The population of obese individuals is increasing in Japan because of sedentary lifestyles characterized by the excessive consumption of energy-dense, nutrient-poor food and the chronic insufficiency of physical activity (5). A growing body of literature well documents the health risks of obesity-related morbidity and mortality and, particularly, an accelerated accumulation of lipids at ectopic sites. Visceral adipose tissue is now generally recognized as a major causal factor in determining the pathophysiology of metabolic, neoplastic, cardiovascular, and other consequences of excess adiposity (3). Recently, a novel condition has arisen: nonalcoholic fatty liver disease (NAFLD) characterized by the excessive accumulation of hepatocellular lipids, for example, steatosis, is strongly associated with obesity and its sustaining conditions and independently increases the risk of serious metabolic disorders (25). Furthermore, Fabbrini et al. (6) reported evidence that hepatic steatosis is the main culprit in the metabolic disorders associated with obesity, rather than any present visceral adipose tissue. These facts indicate that therapeutic strategies that could substantially improve obesity-related liver disease are necessary.
Currently, weight reduction is well established as the most effective therapy advocated for treating obesity-related liver disease. In general, dietary restrictions aimed at weight reduction are recommended as the optimal management strategy for obesity-related liver disease (14). However, weight reduction via dietary restriction is almost always modest and is quite difficult to achieve and maintain. For this reason, therapies that efficiently modulate obesity-related liver disease, but are not contingent upon weight reduction by dietary restriction, are of major pragmatic significance (18).
In that regard, exercise training could potentially be effective for obesity-related liver disease management. Clinical evidence has shown that higher levels of physical activity are associated with lower hepatocellular lipid levels (32,40). In addition, it has been proven that the therapeutic effect of exercise training upon liver function and metabolic disorders in obesity is independent of detectable reductions in body weight (12,18,32).
On the basis of these results, it would be preferable to revaluate strategies by debating the beneficial effect of exercise training in the prevention and treatment of obesity-related liver disease. Further, the molecular mechanism underlying the more desirable effects of exercise training on obesity-related liver disease should be explored.
Thus, we conducted a retrospective analysis of a large number of obese, middle-age men who had completed a 12-wk supervised exercise training program without any dietary restriction to determine whether exercise training without dietary restrictions improves the pathophysiology of obesity-related liver disease. To explore the advantages of an exercise training program without any dietary restriction, we compared these results with those of obese, middle-age men who had completed a 12-wk supervised dietary-restriction program.
MATERIALS AND METHODS
Figure 1 presents a simple flowchart of enrollment in the program. For the current study, which was performed from 2006 to 2010 at the University of Tsukuba in Japan, obese, adult men (BMI > 25 kg·m−2, according to domestic obesity guidelines) were recruited from the Ibaraki prefecture (Kanto region of Japan). Participants were sedentary (defined as exercising <30 min·d−1 over the previous year or more) with no experience of enrollment in any weight-reduction programs within the last year and maintaining a stable weight for at least 12 wk (±3 kg of current body weight). A total of 272 men responded to a general outreach through a local newspaper, distributed fliers, and a public broadcast on local cable TV, asking for a 5-yr commitment to participate in a program of either weight loss via exercise training (E) or dietary restriction (D). The participants trained for 12 wk and were involved in two different intervention groups (E and D). From the accumulated data, we excluded those participants with the following: younger than 35 yr or older than 65 yr, BMI > 35 kg·m−2, withdrawn, low attendance (below 70%), and errors or deficits in data. Finally, of the initial 272 participants, 212 (E total group, ET: n = 108; D total group, DT: n = 104) subjects were recruited, whose data were analyzed and discussed in this study. From among these groups, we classified men with suspicious liver fibrosis (NAFLD fibrosis score2 > −1.455, alcohol consumption < 21 U·wk−1) through further analysis to get a more concrete outcome for the exercise-training effect (ET with abnormal liver function and suspicious liver fibrosis by NAFLD fibrosis score, EN group: n = 42; DT with abnormal liver function and suspicious liver fibrosis by NAFLD fibrosis score, DN group: n = 29).
The protocol of this interventional study was approved by the institutional review board at the University of Tsukuba; written informed consent was obtained from all participants before their entry in the study.
Exercise training program
Subjects in the ET and EN groups of approximately 25 people per class underwent an aerobic exercise-training protocol under the supervision of six skilled professional trainers. The exercise training protocol consisted of (a) 15–20 min warm-up sessions, including stretching; (b) 40–60 min fast walking and/or light jogging sessions; and (c) 15–20 min cool-down sessions that included resistance training. This class was held 90 min·d−1, three times a week for 12 wk. As participants were taking part in the exercise training class, we provided face-to-face individual counseling to help set up the proper intensity and monitor participants’ general state of health. The exercise intensity was set such that it raised the participants’ heart rate to 60%–85% of their maximum heart rate.
The exercise intensities were monitored using short-range telemetry (Polar RS400, Polar Electro Oy, Kempele, Finland) and a single-axis accelerometer (Lifecorder; Suzuken Co. Ltd., Nagoya, Japan). The daily energy expenditure (EE) of participants was assessed every day by using the single-axis accelerometer. EE recording was started 2 wk before the start of the intervention and continued during the intervention period.
Dietary restriction program
Participants in the DT and DN groups were provided dietary restriction (1680 kcal·d−1) through a nutritionally balanced diet program. The program included 12 weekly lectures and small interactive group sessions to help participants maintain their dietary restriction. During the 12 lectures, participants were provided food diaries that included several instructions for creating a daily report, for example, personal goal setting, body-weight change, and the state of their physical and mental condition. After every weekly lecture, skilled dietitians offered detailed comments on the food diaries and one-on-one dietary behavioral counseling to assist the participants to adhere to the proper energy intake and encourage them to maintain the program.
For the daily energy intake (EI) assessment, after receiving detailed instructions, men completed the dietary records for three continuous days at the baseline and at the 12th week, under the supervision of a skilled dietitian. The dietary records were analyzed using Eiyoukun software version 4.0 (Kenpakusya, Tokyo, Japan) by a skilled dietitian.
Anthropometric parameters were measured at baseline and 12 wk. We measured their body weight to within 0.1 kg using a digital electronic scale with the men dressed in light clothing (TBF-551; Tanita, Tokyo, Japan) and the standing height to within 0.1 cm with a wall-mounted stadiometer (YG-200; Yagami, Nagoya, Japan). We used these data to calculate the BMI (kg·m−2). Body composition (fat mass and lean body mass) was evaluated using dual energy x-ray absorptiometry (DXA, Lunar DPX-L, Lunar Corp., Madison, WI; or QDR® 4500, Hologic, Inc., Bedford, MA) and used to calculate the body fat percentage. Abdominal fat area was determined by computed tomography scan (Aquilion 16, Toshiba, Tokyo, Japan; or Somatom AR.C, Siemens, Erlangen, Germany) in which the total abdominal fat area, visceral abdominal fat area, and subcutaneous abdominal fat area were measured at the level of the umbilicus. Waist circumference was measured to within 0.1 cm using a fiberglass tape at the umbilicus level.
Maximal oxygen uptake (V˙O2 max) tests were conducted using a graded direct cycling ergometer (Monark 828E; Monark, Stockholm, Sweden) under the supervision of a medical practitioner and a physical trainer. The workload was increased every 1 min for 15-W intervals after a 2-min warm-up period at 30 W, until volitional exhaustion occurred. Ventilation and gas exchanges were measured using a breath-by-breath gas analyzer (Oxycon Alpha, Mijnhardt, Breda, Netherlands; or Aero monitor AE-280, Minato Medical Science, Osaka, Japan), and heart rate was assessed using an electrocardiograph (Dynascope; Fukuda Denshi, Tokyo, Japan) during the V˙O2 max test. In addition, to calculate physical age (21), subjects performed tests to measure balance, agility, flexibility, muscle strength, and endurance, such as side stepping, one-legged stance with eyes closed, trunk flexion, trunk extension, vertical jump, and grip strength.
Using a butterfly needle, blood samples were drawn from the median cubital vein at the baseline and at the 12th week after a period of no exercise for 48 h and a fasting status of 12 h. The collected blood was placed on ice and transported to the laboratory; then, serum and plasma were separated by centrifugation. The separated fractions were stored at −80°C until further analysis. Levels of triglycerides and HDL-C were analyzed by enzyme method; aspartate aminotransferase, alanine aminotransferase (ALT), and gamma glutamyl transpeptidase levels, by JSCC transferable method; fasting plasma insulin and ferritin levels by chemiluminescent immunoassay method; high-sensitivity C-reactive protein (hs-CRP) level by fixed time assay method; fasting plasma glucose level by enzymatic colorimetric method; and hemoglobin A1c, hyaluronic acid, and plasma type IV collagen levels by latex agglutination method. Commercial ELISA or ECL kits were used to measure serum levels of total adiponectin (Sekisui Medical Co, Ltd., Tokyo, Japan), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and leptin (R&D Systems Inc.; Minneapolis, MN), thiobarbituric acid reactive substances (TBARS) (Cayman Chemical Co.; Ann Arbor, MI), M30 (Peviva AB; Bromma, Sweden), and transforming growth factor beta 1 (TGF-β1) (Meso Scale Discovery; Gaithersburg, MD). The intra-assay coefficients of variation for adiponectin, TNF-α, IL-6, leptin, TBARS, M30, and TGF-β1 in our laboratory are 3.4%, 4.9%, 3.6%, 2.9%, 5.7%, 5.2%, and 4.6%, respectively.
Surrogate markers were calculated from insulin resistance by the homeostasis model assessment (HOMA-IR) derived by Matthews et al. (26) and from NAFLD fibrosis scores according to the equation of Angulo et al. (2), and also from physical age derived by Lee et al. (21).
Statistical analysis was performed using SPSS Statistics for Windows, version 20.0 (IBM Corp.; Armonk, NY). Descriptive parameters show the mean, SE, or proportion (%). Unpaired Student’s t-tests, with the pretreatment score acting as the dependent variable, were performed to examine the differences between the groups before the trials. In case of categorical variables, the chi-square test was used. To compare intragroup changes over time (at the baseline and 12th week), all dependent variables were analyzed by a paired Student’s t-test. We also compared variables between groups that changed from the baseline to the 12th week using either the unpaired Student’s t-test or ANCOVA. A P value of <0.05 was defined as statistically significant.
A total of 212 subjects, 108 of whom participated in an exercise training program and 104 in a dietary restriction program, were entered into the current study. Moreover, among these participants, 71 (n = 42 in EN and n = 29 in DN) had abnormal liver function and suspicious liver fibrosis. These 71 participants were further analyzed to obtain a more solid outcome for determining exercise training effects.
At the baseline assessment, there were no significant differences in the proportion of age, alcohol, smoking, and medication for participants between the ET and the DT groups and between those in the EN and DN groups (data not shown). The mean values of daily EI and EE and of the anthropometric and physical capacity parameters were not significantly different (Table 1). The attendance rate was 80.3% in the ET group, 78.7% in the DT group, 83.2% in the EN group, and 84.6% in the DN group. The difference was not statistically significant. The mean values of the blood test parameters were not significantly different between the EN and the DN groups, whereas there was a statistical difference in the three parameters of hemoglobin A1c, aspartate aminotransferase, and ALT between the ET and the DT groups (P < 0.05).
Tables 1 and 2 show the results of intervention adherence for each group during the intervention periods. According to the 3-d food record, although EI decreased significantly in the DT and DN groups, EI did not change significantly in the ET and EN groups from the baseline to the 12th week. EE assessed using the single-axis pedometer increased significantly in the ET and EN groups but not in the DT and DN groups from the baseline to the 12th week. Collectively, both DT and DN groups showed a significant decrease in EI (P < 0.01), and both ET and EN group showed a significant increase in EE (P < 0.01).
Anthropometric and physical capacity
In the results for anthropometry and physical capacity for the 108 participants in the ET group and the 104 in the DT group (Table 1), all of the parameters except total lean mass (TLM) were improved at the 12th week in both groups, whereas TLM was maintained at the 12th week in the ET group but was decreased in the DT group. When a comparison was made between the groups, the magnitude of the changes in TLM (P < 0.01) and V˙O2max (P < 0.01) was greater in the ET group and that of the changes in the other parameters was greater in the DT group. The results for the 42 participants in the EN group and the 29 in the DN group (Table 2) seemed to copy those for the 108 participants in the ET group and the 104 in the DT group (Table 1).
In the results of the blood test parameters for the 108 participants in the ET group and 104 in the DT group (Table 1), all nine parameters were improved at the 12th week in both the ET and the DT groups. When an intergroup comparison was made, the magnitude of change in only adiponectin was greater in the ET group (P < 0.01) than that in DT group.
Similarly, in the results of the blood test parameters for the 42 participants in the EN group and the 29 participants in the DN group (Table 2), all seven parameters were improved at the 12th week in both groups. However, a comparison between the groups revealed that the magnitude of the changes in these parameters was not significantly different.
With regard to adipokines and inflammatory cytokines (Fig. 2), exercise training for 12 wk significantly reduced the serum levels of TNF-α by 35.0%, leptin by 27.4%, and IL-6 by 26.7%. The training increased the level of adiponectin by 45.0%. By comparison, diet restriction significantly reduced the serum levels of TNF-α by 52.7%, leptin by 54.1%, IL-6 by 28.3%, and TGF-β by 9.4%. Moreover, when a comparison was made between the EN and the DN groups, the magnitude of the increase in adiponectin was greater in the EN group (P < 0.05) but the decrease in TGF-β1 was greater in the DN group (P < 0.05).
Serum markers of oxidative stress (TBARS) and hepatocyte apoptosis (M30), inflammation (hs-CRP and ferritin), and liver fibrosis components (hyaluronic acid and type IV collagen) were determined in 72 subjects in EN and DN groups. Figure 3 shows the results of changes in these parameters in each group from the baseline to the 12th week. In the EN group, significant changes were observed in four parameters: TBARS by −33.5%, hs-CRP by −49.5%, ferritin by −25.0%, and hyaluronic acid by −25.4%. However, changes in the parameters of type 4 collagen by −2.3% and M30 by −2.6% were not significant. In the DN group, significant changes were observed in three parameters: M30 by −17.5%, hs-CRP by −35.3%, and hyaluronic acid by −41.5%. However, changes in the parameters of TBARS by −10.5%, ferritin by +1.1%, and type 4 collagen by −3.0% were not significant. A comparison between the EN and the DN groups revealed that the degree of change from the baseline to the 12th week was statistically significant for TBARS, M30, and ferritin (P < 0.05).
The results of changes in the surrogate markers of physical age, HOMA-IR, and the NAFLD fibrosis score are shown in Tables 1 and 2. The physical age and HOMA-IR markers were improved in all four groups from the baseline to the 12th week (P < 0.01). However, for the NAFLD fibrosis scores, although the EN group showed an improved value (P < 0.05), the other 3 groups, ET, DT and DN, did not show any significant change for that value at the 12th week. A comparison between the ET and the DT groups and between the EN and the DN groups revealed that there was a significant improvement for physical age markers (P < 0.01) but not for HOMA-IR or the NAFLD fibrosis score in the ET and EN groups.
NAFLD covers a spectrum of hepatic pathologies, ranging from simple steatosis, through steatohepatitis to fibrosis and eventually liver cirrhosis (9). It is now considered as the hepatic manifestation of obesity and metabolic syndrome (34), which are closely associated with low-grade chronic inflammation, adipokine imbalance, and impaired redox signaling (35). The results of this study revealed that in the 42 subjects with abnormal liver function and suspicious liver fibrosis, exercise training reduced the elevated levels of ferritin and TBARS (Fig. 3) and also increased adiponectin levels in the serum (Fig. 2). Increased serum levels of ferritin and TBARS are an indicator of hepatic inflammatory condition (20), and an in vivo increased lipid peroxidation (16). To the best of our knowledge, these data provide the first direct evidence that exercise training improve inflammation and its related lipid peroxidation levels, both of which may contribute to enhanced innate immunity and dysregulation of pro-inflammatory signaling pathways in obese subjects with NAFLD (38), demonstrating the advantages of exercise training in comparison with dietary restriction.
In NAFLD, a considerable amount of hepatic iron accumulation has been observed (24) and molecular mechanisms, including the down-regulation of the iron exporter ferroportin-1 (Fpn1), have been described (1). Iron excess, coupled with imbalance in adipokine production (23), will further promotes inflammatory progression that in turn increases oxidative stress levels, hampers insulin extraction, and fibrosis in the liver (11, 20). Moreover, the increased levels of TBARS, a lipid peroxidation marker, indicate high oxidative stress. Lipid peroxidation has been shown to impair nucleotide and protein synthesis in the liver, consequently resulting in apoptosis, inflammation, and fibrosis (22). Therefore, the reduction in levels of serum ferritin and TBARS resulting from the 12-wk exercise training contribute to the improvement in the pathophysiology of obesity-related liver abnormalities, e.g., inflammatory conditions, insulin resistance, and liver fibrosis.
Another important finding of this study is that exercise training in obese subjects with NAFLD decreases the serum levels of leptin, an adipokine, and pro-inflammatory cytokines, for example, TNF-α and IL-6 (Fig. 2), which in turn is associated with the decreases in the serum levels of hs-CRP, ferritin, and TBARS (Fig. 3). It has recently been reported that leptin-mediated signaling regulates hypersensitivity to low-dose endotoxin and enhances inflammation during progression from simple steatosis to NASH (15). In addition, the increased IL-6 level is strongly associated with hepatic steatosis that could potentially induce insulin resistance in subjects with NAFLD (33). Taken together, our results have shown that exercise training improves the inflammatory and oxidative stress conditions in obese subjects, and moreover, this benefit seems independent of detectable weight reduction.
The details of the molecular mechanism by which exercise training reduces hepatic inflammatory conditions and its related oxidative stress levels in obese subjects have not been well understood. However, the experimental results from our laboratories and others can help address these issues. First, exercise-induced oxidative stress, especially during high-intensity exercise, appears to activate a transcription factor termed nuclear factor-E2-related factor 2 (Nrf2) (19,27). Nrf2 restrains the dysregulation of inflammatory signaling pathways by mounting an appropriate innate immune response (36). Nrf2 serves as an oxidative stress sensor and functions as a comprehensive host defense factor (29). Our studies has shown that the activated Nrf2 regulatory pathway can decrease the in vivo levels of pro-inflammatory cytokines and oxidative stress, which in turn are implicated in the pathogenesis of numerous liver abnormalities (29). Second, Nrf2 reportedly regulates iron efflux from macrophages through Fpn1 gene transcription and controls iron metabolism during inflammation (13). Fpn1 is an iron exporter on the surface of absorptive intestinal enterocytes, macrophages, hepatocytes, and placental cells, all of which release iron into plasma (28). As reported by Harada et al. (13), the activation of Nrf2 resulted in increased expression of Fpn1 in macrophages and enhanced the excretion of radioisotope-labeled iron. Third, treatment with potent Nrf2 activators such as 2-cyano-3,12 dioxooleana-1,9 dien-28-imidazolide (31) effectively prevented high-fat diet-induced increases in body weight, adipose mass, hepatic lipid accumulation, and insulin sensitivity in mice. Nrf2 activation may contribute to management of obesogenesis and diabetes. Taken together, Nrf2 is a comprehensive factor that defends the host from oxidative stress in its various aspects.
In addition, the results revealed that serum adiponectin levels were markedly increased by exercise training compared with the increase induced by dietary restriction (Fig. 2). The magnitude of the increase was statistically significant. This offers another advantage of exercise training in comparison with dietary restriction. The decreased adiponectin levels are associated with hepatic steatosis grade and the severity of NAFLD (8). On the other hand, the increased adiponectin improves insulin resistance and enhances the burning of fatty acids, which in turn improves the pathological condition of NAFLD (8). Serum adiponectin levels were inversely correlated with transaminase activity in Japanese male workers (39). Furthermore, hepatic adiponectin signaling played a protective role against progression of nonalcoholic steatohepatitis in an experimental model of NAFLD (37). Thus, although hepatic adiponectin resistance and sensitivity mediated by the adiponectin receptor 2 (37) should be considered, it is likely that the increased serum adiponectin achieved by exercise training without concomitant dietary restriction contributes to the improvement pathophysiology of obesity-related liver disease, according to data from 2006 to 2010.
For clinical relevance, three times a week of exercise training frequency is not an easy task for most individuals with NAFLD, and it is strongly recommended to continue any type of exercise at least once or twice a week. Although the effectiveness of such exercise habituation with lesser frequency is uncertain, similar health benefits to those by three times a week would be obtained according to the findings of recent studies (17). Several the studies have reported evidence indicating the direct effect of exercise therapy on hepatic steatosis independent of weight reduction (12,18), and the greatest hepatic benefit could be obtained with both low- and moderate-exercise volume (i.e., ≤150 min·wk−1), the volume of which is below the current guidelines for health promotion (10) and those for the specific recommendation for managing body weight (i.e., >250 min·wk−1) (4). For example, a low- and moderate-exercise volume of ≤150 min·wk−1 may correspond to the volume provided by once or twice a week of frequency of our exercise training procedure. However, it should be noted that at present, there is much less scientific evidence for benefits of exercise training (and/or physical activity) in isolation and the optimal dose and modality for exercise therapy for NAFLD (7).
In conclusion, the results of this study have shown that exercise training without any dietary restriction provides benefits for the management of obesity-related liver disease through a reduction in inflammation and oxidative stress levels in the liver, which in turn improves insulin resistance (Fig. 4). These conditional changes may arrest the progression of NAFLD from simple steatosis to steatohepatitis. The benefits appear to be unrelated to body-weight reduction. The initial treatment of obesity-related liver diseases uses diet and exercise therapy; although recent large-scale interventional studies have reported the effectiveness of vitamin E, a known antioxidant in adults, in NAFLD (30), no first-line drug therapy has been established thus far. From the viewpoint of increased inflammation and oxidative stress levels in obese subjects, the induction of an in vivo anti-oxidative stress system by exercise training intervention could be a new option for improving the pathophysiology of obesity-related liver disease.
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (grant nos. 23300250, 24390488, 24659104, 24650436, and 25282172).
The authors are grateful to Dr. Miki Eto (Faculty of Health and Sports Science, University of Tsukuba) and Drs. Rina So and Takehiko Tsujimoto (Graduate School of Comprehensive Human Sciences, University of Tsukuba) for their help with part of the intervention trials. They also appreciate Dr. Mijung Kim (Graduate School of Life and Environmental Sciences) and Prof. Randeep Rakwal (Organization for Educational Initiatives) of University of Tsukuba for their critical reading of the manuscript and constructive comments therein.
The authors declare that they have no competing interests.
The results of the present investigation do not constitute endorsement by the American College of Sports Medicine.
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