Open AccessArticle

Potential Effect of Defatted Mealworm Hydrolysate on Muscle Protein Synthesis in C2C12 Cells and Rats

Seo-Hyun Choi

^1,†,

Tae-Hwan Jung

^2,†

and

Kyoung-Sik Han

^1,2,*

Department of Food Science and Biotechnology, Sahmyook University, Seoul 01795, Republic of Korea

Department of Integrative Biotechnology, Sahmyook University, Seoul 01795, Republic of Korea

Author to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Appl. Sci. 2024, 14(24), 11772; https://doi.org/10.3390/app142411772

Submission received: 12 November 2024 / Revised: 12 December 2024 / Accepted: 13 December 2024 / Published: 17 December 2024

(This article belongs to the Special Issue Functional Foods: Bioactivity and Potential Health Effects)

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Figure 1
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile and non-protein nitrogen analysis of DMP and DMH. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (B) non-protein nitrogen. Values are means ± standard error. The data were analyzed using Student’s t-test (* p < 0.05). M, molecular weight marker; DMP, defatted mealworm powder; DMH, defatted mealworm hydrolysate. "> Figure 2
Cell viability of DMH on dexamethasone-induced C2C12 cell viability. (A) Cytotoxicity of DMH; (B) cell protective effect of DMH. Values are means ± standard error. The data were analyzed using the Student’s t-test (* p < 0.05) compared to the group without DMH added. DMH, defatted mealworm hydrolysate. DEX, dexamethasone. "> Figure 3
Body weight change of rats during the experiment. Values are means ± standard error. Different letters (a and b) above the bars indicate significant differences by one-way analysis of variance with Duncan’s multiple range test (p < 0.05). DEX, dexamethasone (2.25 mg/kg, i.p.) + control diet; DEX + DMH, dexamethasone (2.25 mg/kg, i.p.) + defatted mealworm hydrolysate diet; i.p., intraperitoneal injection. "> Figure 4
Grip strength test of rats fed the control and experimental diets for 8 weeks. The data were analyzed using the Student’s t-test (* p < 0.05) compared to control. DEX, dexamethasone (2.25 mg/kg, intraperitoneal injection) + control diet; DEX + DMH, dexamethasone (2.25 mg/kg, intraperitoneal injection) + defatted mealworm hydrolysate diet. "> Figure 5
Weight of muscle tissue in rats. Values are means ± standard error. The data were analyzed using the Student’s t-test (* p < 0.05) compared to control. TA, tibialis anterior muscle; GAS, gastrocnemius muscle. DEX, dexamethasone (2.25 mg/kg, intraperitoneal injection) + control diet; DEX + DMH, dexamethasone (2.25 mg/kg, intraperitoneal injection) + defatted mealworm hydrolysate diet. "> Figure 6
Expression of mRNA for muscle protein synthesis and degradation factors in the muscle tissue obtained from the rats. Values are means ± standard error. The data were analyzed using the Student’s t-test (* p < 0.05) compared to DEX group. DEX, dexamethasone (2.25 mg/kg, intraperitoneal injection) + control diet; DEX + DMH, dexamethasone (2.25 mg/kg, intraperitoneal injection) + defatted mealworm hydrolysate diet; MuRF-1, muscle RING finger-1; MyoD, myoblast determination protein. ">

Versions Notes

Abstract

(1) Background: the objective of this study was to examine the impact of defatted mealworm hydrolysate (DMH), formulated through protein hydrolysis, on muscle protein synthesis in C2C12 cells and rats; (2) Methods: C2C12 cells were treated with dexamethasone and DMH, and cell viability was quantified using the MTT assay. Twenty-four Sprague–Dawley rats were divided into three groups (control, DEX, and DEX + DMH) and treated for 8 weeks. The DEX and DEX + DMH groups were administered intraperitoneal injections of DEX at a concentration of 2.25 mg/kg over a 3-d period. The control and DEX groups were fed a control diet, whereas the DMH group had part of the protein composition of the control diet replaced with 3.5% of DMH. The impact of DMH on muscle protein synthesis was evaluated through the measurement of grip strength, gastrocnemius and tibialis anterior muscle weights, and the investigation of muscle protein synthesis and degradation factor mRNA expression utilising the real-time PCR method; (3) Results: in vitro experiments demonstrated that treatment with DMH at concentrations greater than 5 mg/mL markedly alleviated DEX-induced injury in C2C12 cells. In vivo experiments demonstrated that the mRNA expression levels of myogenin and myoblast determination proteins, which promote muscle protein synthesis, were significantly increased. Furthermore, rats fed DMH exhibited significantly enhanced grip strength and tibialis anterior weight; (4) Conclusions: these findings indicate that DMH may serve as a functional material capable of promoting muscle protein synthesis and that the utilization of proteolytic enzymes is advantageous for the effective utilization of mealworms.

Keywords:

defatted mealworm hydrolysate; dexamethasone; MyoD; myogenin; muscle protein synthesis

1. Introduction

The loss of muscle mass is a gradual process that begins in early adulthood and accelerates with age. Volpi et al. [1] reported a loss of approximately 1–2% per year after age 30 and 2–3% per year after age 60. Furthermore, the rate of loss increases with age. The loss of muscle mass due to aging can result in a reduction in muscle strength, gait speed, and balance, which may subsequently lead to a decline in physical activity and the development of conditions [2,3]. This decline in physical function may contribute to the development of various diseases [4,5]. It is possible that mitochondrial dysfunction, chronic inflammation and neuromuscular damage may be involved in the loss of muscle and the precise mechanisms of muscle synthesis remain unclear [6,7,8].

Nutritional factors, including reduced physical activity and insufficient protein intake, have the potential to impede muscle synthesis [9,10]. Furthermore, muscle degradation factors, such as muscle RING finger-1 (MuRF-1) and atrogin-1, and muscle synthesis factors, including myoblast determination protein (MyoD) and myogenin, are associated with muscle degradation, although individual differences remain [11,12].

Skeletal muscle serves as a health marker, influencing the body’s capacity to maintain and perform activities. Skeletal muscle mass is maintained by an equilibrium between muscle protein breakdown and synthesis. When this equilibrium is disrupted, skeletal muscle mass decreases [13,14]. A reduction in skeletal muscle mass is associated with a decline in exercise capacity, which in turn results in a reduction in physical activity performance [15]. Jung et al. [16] demonstrated that a diet with high protein intake is an effective method for increasing skeletal muscle mass. Conversely, individuals who consumed protein below the recommended intake level had significantly reduced skeletal muscle mass and strength.

Tenebrio molitor L. is the larva of Tenebrio molitor, which belongs to the beetle family Tenebrionidae and is commonly referred to as the mealworm [17]. Mealworms are used as food in many countries, including the United States, Belgium, the Netherlands, France, the United Kingdom, China, Thailand, Japan, and Canada, because of their capacity for mass rearing and efficient production [18]. In Korea, the Ministry of Food and Drug Safety has recognized mealworms as edible food since 2016. Furthermore, mealworms have considerable potential as a food ingredient capable of providing a high-quality protein source [19]. However, mealworms have a high fat content, which can result in quality changes owing to rancidity and poor storage [20]. To address these issues, mealworms are typically defatted and pulverized, and most mealworms employed in the food industry are distributed and utilized as defatted mealworm powder (DMP) [21,22].

DMP is a rich source of essential amino acids necessary for muscle synthesis, including branched-chain amino acids (BCAAs) [23]. BCAAs, which consist of essential amino acids, such as leucine, isoleucine, and valine, serve as precursors for muscle protein synthesis [24]. Moreover, studies conducted on rats have indicated that the oral administration of BCAAs can facilitate the expression of mRNAs linked to muscle protein synthesis, including myogenin. [25,26,27].

Enzymatic protein hydrolysis has been extensively employed in the food industry as a means of more effectively utilizing protein materials [28]. The cleavage sites on raw proteins vary depending on the type of protein-hydrolyzing enzyme used, resulting in various potential final products [29]. Enzymes can be classified into two main categories: exopeptidases and endopeptidases. Based on the manner in which they act on the substrate, these enzymes can be classified as either exopeptidases, which act on the N-terminal α-amino group, or endopeptidases, which act on peptide bonds within the protein [30]. Enzymatic protein hydrolysis can enhance the digestive and absorptive processes of proteins by fragmenting larger molecular weight proteins into smaller peptides. This process also offers the advantage of improved product processability by increasing solubility [31]. Moreover, the primary protein hydrolysates produced by proteolytic enzymes are low-molecular-weight peptides that are readily absorbed by the body and serve as excellent sources of protein [32,33]. Notwithstanding the advantages of enzymatic methods, most studies utilizing mealworms employ mealworm extracts. However, there is a paucity of research on the utilization of mealworm hydrolysate. Accordingly, the present study aimed to develop a defatted mealworm hydrolysate (DMH) by treating DMP with proteolytic enzymes and explore the potential utilization of DMH as a functional protein.

2. Materials and Methods

2.1. Sample Preparation

The enzymes used to develop DMH, alcalase and bromelain, were purchased from Novozymes (Bagsværd, Denmark). Distilled water was added to achieve a 10% weight concentration of DMP, mixed thoroughly, and the pH was adjusted to 7.25. The proteolytic enzymes alcalase and bromelain were added at a concentration of 0.1% each and allowed to react at 50 °C for 1 h. The reaction mixture was then heated at 95 °C for 10 min to inactivate the enzymes, after which it was subjected to centrifugation at 7000 rpm for 10 min. DMH was prepared by spray drying the resulting supernatant.

2.2. Non-Protein Nitrogen (NPN)

To ascertain the extent of DMH protein hydrolysis, the NPN content in the samples was determined, and the requisite reagents were procured from Duksan General Science (Seoul, Republic of Korea). The samples were diluted 10-fold with distilled water, mixed with 24% trichloroacetic acid in equal proportions, and allowed to react for 30 min. The supernatant was recovered by centrifugation at 4000 rpm for 15 min and subsequently used for NPN analysis. To prepare the alkaline copper reagent necessary for the determination of NPN content, solution A, comprising 2% sodium carbonate dissolved in 0.1 N sodium hydroxide, and solution B, consisting of 0.5% copper sulfate mixed with 1% sodium citrate, were prepared. Subsequently, an alkaline copper reagent was prepared by mixing solutions A and B at a ratio of 50:1. A solution of 5 mL of the prepared reagent and 1 mL of the sample was prepared and allowed to react for 10 min at room temperature. Folin–Ciocalteu’s phenol reagent (0.5 mL) was added to induce a color reaction for 20 min. The absorbance was measured at 750 nm using an Optizen 2120UV spectrophotometer (Mecasys, Daejeon, Republic of Korea), and a calibration curve was prepared using bovine serum albumin as the standard.

2.3. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was performed to determine protein hydrolysis in DMH. Samples were quantified for protein by the Bradford method and mixed with 2× Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) at a ratio of 1:1, loading a total of 10 µg per sample. SDS-PAGE was performed using a 12% acrylamide gel. After electrophoresis at 80 V for approximately 1 h, the gel was stained with 0.25% Coomassie brilliant blue solution. The gel was then decolorized using a decolorizing reagent prepared by mixing methanol, acetic acid, and distilled water, and the bands were identified using a ChemiDoc Imager (Bio-Rad).

2.4. Cell Culture

The cell line employed in the experiments, C2C12 myoblast, was obtained from the American Type Culture Collection (Manassa, VA, USA). The C2C12 cell line was cultivated in Dulbecco’s modified Eagle’s medium (GenDEPOT, Barker, TX, USA) supplemented with 10% fetal bovine serum (GenDEPOT) and 1% penicillin-streptomycin (Gibco, MA, USA). The incubator was set to maintain a temperature of 37 °C and a CO₂ concentration of 5%.

2.5. Cell Viability Assays

To ascertain the extent of the cytotoxicity exerted by DMH on C2C12 cells, cell viability was assessed. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The C2C12 cells were seeded in 24-well plates at a density of 1 × 10⁴ cells/well and incubated for 24 h. Subsequently, the cells were treated with DMH at varying concentrations (0, 1, 5, 10, 25, and 100 µg/mL) for 24 h. Furthermore, 1000 µL of 2.5 µg/mL MTT reagent was added to each well and incubated for 3 h at 37 °C with 5% CO₂ in a humidified incubator. Moreover, the supernatant was removed, and 1000 µL of dimethyl sulfoxide was added to each well to dissolve the resulting formazan crystals. The absorbance was then measured at 570 nm using an MMR SPARK^® spectrophotometer (Tecan, Männedorf, Switzerland). Cell viability is expressed as a percentage of the absorbance of the control. We investigated the effect of DMH on the protection of C2C12 cells against damage induced by DEX. C2C12 cells were seeded in 24-well plates at a concentration of 1 × 10⁴ cells/well and incubated for 24 h. Subsequently, the cells were treated with 100 µM DEX and DMH at varying concentrations (0, 1, 5, 10, 25, and 100 µg/mL) for 24 h. Cytoprotective effects of DMH were investigated by measuring cell viability using the aforementioned procedure.

2.6. Animal Studies

The animal experiments were approved by the Chairman of the Animal Ethics Committee at Sahmyook University (SYUIACUC 2023-012) and conducted in accordance with the regulations governing the use of laboratory animals. A total of 24 3-month-old Sprague–Dawley rats were obtained from RaonBio (Gyeonggi-do, Republic of Korea), and chow was manufactured by Duyeol Biotech (Seoul, Republic of Korea). The animals were housed in an animal facility at Sahmyook University, Seoul, Republic of Korea. The facility maintained a temperature of 22 ± 2 °C and a humidity of 50 ± 10% with a 12-h light/dark cycle. The body weight of the rats was measured once per week. Following a 1-week acclimation period, with the exception of the control group, all experimental groups were administered a single intraperitoneal injection of 2.25 mg/kg DEX (Sigma-Aldrich, St. Louis, MO, USA) over 3 d. The experiment was conducted over 8 weeks following the intraperitoneal administration of DEX. The rats were divided into three groups: control, DEX, and DEX + DMH. The control and DEX groups were fed the AIN-93G diet (Envigo, Indianapolis, IN, USA), whereas the DEX + DMH group was fed a diet based on the composition of the control diet, with some protein sources replaced with 3.5% of DMH (Table 1)

2.7. Grip Strength Test and Weight of Muscle Tissue

The objective of the grip strength test was to ascertain the maximum muscle strength of the rats. A grip strength meter (Bioseb, Vitrolles, France) was used to induce the rats to grip a T-bar, and force was measured by pulling the tail at a speed of 2 cm/s. The average value of three replicates was used as the result. At the end of the experiment, the rats were anesthetized with CO₂ gas and sacrificed, and the tibialis anterior (TA) and gastrocnemius (GAS) muscles were harvested and weighed.

2.8. RNA Extraction and Real-Time Polymerase Chain Reaction (PCR)

To examine the expression of mRNAs associated with muscle protein synthesis and degradation factors in each group, RNA was extracted from the muscle tissue obtained from the rats. Reagents used for RNA extraction were obtained from Sigma-Aldrich. Subsequently, 1 mL of TRIzol and 100 mg of the sample were homogenized, followed by the addition of 200 µL of chloroform and vortexing. The supernatant was recovered by centrifugation, and 500 µL of isopropanol was added to the supernatant. The mixture was then cooled on ice for 10 min to precipitate the RNA. The precipitated RNA pellet was washed, and cDNA was synthesized using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s instructions. To confirm the expression of mRNA, real-time PCR was performed using TOPrealv™ SYBR Green qPCR High-ROX PreMIX, and primers were purchased from Enzynomics (Daejeon, Republic of Korea) and Bioneer (Daejeon, Republic of Korea). The primers utilized in the experiments were β-actin (forward, TAT CGG CAA TGA GCG GTT CC; reverse, AGC ACT GTG TTG GCA TAG AGG), MuRF-1 (forward, ATC ACT CAG GAG CAG GAG GA; reverse, CTT GGC ACT CAA GAG GAA GG), atrogin-1 (forward, AGC TTG TGC GAT GTT ACC A; reverse, GGT GAA AGT GAG ACG GAG CA), MyoD (forward, ACT ACA GCG GCG ACT CAG AC; reverse, ACT GTA GTA GGC GGC GTC GT), and myogenin (forward, TGA ATG CAA CTC CCA CAG C; reverse, CAG ACA TAT CCT CC ACC GTG). A total of 20 µL was prepared by combining 10 µL of SYBR Green master mix, 1 µL of each forward and reverse primer, 6 µL of DNase-free water, and 2 µL of cDNA. This solution was subjected to real-time PCR using a DTPrime 5 (DNA Technology, Moscow, Russia). The initial denaturation step was set at 95 °C for 10 min, followed by denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, and extension at 72 °C for 30 s, repeated for 40 cycles. The relative mRNA expression between each group was determined using the 2^−ΔΔCt method, whereby the ΔCt was calculated by subtracting the individual ΔCt values of the experimental group from those of the control group.

2.9. Statistical Analysis

The results are expressed as the mean ± standard error (SEM). Statistical significance between groups was tested using PASW Statistics 18 (SPSS Inc., Chicago, IL, USA). The data were statistically analyzed using the Student’s t-test or one-way ANOVA and Tukey’s Honestly Significant Difference (HSD) test.

3. Results

3.1. Hydrolysis by Enzymatic Treatment

The protein hydrolysate phase of DMH was identified by SDS-PAGE and NPN analysis. The protein composition of DMP without enzyme treatment was found to be in the range of 26 to 70 kDa, whereas the protein composition of DMH treated with proteinase was below 10 kDa, as all high-molecular-weight proteins were hydrolyzed (Figure 1A). NPN measurements confirmed the extent of protein hydrolysis. The results demonstrated that DMH exhibited a significantly higher NPN content than that of DMP, confirming that DMH was effectively hydrolyzed by enzymatic treatment (Figure 1B).

3.2. Cytotoxicity and Protective Effect of DMH

In order to ascertain the cytotoxicity of DHM on C2C12 cells, the cells were treated with varying concentrations of DMH (0, 1, 5, 10, 25, and 100 mg/mL) for a period of 24 h. The cell viability was then determined using the MTT assay (Figure 2A). Compared to the control, there was no statistically significant difference in cell viability when treated with DMH at 1–100 mg/mL. C2C12 cells were then treated with DEX to induce damage, and the effect of DMH on cell viability was investigated. As shown in Figure 2B, cell viability after DEX treatment was approximately 79.88%, however, after DMH treatment, cell viability increased significantly from approximately 87.06% to 104.49% (p < 0.05).

3.3. Changes in Body Weight of Rats

Weight changes of the rats during the experimental period are shown in Figure 3. During the adaptation period of 1 week, there were no significant differences in body weight between the experimental groups. However, following DEX administration, there was a significant decrease in the body weight of the DEX and DEX + DMH groups compared with that of the control group (p < 0.05). Approximately 2 weeks after the final DEX dose, there was no significant difference in body weight between the groups.

3.4. Grip Strength Test

The results of the grip strength test conducted at the conclusion of the 8 weeks experiment are presented in Figure 4. The mean grip strength of rats fed a diet supplemented with DMH was 559.33 g, which was significantly higher than that of rats in the other experimental groups (p < 0.05).

3.5. Weight of Muscle Tissue

The results of normalising the weight of TA and GAS to the body weight of the rats are illustrated in Figure 5. No significant differences were observed in the weights of the between the groups. However, a significant increase in the TA weight of rats fed a diet supplemented with DMH was noted compared with observations of the other groups (p < 0.05).

3.6. Expression of mRNA for Muscle Protein Synthesis and Degradation Factors

MuRF-1 and atrogin-1 facilitate muscle protein degradation, whereas MyoD and myogenin regulate muscle protein synthesis. To investigate the effects of DMH intake on muscle protein synthesis and degradation, we measured the mRNA expression of each factor, with results presented in Figure 6. The results demonstrated that there were no statistically significant differences in the mRNA expression of MuRF-1 or atrogin-1 between the experimental groups. By contrast, the mRNA expression of MyoD and myogenin in the DEX + DMH group was approximately 152.6% and 226.9%, respectively, markedly higher than the mRNA expression of MyoD and myogenin in the DEX + DMH group, which was approximately 70.7% and 52.8%, respectively (p < 0.05).

4. Discussion

The consumption of high-quality proteins is of paramount importance for the maintenance of the skeletal muscle-to-total body weight ratio, as it stimulates the synthesis of muscle proteins (Liao et al., 2019) [34]. The moisture, ash, crude protein, crude fat, and carbohydrate contents of DMP were approximately 4.7%, 4.9%, 68.9%, 7.7%, and 13.7%, respectively. The composition of DMH was found to be similar to that of DMP. However, the present study was limited in that it did not analyze the amino acid composition of DMP and DMH. In this study, the two enzymes were combined to create DMH, which has the potential to be used as a high-quality protein material. The present study revealed that DMP is comprised of a considerable proportion of proteins with a molecular weight below 15 kDa. Despite the utilisation of SDS-PAGE with increasing concentrations of acrylamide, proteins with a molecular weight below 15 kDa remained unidentified, indicating a potential limitation. However, the results of protein hydrolysis of DMH using SDS-PAGE demonstrated that alcalase and bromelain were effective in hydrolysing the polymeric proteins in DMP. Furthermore, the NPN content, an indicator used to assess protein hydrolysis, was quantified, and it was found that the NPN content of DMH was notably higher than that of DMP.

Bromelain is a plant-derived proteolytic enzyme from the stem and juice of the pineapple plant (Ananas comosus) and is categorized as a cysteine endoprotease [28]. It has been reported that protein hydrolysates prepared using bromelain have the capacity to increase the content of low-molecular-weight peptides [35]. Alcalase is a microbial proteolytic enzyme derived from the fermentation of Bacillus licheniformis and is a serine endopeptidase that cleaves peptide bonds [36]. Alcalase is widely used for the production of bioactive peptides through the cleavage of peptide bonds located midway along the peptide chain [37]. Alcalase effectively hydrolyzes rice bran proteins, resulting in peptides with enhanced bioactivities [38]. The hydrolysis of proteins by enzymatic treatment can increase their digestibility by breaking down high-molecular-weight proteins into low-molecular-weight peptides [39]. Furthermore, Cho et al. [40] reported that growing pigs fed a diet supplemented with mealworm hydrolysate exhibited enhanced protein digestibility compared with those receiving other supplements.

DEX, a synthetic glucocorticoid, was used to induce damage in C2C12 cells by inhibiting protein synthesis and promoting protein degradation [41]. In the present study, treatment of C2C12 cells with 100 µM DEX resulted in cell damage and reduced cell viability. However, the viability of C2C12 cells was restored when DMH was added at a concentration of 5 mg/mL or higher, yielding results analogous to those observed by Choi et al. [42]. These authors demonstrated that the addition of mealworm extract restored the viability of C2C12 cells after DEX-induced cell damage. DEX has also been employed in vivo to induce a reduction in muscle mass and strength in rats, as it activates the ubiquitin-proteasome system [43]. Furthermore, the administration of DEX intraperitoneally has been observed to induce weight loss in rats when the concentration exceeds a certain threshold. Based on the findings of preliminary experiments, the concentration of intraperitoneal DEX was set at 2.25 mg/kg. The results demonstrated that DEX administered intraperitoneally markedly reduced the body weight of rats, consistent with the observations reported by Seo and Lew [44], who used 2 mg/kg of DEX (i.p.) to induce weight loss in rats. A limitation of this study is that although dexamethasone administration intraperitoneally resulted in a transient reduction in body weight, it did not lead to a corresponding reduction in muscle mass in the rats. Although the muscle protein synthesis factors MyoD and myogenin were reduced in comparison to the control group, it is speculated that the duration of DEX (i.p.) should have been extended in order to achieve a reduction in muscle mass.

MuRF-1 and atrogin-1 have been demonstrated to induce muscle protein degradation, which can lead to the development of muscle degradation. In addition, DEX has been shown to activate the UPS, thereby promoting the expression of MuRF-1 and atrogin-1 [45]. Myogenic regulatory factors, including myogenin and MyoD, are involved in muscle cell proliferation and differentiation. These factors promote the synthesis of muscle cells by differentiating myoblasts into myotubes [46]. Myogenin and MyoD induce skeletal muscle development by forming myofibrils. In addition, DEX reduces the expression of myogenic regulatory factors, including myogenin and MyoD, which are involved in myogenic differentiation [47,48,49]. The mRNA expression of MyoD and myogenin in the tibialis anterior (TA) was found to be significantly increased in the DMH group. It was hypothesised that the elevation in MyoD and myogenin levels led to a notable increase in TA weight. The present study revealed no significant differences in MuRF-1 or atrogin-1 mRNA expression between the groups. However, myogenin and MyoD mRNA expression levels were significantly elevated in rats fed the DMH-supplemented diet in comparison to those in the DEX group. Furthermore, the DEX + DMH group exhibited a notable increase in grip strength and TA weight compared with the other groups. Despite an increase in mRNA expression of MyoD and myogenin in the DMH group, there was no corresponding increase in the weight of GAS. Despite an increase in the weight of the TA, its proportion of total body weight remained relatively insignificant. Consequently, we speculate that there was no significant difference in weight recovery between the DEX and DEX + DMH groups.

The present study was limited in that it was not possible to determine the direct mechanism by which DMH induced increased mRNA expression of myogenin and MyoD by western blot. Furthermore, it should be noted that the histological sections of muscle tissue were not identified through tissue staining, which represents a limitation of the study. The increase in myogenin and MyoD levels following DMH consumption is due to the activation of protein kinase B, also known as Akt [50]. Akt is a serine-threonine kinase that promotes myogenesis by regulating protein synthesis. Akt activation promotes muscle differentiation by increasing the expression of myogenin and MyoD [51,52]. A recent study on mealworms reported that rats fed mealworm-derived protein supplements exhibited activated Akt and ameliorated skeletal muscle atrophy [53]. Another study reported that orally administered mealworm extracts activated Akt, increased myogenin expression, and improved grip strength [54].

Edible insects have recently emerged as potential alternative sources of animal protein owing to their nutritional profiles and environmental benefits. They are not only a rich source of protein and essential amino acids but also offer a cost-effective and sustainable alternative to conventional animal farming, which has been linked to high greenhouse gas emissions [55,56]. In particular, mealworms are a rich source of minerals, including calcium and magnesium, and are highly nutritious owing to their high protein content and abundance of essential amino acids, which can contribute to the development of muscle tissue [57]. Moreover, it has been demonstrated that mealworms possess the capacity to facilitate the growth of muscle cells and to avert the development of musculoskeletal ailments in older adults [58]. The present study demonstrated that DMH protected C2C12 cells from DEX-induced damage and that DMH-fed rats exhibited increased grip strength and TA weight. MyoD is associated with an increase in myoblasts, while myogenin is a factor associated with the differentiation of myoblasts into myotubes. It can therefore be postulated that DMH may induce muscle protein synthesis by promoting the differentiation of myoblasts into myotubes. However, the findings of this study can only be interpreted as indicative of a potential effect of DMH on muscle synthesis, given that the study was conducted at the mRNA expression level only. These findings may be attributed to DMH-induced elevation in the expression levels of myogenin and MyoD, which have been demonstrated to facilitate muscle protein synthesis. Choi et al. [42] reported that mealworm extracts enhanced myogenin mRNA expression levels in C2C12 cells, thereby promoting myogenic differentiation.

5. Conclusions

The objective of this study was to examine the potential effects of DMH on muscle protein synthesis in C2C12 cells and in rats. The proteolytic enzymes alcalase and bromelain were employed to synthesize DMH, which is predominantly composed of low-molecular-weight peptides. The proteolytic nature of DMH was substantiated through the utilization of SDS-PAGE and NPN content determination. The in vitro experiments demonstrated that DMH protected C2C12 cells from DEX-induced damage. In vivo, rats fed DMH exhibited increased grip strength and TA weight, accompanied by elevated mRNA expression levels of myogenin and MyoD, which are known to promote muscle protein synthesis. The findings indicate that DMH, developed through enzymatic processing, has the potential to serve as a functional material to facilitate muscle growth.

Author Contributions

Conceptualization, K.-S.H.; methodology, S.-H.C. and T.-H.J.; software, S.-H.C. and T.-H.J.; validation, K.-S.H.; formal analysis, S.-H.C., T.-H.J. and K.-S.H.; investigation, S.-H.C. and K.-S.H.; data curation, S.-H.C. and T.-H.J.; writing—original draft preparation, S.-H.C. and T.-H.J.; writing—review and editing, K.-S.H.; supervision, K.-S.H.; project administration, T.-H.J. and K.-S.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Informed Consent Statement

Not applicable.

Data Availability Statement

The data generated for this study are available upon reasonable request from the corresponding author. The data are not publicly available due to privacy.

Acknowledgments

We appreciate to the student researchers who assisted with the animal experiments in this study.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile and non-protein nitrogen analysis of DMP and DMH. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (B) non-protein nitrogen. Values are means ± standard error. The data were analyzed using Student’s t-test (* p < 0.05). M, molecular weight marker; DMP, defatted mealworm powder; DMH, defatted mealworm hydrolysate.

Figure 2. Cell viability of DMH on dexamethasone-induced C2C12 cell viability. (A) Cytotoxicity of DMH; (B) cell protective effect of DMH. Values are means ± standard error. The data were analyzed using the Student’s t-test (* p < 0.05) compared to the group without DMH added. DMH, defatted mealworm hydrolysate. DEX, dexamethasone.

Figure 3. Body weight change of rats during the experiment. Values are means ± standard error. Different letters (a and b) above the bars indicate significant differences by one-way analysis of variance with Duncan’s multiple range test (p < 0.05). DEX, dexamethasone (2.25 mg/kg, i.p.) + control diet; DEX + DMH, dexamethasone (2.25 mg/kg, i.p.) + defatted mealworm hydrolysate diet; i.p., intraperitoneal injection.

Figure 4. Grip strength test of rats fed the control and experimental diets for 8 weeks. The data were analyzed using the Student’s t-test (* p < 0.05) compared to control. DEX, dexamethasone (2.25 mg/kg, intraperitoneal injection) + control diet; DEX + DMH, dexamethasone (2.25 mg/kg, intraperitoneal injection) + defatted mealworm hydrolysate diet.

Figure 5. Weight of muscle tissue in rats. Values are means ± standard error. The data were analyzed using the Student’s t-test (* p < 0.05) compared to control. TA, tibialis anterior muscle; GAS, gastrocnemius muscle. DEX, dexamethasone (2.25 mg/kg, intraperitoneal injection) + control diet; DEX + DMH, dexamethasone (2.25 mg/kg, intraperitoneal injection) + defatted mealworm hydrolysate diet.

Figure 6. Expression of mRNA for muscle protein synthesis and degradation factors in the muscle tissue obtained from the rats. Values are means ± standard error. The data were analyzed using the Student’s t-test (* p < 0.05) compared to DEX group. DEX, dexamethasone (2.25 mg/kg, intraperitoneal injection) + control diet; DEX + DMH, dexamethasone (2.25 mg/kg, intraperitoneal injection) + defatted mealworm hydrolysate diet; MuRF-1, muscle RING finger-1; MyoD, myoblast determination protein.

Table 1. Composition of control and experimental diet.

	Diet ⁽¹⁾
Composition (g/kg)	Control	DEX	DEX + DMH
Corn starch	397.486	397.486	394.596
Maltodextrin	132	132	132
Cellulose	50	50	50
Sucrose	100	100	100
Soybean oil	70	70	66.15
Casein	200	200	171.74
Mealworm hydrolysate	0	0	35
L-cystine	3	3	3
Vitamin mix	10	10	10
Mineral mix	35	35	35
Choline bitartrate	2.5	2.5	2.5
t-Butylhydroquinone	0.014	0.014	0.014
Total (g)	1000	1000	1000

⁽¹⁾ DEX, dexamethasone (2.25 mg/kg, i.p.) + control diet; DEX + DMH, dexamethasone (2.25 mg/kg, i.p.) + defatted mealworm hydrolysate diet. i.p., intraperitoneal injection.

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Choi, S.-H.; Jung, T.-H.; Han, K.-S. Potential Effect of Defatted Mealworm Hydrolysate on Muscle Protein Synthesis in C2C12 Cells and Rats. Appl. Sci. 2024, 14, 11772. https://doi.org/10.3390/app142411772

AMA Style

Choi S-H, Jung T-H, Han K-S. Potential Effect of Defatted Mealworm Hydrolysate on Muscle Protein Synthesis in C2C12 Cells and Rats. Applied Sciences. 2024; 14(24):11772. https://doi.org/10.3390/app142411772

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Choi, Seo-Hyun, Tae-Hwan Jung, and Kyoung-Sik Han. 2024. "Potential Effect of Defatted Mealworm Hydrolysate on Muscle Protein Synthesis in C2C12 Cells and Rats" Applied Sciences 14, no. 24: 11772. https://doi.org/10.3390/app142411772

APA Style

Choi, S.-H., Jung, T.-H., & Han, K.-S. (2024). Potential Effect of Defatted Mealworm Hydrolysate on Muscle Protein Synthesis in C2C12 Cells and Rats. Applied Sciences, 14(24), 11772. https://doi.org/10.3390/app142411772

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