Volume 27, Issue 4 (Autumn 2021)                   Intern Med Today 2021, 27(4): 502-517 | Back to browse issues page

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Dehbashi M, Fathi M, Attarzadeh Hosseini S R, Mosaferi Ziaaldini M. The Effect of Eight Weeks Endurance Training, Somatropin Injection, and Its Lipolytic Fragment (AOD9604) on Cytokeratin-18 and Liver Enzymes of Mice Induced Liver Damage Due to a High-Fat Diet. Intern Med Today 2021; 27 (4) :502-517
URL: http://imtj.gmu.ac.ir/article-1-3621-en.html
1- Department of Sport Physiology, Faculty of Sport Sciences, Ferdowsi University of Mashhad, Mashhad, Iran.
2- Department of Sport Physiology, Faculty of Sport Sciences, Ferdowsi University of Mashhad, Mashhad, Iran. , mfathei@um.ac.ir
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1. Introduction
For years, medical scientists have been looking for ways to overcome health-related abnormalities, among which inactivity combined with a high-fat diet is one of the most important drivers of obesity and diseases, such as the fatty liver. Nonalcoholic Fatty Liver Disease (NAFLD) is the most common cause of chronic liver disease. It is the main indication of liver transplantation in developed countries, and its global prevalence is estimated at 25% [1 ,2]. NAFLD includes a wide range of liver damages that progresses from simple steatosis to steatohepatitis, fibrosis, cirrhosis, and eventually hepatocellular carcinoma [3]. This disorder results from the deposition and accumulation of fat microvesicles in the cytoplasm of hepatocytes. This accumulation accounts for more than 5% to 10% of the liver weight and severely damages hepatocytes [4]. Insulin resistance, inactivity, and overweight increase the secretion of free fatty acids from exogenous and endogenous sources [5]. Increased re-lipogenesis and abnormalities in beta-oxidation [6] can increase fat deposition in the liver. In 90% of cases, the general symptom of fatty liver disease is elevated serum aminotransferase levels [7]. Maximus et al. recently showed that the increase in plasma Alanine Transaminase (ALT) is mainly due to insulin resistance in adipose tissue and liver triglyceride content. Therefore, improving NAFLD can effectively reduce liver enzymes [8]. Also, there is a direct relationship between Cytokeratin-18 (CK18) and liver enzymes [9]. CK18 is considered one of the most accurate serum markers in diagnosing patients with fatty liver. It is the major liver protein strand involved in hepatocyte damage and cell death, and is released into the bloodstream by caspases during apoptosis [10]. This cytokeratin is broken down by caspases 3 and 6 at two sites (ASP238 and ASP396) during apoptosis. The broken CK18 fragments are stable, resistant to proteolysis, and can be measured in plasma and serum [11].
The effects of NAFLD in patients with the disease cause a wide range of problems for the individual, including impaired growth hormone secretion [12]. In a cross-sectional study, low Growth Hormone (GH) levels were associated with a higher prevalence of NAFLD [13]. However, GH, as a single-chain polypeptide with 191 amino acids, has a two-way function in the face of reduced visceral fat: it promotes increased insulin resistance while stimulating lipolysis [14].
 Insulin resistance is one of the most important primary pathophysiological mechanisms in developing this disease. This mechanism is associated with ectopic accumulation of fat in the liver, causing an increase in fatty acids in this organ [15]. Many researchers believe in the key role of insulin resistance in stimulating the accumulation of fatty acids in liver cells and consider it as the most important primary pathophysiological mechanism in the development of fatty liver disease. Insulin resistance increases fatty acid invasion in the liver in favor of lipogenesis and prevention of fats lipolysis [16]. However, growth hormone inhibits adipocyte differentiation, reduces triglycerides, and increases lipolysis through the G protein signaling pathway [17]. The 15 GH terminal amino acids stimulate lipolysis, which was first documented by pharmacological researchers at Monash University in Australia. AOD9604 is a peptide fragment of C-terminus HGH (Tyr-hGH177-19-19), a polypeptide available as AOD9604 (Fragment); it contains only 176 to 191 GH amino acids [18]. Importantly, the fragment does not stimulate insulin resistance, so its lipolytic response is higher than the growth hormone. It is stated that it has no growth and synthesis effects like growth hormone [17]. 
It is well known that physical activity is an excellent way to improve health, prevent, and treat obesity-related diseases. Gharghani et al. examined the effect of eight weeks of aerobic exercise on the glucose and liver enzymes response of NAFLD-induced mice. This study showed an improvement in glucose and liver enzyme responses [19]. Based on studies, exercise improves insulin resistance, increases receptor sensitivity to insulin, and generally improves glucose and fatty acid intake. All of these properties are major pathogenesis pathways of NAFLD [20]. Exercise also enhances the GH response [21]. However, despite the importance of this hormone, research on the effect of GH in the face of NAFLD is minimal and is unknown mainly due to the duality of its impact. In this study, by isolating the lipolytic fragment of growth hormone, for the first time, we could examine the effects of insulin resistance and the lipolytic fragment of this hormone both separately and in combination (GH and AOD9604). We also investigated the impact of exercise activity on nonalcoholic fatty liver-dependent factors.
2. Materials and Methods
The current study is an experimental study. The animal model consisted of adult male mice weighing 20±3 g. After ten weeks of mice feeding with a high-fat diet and confirmation of fatty liver induction in them, 28 mice were randomly divided into four groups of seven each: Control group (C), Exercise (E), Exercise + Fragment (EA), exercise + growth hormone (EGH). All groups underwent a high-fat diet until the end (Table 1).

During this time, the mice were kept under controlled conditions of light (12:12 h light:dark), temperature (22°C±3°C), and humidity (about 45%). It should be noted that the mice entered the study after initial confirmation of the damage by an ultrasound specialist and examination of liver enzymes. The mice under exercise had five training sessions per week, according to Table 2.

At the end of the study, 48 hours after the last training session, their weights were assessed, and the rats were anesthetized. The animal’s chest was then dissected, and direct blood samples were taken from the animal’s heart to ensure minimal harm to the animal.
High-fat diet
The high-fat diet consisted of the rodent-based diet, which was developed by the researcher with the addition of 12% animal fat, 2% cholesterol (German Merck), and 1% colic acid (Sigma America Co). This formula is suitable in terms of calories and energy needed to induce fatty liver (Table 1) [2223].
Endurance training
According to Table 2, the aerobic training program was performed for eight weeks using a special rodent machine made in Iran. First, the conveyor belt was measured with a meter, and then a cutoff point was identified as a criterion. Then, the elapsed time for a complete lap of the belt was inserted into the velocity formula and matched with the monitoring of the device. At first, a week of familiarity with the sports environment was on the agenda, and the mice worked out for 10 to 15 min every day at a speed of 10 m/min for adaptation. From the beginning of the second week, the endurance training program was carried out (Table 1). The control group was kept with only a high-fat diet without any activity [24].
Injection protocol
Injection protocol includes daily intraperitoneal injection of Cinnadaro somatropin (Sinagen made in Iran) 1 mg/kg of animal body weight and lipolytic component (AOD9604) (manufactured by Auspep Australia) 250 mc/Kg of body weight in mice [25]. It should be noted that the animal sample of the mentioned derivatives was not available for the animal model.
Blood sample collection and measurement
To avoid misinterpretation of the data, the mice were injected 48 hours after the last training session. After a night fast, the mice were anesthetized intraperitoneally with a combination of xylazine (8 mg/kg) and ketamine (75 mg/kg). Then, direct blood samples were taken from the animal’s heart at the rate of 1.1±2 mL. After 15 min of centrifugation at 3000 rpm, their serum was separated and frozen at -80°C until further analysis.
The Amount of Aspartate Transaminase (AST) (sensitivity: IU/L2), Alanine Aminotransferase  (ALT) (sensitivity: IU/L4), and glucose (sensitivity: 5 mg/dL) were measured using Pars Azmoun measuring kit and calorimetric method by autoanalyzer (Hitachi 912 made in Japan). Also, the ELISA method was used to measure the levels of insulin and CK18 (Biological Assays kit made in China with a sensitivity of 0.031 ng/mL and 0.023 ng/mL, respectively). To evaluate insulin resistance, the evaluation model method (Homeostatic Model Assessment for Insulin Resistance [HOMA-IR]) was used according to the following formula [26]:
HOMA-IR=fasting insulin(L/mU) × fasting glucose(L/mol) / (1.14)
Statistical analysis
After collecting and entering the data into SPSS version 26, descriptive statistics were used to calculate the central tendency and dispersion indices. The Shapiro-Wilk test was used to ensure the normal distribution of data. Also, the inter-group difference means were analyzed by 1-way ANOVA and Tukey’s post hoc test. The significance level was considered less than 0.05.
3. Results
Table 3 presents the weight changes of the mice after induction and intervention.

Based on Table 4, the results of comparing the means and Tukey test showed that the intergroup changes of the HOMA-IR were statistically significant in the two groups of E (P=0.00) and EA (P=0.03) and lower compared with the control group, but the changes in this factor in the EGH group were not significant compared to the control group (P=0.37).

Also, the intergroup differences of insulin resistance index between the two groups of EGH and EA was significant (P=0.00). Still, the difference between the two groups of E and EA (P=0.09) was not significant.
Our results showed that CK18 showed a significant decrease only in group E (P=0.00) compared to the control group and was not significant in the two groups of EA (0.17) and EGH (0.61). Also, the inter-group comparison of CK18 values in the EGH group was significantly higher than the two groups of E (P=0.00) and EA (P=0.04). Also, the difference between groups E and EA was not significant (P=0.28).
Changes in AST were not significant in either group compared to the control group and inter-groups (P> 0.05). ALT values in all three groups of E (P=0.00), EGH (P=0.03), and EA (P=0.00) were significantly lower than the control group. However, the difference between groups E and EA (P=0.89) and groups E and EGH (P=0.99) and also groups EA and EGH (P=0.81) were not significant.
4. Discussion
Growth hormone deficiency in adults is associated with decreased muscle mass, increased visceral fat, abnormal fat characteristics, and insulin resistance. In addition, functional dysfunctions of the liver with hyperlipidemia and NAFLD are often seen in these individuals, usually accompanied by metabolic syndrome [12]. Some human studies have shown that GH prescription and its derivatives in adults may reduce visceral fat and improve cardio-metabolic disorders [27]. However, some studies have raised concerns about increased insulin resistance and impaired fasting glucose during GH treatment, especially in obese and elderly patients [28]. Insulin resistance and obesity are two important elements in the pathogenesis of NAFLD; both increase the flow of free fatty acids from subcutaneous and visceral fat to the liver and increase the intrahepatic synthesis of fats. The current study results indicate the reduction of HOMA-IR levels in groups E and EA, consistent with Chang et al. and Lambert et al.’s studies [29]. The effect of 12 weeks of the exercise was assessed in Chang’s study on male C57BL6 mice. The study mice were divided into control and high-fat diets and performed five sessions of endurance training each week. Their results showed that exercise reduced insulin sensitivity and increased pancreatic beta-cell function [30].
 Increased defects in glucose metabolism and insulin resistance as one of the main preconditions for NAFLD are associated with disease progression and fibrosis. Correcting these conditions is an essential part of NFALD treatment [31]. In general, the insulin messaging pathway can be examined in two main metabolic and non-metabolic pathways. In the non-metabolic pathway, mediators are activated that eventually enter the nucleus and cause the expression of some genes. However, the metabolic pathway activates storage pathways, such as lipogenesis and glycogenesis, and locate the glucose transporter in the membrane. Thus, glucose enters the insulin target cells and is stored in the cell [32]. Exercise increases insulin function by reducing intracellular triglyceride accumulation and increasing fatty acid oxidation [33]. Other mechanisms that may increase insulin action after aerobic exercise include increased insulin receptor signaling, increased GLUT4 glucose transporter protein, increased glycogen synthetase and hexokinase activity, decreased release, increased clearance of free fatty acids, increased release of glucose from the blood to muscle due to increased muscle capillaries and changes in muscle composition to increase glucose uptake [20].
 Regarding the results obtained in the EA group, no study was found that measured the effect of the fragment on insulin resistance with and without exercise. The decrease in insulin resistance in this group can be attributed to the impact of exercise because the lipolytic effect of the fragment is estimated to be several times GH. So, the amount of free fatty acid due to lipolysis of this peptide fragment is higher, and the fragment increases β3-AR receptor expression in rodents, which increases fat metabolism [25]. It also stimulates lipolysis in visceral adipose tissue by activating hormone-sensitive lipase, leading to the movement of free fatty acid (FFA) into the bloodstream. Increasing plasma fatty acids by various methods may affect the pathway of insulin message transmission and cause dysfunction of molecules effective in insulin message transmission [28]. So one of the ambiguities that was answered in this study was the lack of development of insulin resistance due to fragment injection, while the HOMA-IR in the EGH group was significantly higher than that in the E and EA groups. This result is consistent with the findings of Healy et al. and, in contrast with Matsumoto et al. Healy in a double-blind study, measured the effect of high-dose somatropin injections in 11 male athletes with four weekly endurance training sessions. The groups were divided into control and intervention groups, and the daily injection of somatropin in the intervention group was 0.067 mg/kg body weight. They reported that after the first and fourth weeks of fasting, insulin and HOMA-IR levels increased significantly [34]. In the Matsumoto study, patients were treated for the fatty liver with GH deficiency. It was shown that after 24 months of growth hormone injection, changes in the HOMA-IR index were not significant despite the relative increase and NAFLD-dependent liver factors decreased. In justifying these differences, it can be stated that in our and Healy’s research, the study population used was engaged in exercise, and that excess injection of somatropin peptide may have produced these results. According to exercise reports, exercise alone stimulates GH [21], and injecting this hormone can have dual effects. While the study population used in the second study, had growth hormone secretion defects, and its exogenous compensation did not alter the effects of induction of this hormone on the HOMA-IR index. It should be noted that this factor did not increase in the EGH group compared to the control group, but despite exercising in this group, HOMA-IR did not show a positive change. To justify this result, we can say that GH increases glucose production through gluconeogenesis and glycogenolysis from the liver and kidneys while simultaneously suppressing the levels of glucose carriers (GLUT1) and GLUT4 in the plasma membrane of adipocytes. So unlike exercise, GH has diabetogenic effects [35 ,28].
 Decreased CK18 levels in group E are in line with the studies of Takahashi et al. (2020) and Philly et al. (2012). In the Takahashi study, 50 patients with NAFLD were divided into two groups of resistance training and control for a 12-week trial period. During the study, the exercise group performed three times a week on non-consecutive days, and their results showed that the serum levels of CK18 and FGF21 fragments decreased due to exercise in these individuals [36]. Philly’s study evaluated the effect of seven days of running training on CK18 in 13 overweight people with NAFLD. They found that 60 minutes of short-term, aerobic exercise reduced CK18 maximal heart rate by 80% [36]. In justification of this result, it has been shown that hepatocyte apoptosis indices are reduced by exercise due to its antioxidant activity and increased insulin sensitivity, so exercise provides an anti-apoptotic stimulant that leads to a decrease in CK18 fragments. In addition, this effect can be modulated by a decrease in the pathway of fatty acid synthase (FAS1). FAS, as a glycosylated protein, is expressed in the liver. It is activated by FAS ligand binding, leading to liver cell death complex (apoptosis), and FAS expression is increased in patients with fatty liver [3]. The inter-group differences of CK18 in the EGH group were higher than those of E and EA groups. A study that examined the effect of GH training and injection on CK18 was not found, but based on the relationship between apoptosis and insulin resistance, this effect can be considered one of the reasons why CK18 is higher in this group.
Regarding liver enzymes, a decrease in ALT was observed in the intervention groups compared to the control group, while changes in AST were not significant. Regarding ALT, our results agree with some studies, such as Takahashi et al. (2017) and Farzanegi et al. (2019). In the Takahashi study, the effect of both aerobic and resistance training was evaluated in 103 patients with NAFLD for 22 weeks. Their results showed that ALT enzyme and TG levels were reduced in the aerobic exercise group compared to the control group, while these changes were not significant in the resistance training group [37]. A significant decrease in ALT enzyme due to exercise can be attributed to increased liver oxidation, decreased activity and inhibition of lipogenic enzymes, increased sensitivity to tissue and liver insulin and thus reduced liver fat [38]. Exercise also protects the liver against oxidative stress and endoplasmic reticulum stress [39] and improves autophagy, [40] which are all mechanisms representing cell liver damage in NAFLD. However, ALT results were not significant between the three groups of E, EA, EGH. It seems that in all three groups, the positive effects of exercise on other independent variables are dominant. Regarding AST, despite the relative decrease in the two groups of E and EA, the changes were not significant. Our results contradict the research of Farzanegi et al. [41] and Huber et al. [42]. In justifying this effect, we can point out the differences between the intervention in our research and other inconsistent research. On the other hand, the release of AST from organs, such as the heart and muscle, makes ALT more specific for the liver than AST. Such factors as differences in diets, the effects of exercise, and the effects of fatty liver induction with peptide injections may affect other organs releasing this enzyme, which need to be studied separately.
5. Conclusion
Exercise has a greater response to improve NAFLD markers than the two peptides of GH and fragment. The use of growth hormone may have negative consequences for some indicators of this abnormality, a response not found in its lipolytic fragment. We found that the role of insulin resistance in apoptosis may be greater than the reduction of hepatic fat oxidation pathways. Also, the fragment did not have a greater positive effect on exercise, despite the greatest reduction in liver enzymes and the lack of insulin resistance. Overall, given that numerous non-clinical studies have shown no evidence of genotoxic or toxicological concerns about the safety of AOD9604 [35, 43], it may have positive biological effects in humans. However, this effect requires further research to prove.
Research limitations
 Among the limitations of this research are the unavailability of animal growth hormone samples and their lipolytic fragment and the use of human derivatives for the animal model. Also, minimal research on AOD9604 was another limitation of this research.
Study suggestions 
Due to the importance of the subject and the results of this study, it can be helpful to study the effects of AOD9604 and various sports exercises on factors related to the fat profile (adipokines, hormones).

Ethical Considerations
Compliance with ethical guidelines

All ethical principles of the Helsinki Declaration were observed, and the research was approved by the ethics committee of the Ferdowsi University of Mashhad (Code: IR.UM.REC.1399.070).

This article was extracted from the PhD. dissertation of The last author at the Student Research Committee of the Faculty of Sports Sciences of the Ferdowsi University of Mashhad.

Authors' contributions
Study concept: Mohsen Dehbashi and Dr. Mehrdad Fathi; Writing the manuscript: Mohsen Dehbashi; The article confirmation and data analysis: Dr. Mehrdad Fathi, Professor Seyed Reza Attarzadeh Hosseini, and Dr. Mohammad Mosaferi Ziauddin.

Conflicts of interest
The authors declared no conflict of interest.

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Type of Study: Original | Subject: Physiology
Received: 2020/12/1 | Accepted: 2021/02/17 | Published: 2021/09/23

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