Effects and mechanisms of creatine monohydrate and conjugated linoleic acid on rat muscle fiber related genes and enzyme activities

The growth and development of muscle fibers is an important factor in determining the yield and quality of livestock meat, and the heterogeneity of fiber types is an important characteristic of animal muscle tissue. Muscle fibers can be divided into four types according to the polymorphism of myosin heavy chain (MyHC). Type I (slow oxidative muscle fibers) have a large number of mitochondria, a high myoglobin content and aerobic metabolic enzyme activity, but low ATPase activity, so the contraction rate is slow and sustained; Type IIa (fast oxidative muscle fibers) contain a certain amount of myoglobin, a high glycogen content, and have two energy supply pathways: aerobic metabolism and glycolytic metabolism; Type IIb (fast glycolytic muscle fibers) have a small number of mitochondria, a high glycogen content, high ATPase activity, high glycolytic enzyme activity, and a fast and short contraction rate; Type IIx (intermediate muscle fibers) have a number of mitochondria, a high myoglobin content, a series of enzyme activities, and metabolic and contractile characteristics between type IIa and type IIb muscle fibers. Therefore, when the proportion of oxidative muscle fibers is high, the muscle color, pH value, marbling score and intramuscular fat content are all high, the muscle is tender, the water retention performance is high, and the meat quality is good.

Adenosine monophosphate-activated protein kinase (AMPK) is called the "energy monitor" of the cell, which can promote mitochondrial biosynthesis and metabolic changes in oxidative muscle phenotypes. At present, there are three main ways to activate AMPK: exercise activation, activator, and special feed-induced activation. Nutrition is the basis for ensuring animal muscle development, and how to improve meat quality through dietary nutrition is a problem that people are more concerned about. Therefore, Su Lin, Li Huijiao, Jin Ye* and others from the College of Food Science and Engineering of Inner Mongolia Agricultural University fed rats with creatine monohydrate (CMH) and conjugated linoleic acid (CLA), and measured the AMPK activity and the relative expression of muscle fiber-related gene mRNA to explore the effect of adding CMH and CLA to the diet on the composition of rat muscle fiber types and its relationship with the AMPK metabolic pathway.
1. Effects of dietary supplementation of CMH and CLA on growth performance of rats

As shown in Figure 1, after CMH and CLA were added to the diet, the growth rate of rats in the low- and high-dose CMH groups was higher than that in the control group, while that in the low- and high-dose CLA groups was lower than that in the control group, but there was no significant difference between the groups. As shown in Figure 2, after CMH and CLA were added to the diet, the daily intake of rats was significantly lower than that in the control group (P < 0.05), but there was no significant difference between the low- and high-dose CMH groups and the low- and high-dose CLA groups (P > 0.05). Studies have found that dietary supplementation of CLA has no significant effect on the average daily weight gain of pigs, but in general, the daily intake decreases linearly with the increase in the amount of CLA added, which is consistent with the results of this experimental study.
2. Effects of dietary supplementation of CMH and CLA on rat muscle fiber type composition

As shown in Figure 3, compared with the control group, the relative expression of MyHC IIa in the low-dose CMH group and MyHC IIb in the low- and high-dose CMH groups increased significantly (P < 0.05); at the same time, the relative expression of MyHC IIx mRNA in each experimental group decreased, and there was no significant difference between the low- and high-dose CMH groups (P > 0.05). Therefore, dietary supplementation of CMH increased the relative expression of MyHC IIa and MyHC IIb mRNA in rat gastrocnemius muscle, and the effect of low-dose CMH on muscle fiber type composition was more obvious than that of high-dose CMH.
3. Effects of dietary supplementation of CMH and CLA on LDH, SDH, and MDH activity in rat gastrocnemius muscle

As shown in Figure 4, compared with the control group, dietary supplementation of high-dose CMH and CLA can reduce LDH activity in rat gastrocnemius muscle, among which the high-dose CLA group was significantly different from the control group (P < 0.05). This indicates that the activity of glycolytic enzymes in rat gastrocnemius muscle is weakened after dietary supplementation of CMH and CLA. SDH and MDH are the rate-limiting enzymes in the aerobic oxidation of glucose, which can reflect the degree of mitochondrial aerobic metabolism and evaluate muscle oxidative activity. As shown in Figure 5, the SDH activity of the low-dose CMH group and the low- and high-dose CLA groups was significantly higher than that of the control group (P < 0.05). As shown in Figure 6, the MDH activity of the low-dose CLA group was significantly higher than that of the control group (P < 0.05). Overall, after adding CMH and CLA to the diet, the SDH and MDH activities of the gastrocnemius muscle of rats increased to varying degrees, indicating that the oxidative enzyme activity of the gastrocnemius muscle of rats increased. This is consistent with the result that the proportion of oxidative muscle fibers increased and the proportion of glycolytic muscle fibers decreased after adding CMH and CLA.

4. Effect of adding CMH and CLA to the diet on the AMPK activity of the gastrocnemius muscle of rats

As shown in Figure 7, after adding CMH and CLA to the diet, the AMPK activity of the gastrocnemius muscle of rats in the low- and high-dose CMH and CLA groups was higher than that of the control group, and the low-dose CLA group was significantly higher than the control group (P < 0.05). It has been reported that adding polyunsaturated fatty acids to the diet can promote the expression of the AMPK gene. After feeding mice with unsaturated fatty acids, it was found that the expression of the AMPK gene increased significantly.
5. Effects of adding CMH and CLA on the relative expression of PGC-1α, MEF2C and GLUT4 mRNA in rats

As shown in Figure 8, the relative expression of PGC-1α mRNA in the gastrocnemius muscle of rats in the low-dose CMH group and the low- and high-dose CLA groups was significantly higher than that in the control group (P < 0.05); for the relative expression of MEF2C mRNA, compared with the control group, the low- and high-dose CMH groups increased significantly (P < 0.05), and the high-dose CLA group decreased significantly (P < 0.05); compared with the control group, the relative expression of GLUT4 mRNA in the gastrocnemius muscle of rats in the low-dose CMH group, the high-dose CLA group and the control group increased significantly (P < 0.05). In general, except that high-dose CLA significantly reduced the relative expression of MEF2C mRNA, dietary supplementation of CMH and CLA increased the relative expression of PGC-1α, MEF2C and GLUT4 mRNA, which is consistent with the above-mentioned result of increased AMPK activity.
6. Correlation analysis results between MyHC expression level and AMPK activity, expression level of myofiber transformation-related genes and enzyme activity

As shown in Table 2, AMPK activity was positively correlated with the relative expression of MyHC I, MyHC IIa and MyHC IIx, and negatively correlated with the relative expression of MyHC IIb; LDH activity was positively correlated with the relative expression of MyHC I, MyHC IIb and MyHC IIx, and significantly negatively correlated with the relative expression of MyHC IIa (P < 0.05); SDH and MDH activity were positively correlated with the relative expression of MyHC I and MyHC IIa, and negatively correlated with the relative expression of MyHC IIb. PGC-1α, MEF2C and GLUT all have positive regulatory effects on MyHC I and MyHC IIa muscle fibers, and negative regulatory effects on MyHC IIb and MyHC IIx muscle fibers.
Conclusion
The growth performance of rats supplemented with CMH and CLA in the diet was measured and analyzed. The results showed that the addition of CMH and CLA to the diet had no significant effect on the growth rate of rats, but could significantly reduce the daily intake of rats, which indicates that CMH and CLA have a positive effect on improving feed conversion rate. The results of this study showed that the addition of CMH to the diet upregulated the relative expression of MyHC I, IIa, and IIb mRNA in the gastrocnemius muscle of rats, while the activity of oxidase (SDH, MDH) and AMPK activity and the relative expression of PGC-1α, MEF2C, and GLUT4 mRNA in the muscle increased, but reduced the relative expression of MyHC IIx mRNA and the activity of glycolytic enzyme (LDH). The addition of CLA to the diet increased the proportion of oxidative muscle fibers in the gastrocnemius of rats, and at the same time increased the activity of MDH, SDH, AMPK and the expression of PGC-1α and GLUT4 mRNA genes, but reduced the proportion of glycolytic muscle fibers and LDH activity. This shows that the addition of CMH and CLA to the diet can increase AMPK activity, upregulate the expression of genes related to the AMPK pathway, enhance muscle oxidative metabolism, and transform muscle fiber types. Therefore, in the future, the livestock breeding process can regulate the transformation of muscle fiber types by feeding CMH and CLA and activating the AMPK signaling pathway, thereby improving the quality of livestock meat.
This article "Effects and Mechanisms of Creatine Monohydrate and Conjugated Linoleic Acid on Rat Muscle Fiber-Related Genes and Enzyme Activities" comes from "Food Science" Volume 42, Issue 7, 2021, Pages 149-155, Author: Su Lin, Li Huijiao, Hou Yanru, Zhao Yajuan, Bai Yanping, Sun Bing, Zhao Lihua, Jin Ye. DOI:10.7506/spkx1002-6630-20200915-183. Click the original text below to view the relevant information of the article.
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