results of this study suggest that the circadian rhythm disturbance associated with shift work may contribute to an increased risk of sarcopenia.
To our knowledge, this is the first study to reveal an association between shift work, which is associated with circadian rhythm disturbance, and sarcopenia in a large nationally representative population. Although previous studies have shown that sleep duration is related to sarcopenia, the effect of circadian rhythm disturbance on an increased risk of sarcopenia has not been studied in human populations, although it has been examined on an endocrinological basis in molecular studies [24][25][26] .
Interestingly, irregularly scheduled shift work was significantly associated with a higher prevalence of sarcopenia compared to regularly scheduled work, even after adjusting for age, sex, diet, physical activity, alcohol intake, and smoking. No previous studies have directly compared the prevalence of sarcopenia between subjects engaged in regular work and those with irregular shift work. Several preliminary reports indicated a higher prevalence of metabolic syndrome among irregularly scheduled shift workers than among regular shift workers. Sun et al. reported that irregular and unscheduled shift workers had higher rates of obesity than did those engaged in regular rotating night shift work 27 . Further large-scale prospective studies are needed to clarify this issue.  www.nature.com/scientificreports www.nature.com/scientificreports/ Several pathophysiological mechanisms may explain the relationship between shift work and increased risk of sarcopenia 24 . There is emerging evidence of crosstalk between circadian cycling and skeletal muscle metabolism 24 . A preliminary study suggested that the molecular clock, MyoD, and metabolic factors represent a potential system of feedback loops 28,29 . Beta-adrenergic signaling is considered a pathway involved in coordinating communication between circadian clocks in skeletal muscle 24 . Given that circadian rhythm is associated with skeletal muscle recovery and build-up and that shift work is associated with disruption of the circadian rhythm 21,27,30 , shift work and skeletal muscle mass index could be closely associated. However, to our knowledge, no human population-based studies have addressed this issue.
Disruption of the circadian rhythm is associated with the development of a variety of diseases in humans, including metabolic syndrome, cardiovascular disorders, and malignancy 6,7,9,15,31 . Circadian rhythm disruption may lead to disturbance of internal homeostasis for physiological processes and result in the development of pathologies 29 . Indeed, shift workers show high prevalence rates of the diseases outlined above 32 . Moreover, sarcopenic status is known to be closely associated with metabolic syndrome and its complications. The results of the present study indicated that shift work, which disrupts the circadian rhythm, may increase the risk of sarcopenia.
There were several reports that sleep duration <6 hours or >8 hours tended to be associated with sarcopenia. Kwon et al. reported that long sleep duration (≥9 hours) was associated with an increased risk of sarcopenia compared to 7 hours of sleep 19 26 . They revealed that more than 9 hours of sleep duration is independently associated with sarcopenia, especially in women aged > 40 years. In our study, even though <5 hours or > 9 hours of sleep duration were more prevalent in the sarcopenia group compared with the non-sarcopenia group among shift workers, the relationship between sleep duration and sarcopenic status was not statistically significant.
Our study results showed that circadian rhythm disruption is independently associated with sarcopenia even after adjusting for sleep duration. In the present study, we adjusted for sleep duration to examine the association between shift work and the risk of sarcopenia, and we found that circadian rhythm disruption was independently associated with an increased risk of sarcopenia even after adjusting for sleep duration. Sleep duration and circadian rhythm disruption may be associated with sarcopenia in different manners.
This study has several limitations. First, although our results indicated a strong relationship between shift work and sarcopenia, this study should be interpreted cautiously because of its cross-sectional nature. Further large-scale population-based prospective studies are therefore required. Second, although we assessed sleep quality with an extensive questionnaire, other factors may be associated with sleep hygiene, such as obstructive sleep apnea. Obstructive sleep apnea is known to be a determinant of sleep quality and circadian rhythm disruption. Despite these limitations, this study had a number of strengths in that sarcopenia was assessed by dual-energy X-ray absorptiometry in a large nationally representative study population, and we measured extensive potentially confounding factors at baseline, including dietary patterns, leisure time, and physical activity, which are major determinants of sarcopenia.
In conclusion, we evaluated the association between shift work, which is known to disrupt circadian rhythm, and the risk of sarcopenia. As sarcopenia is a well-known risk factor for a variety of diseases, careful examination of sarcopenic status is required among shift workers and other groups with circadian rhythm disruption. Further prospective studies are needed to clarify the causal nature of the relationship between shift work and sarcopenia.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request. Sarocopenia Sarcopenia_ht (N = 1,037, 11.4%) Sarcopenia_wt (N = 1,802, 19