Alteration of Skeletal Muscle’s Satellite cell Differentiation Gene in Young Rats by Nutmeg Supplementation

1Physiology Division, Department of Biomedical Science, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia, 40124. 2Biomedical Sciences Master Program, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia 40124. 3Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran, Jatinangor , Indonesia, 45363. 4Central Laboratory, Universitas Padjadjaran, Bandung, Indonesia. *Corresponding Author Email: hanna@unpad.ac.id

Skeletal muscle plays a major role in physical activities, another important skeletal muscle role is maintaining metabolic homeostasis especially glucose metabolism 1 . Research in the elderly community in Korea showed a greater risk of developing type 2 diabetes mellitus in groups with lower fat-muscle mass ratio [2][3][4] . Skeletal muscle mass affected by various conditions such as aging, cachexia, physical activity, denervation and burning. Gradual and complete reduction in skeletal muscle mass and strength associated with physical disabilities, decrease independence and increasing morbidity and mortality [5][6][7] . Therefore, skeletal muscle mass is an important factor that can affect quality of life. Skeletal muscle mass changed by various stimuli such as exercise, nutrition and micronutrient balance 8,9 . The combination of exercise and proper nutrition proved can induce beneficial molecular signaling pathways to increase and maintain muscle mass. Skeletal muscle mass regulated by the processes of muscle protein synthesis, which lead to muscle hypertrophy and muscle protein breakdown (atrophy) and this process is influenced by physical activities, nutrition such as amino acids, growth factors, glucose and insulin [10][11][12] .
IGF-AKT/mTOR is the main pathway to regulate the skeletal muscle mass by increasing protein synthesis and suppress the protein degradations 13 . Previous studies showed that various physical activities can increase the IGF-1, Akt and mTOR expression in human and its downstream pathways to induce skeletal muscle hypertrophy 14,15 . Notably, the mTOR is known as the main regulator in skeletal muscle regulation. The mTOR regulate the process of protein translation and also known as one of the major pathways to regulate autophagy 16 . Autophagy is a process of self-eating the damaged cellular organs, recycling proteins, protect cells from cellular stress or nutritional limitations and to regulate cell death pathway. Therefore, the autophagy prevents the accumulation of damaged organelles and maintain the myofiber homeostasis.
In skeletal muscles, autophagy needed to maintain the muscle mass and the myofiber integrity 17,18 . However, excessive autophagy can lead to muscle wasting 19 . The activated mTOR, through AKT and AMPK signaling can suppress the autophagy process 20,21 . Previous studies showed resistance training combined with endurance exercise effective to prevent autophagy.
Akt pathways also involved in the satellite cell activation by increasing the expression of MyoD 22,23 .MyoD is a family member of myogenic regulatory factors (MRF) that play a role in regulating the process of myogenesis and muscular hypertrophy. MyoD is highly expressed in satellite cells during differentiation period. Satellite cells activated when muscle regenerated and differentiated, thus satellite cells play an important role in the process of muscle regeneration and maintain muscle mass by producing new fiber muscle 24 .
Recent studies in natural components showed a promising effect to stimulate the IGF-Akt pathways as alternative supplements to increase and maintain skeletal muscle mass. Nutmeg or often called Nutmeg with the scientific name Myristica Fragrans is a typical Indonesian plant that is from the Maluku islands, has many benefits such as anti-inflammatory, analgesic, anticancer, antioxidant, hypoglicemic and antidiabetic [25][26][27][28][29][30] . Nutmeg seeds have good potential for the treatment of type 2 diabetes with increasing activity through the mechanism of PPAR g agonist 31,32 . The activity of PPAR g agonist in Nutmeg seeds by increasing insulin sensitivity is expected to activate the AKT and mTOR signaling pathways to increase protein synthesis and muscle mass. Previous research showed that the Nutmeg extract treatment able to maintain muscle mass and prevent sarcopenia in old rats by increasing IGF-1 expression followed by a cascade of Akt / mTOR signaling pathway which leads to increase myogenesis and muscle regeneration. 33 Age differences affect the regulation of skeletal muscle protein synthesis process. Changes in metabolism process such as higher fat deposition repress myogenesis in elder age. 34 Consequently, effect of administration of nutmeg in young rats' skeletal muscle needs to be determined. Therefore, we investigate the Nutmeg regulation on the skeletal muscle hypertrophy through the Akt/ mTOR pathways and the involvement of satellite cell in skeletal muscle hypertrophy.

Animal experiments
12 male Wistar rats aged 6 weeks, obtained from the Animal Facility of PT Biofarma Indonesia. Rats were kept at 24°C under a 12-hour light -dark cycle, and humidity were adjusted to 55%, with food and water ad libitum for 12 weeks in Animal Laboratory, Physiology Division, Faculty of Medicine, Universitas Padjadjaran. All experimental protocols and methods were approved by the Ethics Committee, Faculty of Medicine, Universitas Padjadjaran Number 28/UN.6.KEP/ EC/2019.
The rats were randomly divided into control and treatment group. The Nutmeg extract was given to the treatment group for 12 weeks by gavage. The Nutmeg extract used in this study was free safrole and myristicin. Nutmeg extract as Glucopala Caplet were obtained from the Faculty of Pharmacy Laboratory, Padjadjaran University batch number FP08.A1604.001 31,[35][36][37] . The dose given to the rats in the treatment group was converted from human dose (300 mg / day) with final dose 8.1 mg/kg/day.Nutmeg seed extract was given once a day. The rat sacrificed using overdose isoflurane, then the soleus and gastrocnemius

RNA extraction and semi-Quantitative PCR
The RNA isolated from ± 2 mg muscles tissue with Trizol reagent (from Invitrogen, USA). Polymerase Chain Reaction (PCR) using One Step PCR kit (Bioline, USA). Primers for AKT, MyoD, Myf5 and GAPDH were presented in Table 1.The gene expression results were normalized with GAPDH. electrophoresis Analysis of the results of RT-PCR was carried out using 2% agarose electrophoresis gel. The PCR band were visualized using BluePAD (Biohelix, Taiwan) and band image were quantified using ImageJ software (NIH, USA).

statistical Analysis
The Data were analyzed by comparing the mean with Independent t Test using SPSS v.25 with significant value if p<0.05. Data were presented as mean ± standard deviation (SD).

Body weightand skeletal muscle mass
During the treatment period, body weight were measured weekly to analyze whether skeletal muscle weight increase due to difference body weight. Rats body weight were increase in both groups ( Figure 1A). But there was no difference of body weight among groups. By the end of treatment period, rats were euthanized and skeletal muscle were isolated. Soleus and Gastrocnemius muscle were isolated and measured. Muscle weight were normalized with rats' body weight. Soleus muscle and gastrocnemius muscle mass were higher in Nutmeg group compared with control, p<0.05 ( Figure 1B and 1C).

Nutmeg extract stimulate satellite Cells Proliferation through Akt Pathway
The Akt pathway is an important pathways to increase the protein synthesis. Moreover, the Akt involved in the cells survivals, differentiation and proliferation. In this study we observed the soleus and gastrocnimeus Akt expression in the treatment group was increase significantly p<0.01 (Figure 2). Akt expression were increased 1.37 fold in Soleus and 2.9 fold in Gastrocnemius.
Interestingly, MyoD expression were increase only in soleus of the Nutmeg group (2 fold, p<0.01, Figure 2A). While in gastrocnemius muscle, no difference of MyoD expression were found in control and Nutmeg group (p=0.43). Myf5 is the earliest Myf that expressed in early myogenesis. We measured Myf5 gene expression in Soleus and Gastrocnemius muscle. We found no changes of Myf5 mRNA expression in both muscle (Figure 2A,B).

Nutmeg extract Inhibit Autophagy in soleus Muscle
The autophagy is involved in the balance of protein synthesis and degradation through the AKT-mTOR pathways. The activation of AKT blocks the FoxO and inhibit protein degradation.  The previous study showed the mTOR inhibits autophagy by increase the autophagy mediator which is known to indicate inhibition of autophagy 39 .
As shown in Figure 3A,Nutmeg extract increase mTOR 1.25 fold and P62 gene 1.3 fold compared to control (p<0.05) but did not increase the Igf-1 in soleus muscles. While in gastrocnemius muscle,no significant difference on autophagy pathway were observed between nutmeg and control group (p>0.05; Figure 3B).

dIsCussIoN
Nutmeg seeds have various active components, including antioxidants, antibacterial, analgesic and antidiabetic effects. Nutmeg seed has potential role as antidiabetic agent by increasing insulin sensitivity through PPARg agonists. PPARg agonist increasing insulin sensitivity and involves in phosphorylation of insulin receptors (IRS-1) to activate the PI3K/AKT pathway cascade 31,40 .
The molecular mechanism of muscle mass regulation involves various signal transduction pathways. Increasing skeletal muscle mass in the post-natal period is related to the process of hypertrophy of muscle fibers through the regulatory pathway between protein synthesis and degradation which are carried out through the PI3K/AKT pathway 10 . Akt pathway activate satellite cells differentiation through MRFs expression. MRFs including MyoD and Myf5 induce in satellite cell differentiation and proliferation.
In this study, we found that Nutmeg extract increased soleus and gastrocnemius muscle mass supported by increased of AKT gene expression. It suggest that nutmeg extract increased skeletal muscle mass through activation Akt signaling. Activation of the PI3K/Akt pathway causes phosphorylation of mTOR and GSK3â which are important regulators of the protein translation process. mTOR has two different complexes that work through regulation of protein metabolism and autophagy 16 . Increased expression of Akt was observed in our previous study using old mice that show the effect of Nutmeg on increasing soleus muscle mass, through the IGF-AKT-mTOR pathway thereby increasing protein synthesis and inhibiting autophagy in aging 33 . This study showed similar results, Akt-mTOR pathway were increasing while autophagy process were inhibited in soleus muscle. However, in young rat, we observed no significant change in gastrocnemius mTOR expression. Possibly, this phenomenon was due to activation of FoxOs as the balancer of mTOR and Akt pathway 51 .
The pathway to increase protein synthesis is carried out through IGF1 / Akt / mTOR. However, in this study, the Akt and mTOR increase did not preceded by change in IGF1 expression. Upregulated of Akt / mTOR expression might be caused by the improvement of glucose uptake by PPAR agonists in Nutmeg seed extract especially in soleus muscle 31 .
Correlated with Akt/mTOR gene alteration in soleus muscle, we observed significant changes in autophagy related gene in nutmeg extract treated rats. Our results showed autophagy related gene were increased in nutmeg group. Increased in p62 gene expression together with trend of LC3 upregulation ( Figure 3A) indicated the inhibition of autophagy in soleus muscle. This results was consistent with our previous study in aging rats, that nutmeg extract decreased autophagy in 80 weeks rat' soleus muscle 33 . In gastrocnemius muscle, we found the similar trend of autophagy inhibition although not statistically significant ( Figure 3B) 38 .
Satellite cells mainly known for their contributions to regenerate the muscle cells while injury, the maintenance of muscle mass and hypertrophy. 41,42 Satellite cells suggested promoting the muscle fibres hypertrophy by mediating myonuclear addition and sustains muscle growth. MyoD and Myf5 expression in adult muscles increases when satellite cells are activated. This marker shows the satellite cells differentiation, followed by myogenic determination and finally directing progenitor cells to form skeletal muscle lines. Several studies have shown the role of MyoD and Myf5 in response to muscle hypertrophy, denervation, disease, and atrophy. 11, 43 Our results showed an increase in the expression of MyoD in the soleus muscles but not in gastrocnemius muscle. This could be relates to the type of muscle fiber, where the soleus muscle is dominated by type I muscle fibers (oxidative), while the gastrocnemius muscle is dominated by type II fibers (glycolytic). Oxidative muscle fibers are known to have a greater capacity of protein synthesis and autophagy process. This high protein turnover process, in type I muscle fibers showed higher potential for adaptation and regeneration in this type of fiber [44][45][46][47] .However, both types of muscle fibers have the same sensitivity to the process of insulin phosphorylation. This statement is in line with the results of this study, the Aktas the insulin signaling downstream-expression was increased in both muscle type 48 .
The increasing of MyoD expression was not accompanied by an increase in Myf5.This results might be associated with the different role of the two genes during satellite cell activation. MyoD and Myf5 expression in adult muscles increases when satellite cells are activated. Several studies have shown the role of MyoD and Myf5 in response to muscular hypertrophy, denervation, disease, and atrophy 43,49 . Research on transgenic mice that have been omitted by the expression of Myf5 and MyoD shows failure of regeneration and differentiation. This shows the role of Myf5 in the process of activation and differentiation of satellite cells 24 . However, other studies have shown different roles between Myf5 and MyoD. Myf5 involved in proliferation prior differentiation of myoblast while MyoD increased during early differentiation of satellite cells 50 .
It suggest that nutmeg extract might increase skeletal muscle mass through satellite cells differentiation. This results was consistent with our previous results in elderly rats 33 . However, nutmeg crude extract that has some different effect on young and elder rat. Discrepancies in young rats might be caused by age difference between subjects (young and elderly rats) and post transcriptional modification, enzymatic reactions and hormonal modulation. In both studies, we use crude extract of nutmeg. The nutmeg extract contain another active compound that might have different effect on young and aging rats skeletal muscle.Another studies to characterized the active compound in nutmeg extract that is essential for skeletal muscle differentiation need to be conducted.
Limitation of this study was we only use gene expression levels to detect early changes in skeletal muscle. Future study to analyze the protein levels is needed to address discrepancies of soleus and gastrocnemius response to Nutmeg administration.

CoNClusIoN
Taken together, we conclude the Nutmeg extract increase skeletal muscle mass in young rats through alteration satellite cells differentiation gene and autophagy gene expression in soleus muscle. However, although gastrocnemius muscle cell mass was increased by Nutmeg extract administration, no changes was observed in MyoD, Myf5 and autophagy gene expresssion. Thus, Nutmeg extract supplementation has potential effect on increasing skeletal muscle mass in young individual.