Various restricted feeding strategies have been adopted for weight loss in recent years, among them intermittent fasting or intermittent energy fasting is one of the most accepted [22, 23]. The fasting concept relies on maintaining a minimum feed intake without specifying a specific way to lose weight [24]. For example, fasting days and limited energy intake. The metabolism of skeletal muscle of young rabbits at 70 days of age is in a relatively vigorous period, and fat deposition increases in leg muscles increases gradually after 70 days of age [25]. Hence, skeletal muscle of rabbits were studied in CG_M and EG_M rabbits from 76 days to 90 days of age to assess their metabolic characteristics during a 15-day weight loss period. Previous studies found that dietary restriction significantly reduced perirenal fat and scapular fat content in rabbits [26]. Body weight and visceral fat of rabbits were significantly reduced at the end of weight loss period, and they drank larger quantities of water during the weight loss period, thus their urine was light yellow. These results indicated that dietary restriction caused phenotypic differences in CG_M and EG_M rabbits.
Skeletal muscle is affected by a variety of factors during the process of weight loss, such as hormones, disease, sex, and age [27]. Energy-restricted diets can activate insulin signaling pathways and reduce abnormal reactions of mTORC1 and ERK1/2 signaling pathways and expression of negative growth regulatory factors in skeletal muscle of obese mice [28, 29]. Moreover, caloric restrictions can reduce skeletal muscle mass, particularly muscle fiber length and cross-sectional area, and inhibit the expression of the PGC- 1α/Expression of IGF-1/AKT1 signaling pathway [30]. We found that functional pathway enrichment of DEGs in the transcriptome was mainly related to metabolism and growth signaling pathways such as the FOXO signaling, glutathione metabolism, HIF-1 signaling, AMPK signaling, and PPAR signaling pathways. The FOXO signaling pathway mediates insulin and insulin-like growth factors that inhibit cell metabolism, growth, differentiation, oxidative stress, and autophagy [31]. Under insufficient nutritional conditions, skeletal muscle will undergo proteolysis and amino acid degradation to maintain glycolipid metabolism in the liver. This degradation pathway is closely related to the regulatory pathway mediated by Forkhead Box (Fox) O transcription factors [32]. Conversely, the differentiation of skeletal muscle is also associated with the insulin pathway that inhibits signaling factors in the FOXO signaling pathway that mediates autophagy regulation and transcriptional protein degradation [33]. Our study also indicated that dietary restriction causes skeletal muscle loss through the insulin-mediated FOXO signaling, AMPK signaling, and PPAR signaling pathways by degradation of amino acids to provide nutrients for the liver and sustain the normal metabolic state of the organism.
Proteins are the embodiment of biological functions. The biological process from gene to protein is influenced by a variety of cell growth and metabolism regulatory factors. The proteomic analysis of skeletal muscle after weight loss indicated that the biological pathways involved in DEPs were mostly related to cellular protein metabolic processes, confirming the crucial role of protein metabolism in skeletal muscle. However, the KEGG enrichment analysis showed that these DEPs were also related to the ribosome, complement cascade, coagulation cascade, and legionellosis pathways, which are closely associated with immune pathways. The complement and coagulation cascade pathways are involved in the metabolic processes of human obesity and diabetes mellitus that influence a variety of human diseases [34]. Our results suggested that there was also an inflammatory response in rabbit skeletal muscle after weight loss, which may be caused by lower nutrient levels after dietary restriction.
Comparative omics analyses can demonstrate potential mechanisms for biological processes [35]. Consistent with previous research, a low Pearson correlation coefficient (0.146) existed between comparative genomic and proteomic analyses indicating the existence of complex regulatory mechanisms [36]. Interestingly, eight genes were involved in chronic muscle fiber formation. Collagen V [col(V)] is the main connective tissue protein, which plays an important role in the formation of muscle fibers. The majority of intramuscular collagen is located around the muscle bundle. Different types of collagen play key roles in the regulation of the homeostasis of skeletal muscle, and are involved in many biological functions including signal transduction, muscle fiber formation, and immunity [37]. Two types of COL5A3 and Collagen VI are involved in the development and metabolism of skeletal muscle, and they are also closely related to insulin resistance in obese individuals [38, 39]. The COL5A2 and COL3A1 genes in CG_M and EG_M rabbits were highly correlated with transcription and translation levels, suggesting a vital role in the regulation of muscle fiber formation. Muscle fiber composition is closely associated with insulin resistance. Two important muscle fiber types (type I and type II) exist in skeletal muscle. Type I muscle fibers are a chronic muscle fibers that have many mitochondria for oxidative metabolism and can increase their number after exercise or weight loss. Type II are a fast muscle fibers that contain few mitochondria and are mainly involved in glycolytic metabolism of skeletal muscle. The number of type I slow muscle fibers is lower than the number of type II fast muscle fibers in obese individuals [40, 41]. Thus, slow muscle fibers are important for muscle metabolic processes during obesity. The ATP2A2 protein is an ATPase in the sarcoplasmic reticulum that is closely related to Ca2 + transport in the regulation of Ca2 + signaling pathway. It is also a regulator of slow muscle fiber formation [42, 43]. The PDLIM3 gene contains a NH2-terminal PDZ domain and a COOH-terminal LIM domain, both of which can enhance the connections between alpha-actinin and actin filaments to regulate muscle differentiation [44]. Similarly, its alternative splicing form can cause muscle atrophy[45]. The Myosin light chain 3 (MYL3) and the Ubiquitin C-terminal hydrolase L1 (UCHL1) are involved in the formation of slow muscle fibers. Specifically, MYL3 is considered a biomarker protein associated with myopathy [46–48]. Notably, the transcriptome and proteome analyses in our study showed a strong correlation between gene and protein expression levels of ATP2A2, PDLIM3, MYL3, and UCHL1, which are likely involved in the regulation of the development of slow muscle fibers. The GPX7 protein is a member of the Glutathione peroxidase family and plays an important role in the antioxidant reaction of cells. It catalyzes Glutathione (GSH) to oxidized Glutathione (GSSG), reducing toxic hydrogen peroxide and lipid peroxides to non-toxic hydroxyl compounds [49] [50]. Obese patients also have lower serum GPX expression levels related to the proportion of body fat [51]. Synchaperone FKBP5 is a protein that regulates cell stress response. Its genetic variants are associated with T2D-related traits and other stress-related diseases [52]. Obese mice with the FKBP5 knockout gene show improved glucose tolerance and enhanced insulin signaling in skeletal muscle [53]. It is highly expressed in the hypothalamus and hippocampus tissues in response to a high-fat diet [54]. Lastly, the GPX7 and FKBP5 genes are involved in metabolic processes associated with oxidative stress in skeletal muscle. The mechanism of insulin resistance in skeletal muscle is always associated with regulatory factors of fat metabolism [55]. Thus, we speculated that these proteins may play an important role in the regulation of cell membrane signal transduction in rabbit skeletal muscle after weight loss, and indirectly promote the oxidative metabolic capacity of muscle cells.