Type 2 diabetes mellitus (T2DM) is a chronic multifactor metabolic disease. The associated chronic hyperglycemia and insulin resistance can induce downstream metabolic changes, resulting in the imbalance of protein, lipid and carbohydrate metabolism, resulting in cardiovascular and cerebrovascular diseases (1), neurodegenerative diseases (2), muscle atrophy (3) and other systemic multi-organ chronic damage and even failure. Consequent detrimental effects occur in muscle mass and muscle strength, thus, significantly impacting the musculoskeletal system and leading to the development of sarcopenia (4, 5). Indeed, the prevalence of sarcopenia is high in patients with T2DM, ranging from 7% to 29.3% (6). However, the pathogenesis and etiology of diabetic sarcopenia (DS) are complex (7), with nutritional deficiency (8), muscle fat accumulation (9, 10), inflammation (11), oxidative stress (9, 12), lack of exercise (13), and diabetic complications such as diabetic microangiopathy (14), diabetic neuropathy (15), and diabetic foot disease (16) reported as potential etiologic factors. DS is characterized by muscle atrophy, slower nerve conduction, reduced maximum tension generated by skeletal muscle contraction, and gradually slower contraction rates. Moreover, with an increase in age, middle-aged and elderly individuals with DS further experience limb movement degeneration, reduced balance, and metabolic rate, as well as increased risk of falls, fractures, etc. Furthermore, although DS is a complication of T2DM with high incidence and different treatment methods (11), diagnostic criteria have only been established for sarcopenia, while that for DS remains unclear.

Metabolomics analysis effectively detects and quantifies small molecules, including metabolites, within samples to identify differentially abundant metabolites across various disease states or physiological periods (17). In particular, it can be applied to detect early markers of disease, discover drug targets, elucidate disease mechanisms, and facilitate improved disease diagnosis. Currently, the diagnostic criteria for DS primarily focus on assessing skeletal muscle strength, mass, etc., with no established serological indicators to guide diagnosis. In this study, metabolomic analysis is employed to screen serum metabolomic markers of DS and elucidate potential underlying mechanisms associated with DS development and pathogenesis. Hence, the primary aim of this study is to identify targets for early clinical diagnosis and treatment of DS, as well as potential markers capable of predicting disease prognosis and development.

In this study, we randomly selected ten DS and ten DM patients for serum untargeted metabolomics analysis. The concentrations of alkanoic acid, 5 ‘-MTA, ADMA, and glutamine were significantly increased in the DS group compared with the DM group, while that of IX was significantly decreased. KEGG analysis further identified ABC transporters and the mTOR signaling pathway as significantly different between the two groups. To our knowledge, this is the first study to report biomarkers for DS using a metabolomics approach. To identify serum metabolomic markers of DS and explore the related mechanisms, muscle testing was performed on enrolled patients and clinical characteristics were statistically analyzed. The results showed that, compared with the DM group, DS patients were older, had lower BMI, and significantly lower grip strength and muscle mass. Moreover, serum laboratory tests showed significant differences in HbA1c, blood glucose, and blood lipid levels between the two groups, which is consistent with previously reported results (21).

Additionally, higher levels of pentadecanoic acid were detected in the DS group compared with the DM group. As a long-chain saturated fatty acid (LCFA), pentadecanoic acid is a potent biological marker of dairy fat intake (22). As LCFAs accumulate, they become deposited in muscle, consequently compromising muscle integrity. In fact, within muscle tissue, continuous accumulation of LCFAs can lead to disordered protein synthesis and lipotoxicity, activation of immune cells, and induction of muscle cell inflammation, resulting in muscle atrophy and apoptosis of muscle proteins (23). Meanwhile, in non-adipose tissue, ectopic accumulation of LCFAs leads to metabolic dysfunction; increased lipid content causes increased lipid load, muscle atrophy, and a corresponding decrease in muscle content and mass (24). It has been suggested that damage to muscle integrity may be related to intramuscular fat (peroxisome proliferator-activated receptor γ)(25) and intramuscular components associated with the resulting muscle inflammation (cyclo-oxygen-ase-2, inducible nitric oxide synthase) (26). This further suggests a relationship between ectopic intramuscular adipose and muscle loss. In addition, LCFAs can lead to insulin resistance, which is one of the primary etiologic factors of sarcopenia (27). Hence, the LCFA pentadecanoic acid accumulates in skeletal muscle, where it promotes the production of inflammatory mediators, insulin resistance, skeletal muscle protein consumption, and muscle fiber atrophy, and decreases skeletal muscle mass and content, which reflects the etiology and pathogenesis of sarcopenia. Accordingly, pentadecanoic acid may represent a predictive biomarker for DS.

5’-MTA levels were also elevated in the DS group compared to the DM group. MTA is a naturally occurring hydrophobic thioadenine nucleoside (28), which is an intermediate metabolite or by-product of the methionine (MET) cycle and polyamine synthetase, both of which are intricately associated with key cellular pathways, including gene expression regulation, proliferation, differentiation and apoptosis (29). Although MET is a substrate for homocysteine, elevated levels of MET can cause hyperhomocysteinemia (30), which has been shown to reduce skeletal muscle cell viability and produce an energy imbalance (31–33). Moreover, elevated plasma homocysteine can lead to different systemic or local neurological diseases and promote inflammation (34). Hence, hyperhomocysteinemia can cause skeletal muscle injury and dysfunction, as well as reduce skeletal muscle development and growth (35). Therefore, an increase in MET increases sarcopenia to a certain extent, which is consistent with our experimental results.

In our study, ADMA concentrations were higher in DS patients than in DM controls. ADMA, a naturally occurring chemical found in the plasma, is a metabolic by-product of the protein modification process in the human cytoplasm and is closely related to L-arginine (L-Arg) (36). L-Arg is a conditionally essential amino acid and precursor of nitric oxide (NO) (37). As a key endogenous regulator of vascular tone and endothelial function, NO plays an important role in the regulation of sympathetic vasoconstriction in the microcirculation of exercise muscles (38). ADMA has also been shown to inhibit NO production by L-arginine via inhibition of NO synthase production (39), thus leading to an imbalance in vascular contraction and diastolic function of skeletal muscle, blood hypoperfusion, tissue ischemia, and hypoxia. Consequently, skeletal muscle protein formation is negatively impacted, cell and skeletal muscle cells begin to atrophy, and the occurrence and development of sarcopenia is exacerbated. In addition, ADMA can promote the development of chronic inflammation, which can cause damage to skeletal muscle cells. Meanwhile, inflammatory factors also impact ADMA homeostasis (40). Regarding DS, in particular, the enhanced inflammatory response and increased activity of inflammatory factors lead to a decrease in the Arg/ADMA ratio (41), which increases ADMA synthesis and exacerbates the development of diabetes-related muscle mass, strength, and functional decline.

We also found that glutamine concentrations were higher in DS patients than in DM controls. Glutamine is the most abundant amino acid in the blood. As a non-essential amino acid, it is mainly catalyzed by glutamine synthase to synthesize ammonia (NH3) and glutamate in skeletal muscle and is involved in myriad metabolic pathways, including signal transduction, nitrogen transport, energy metabolism, etc. (42, 43). Under stimulation by sarcopenia and hyperglycemia, the body enters a state of long-term, low-level inflammation, causing the abundance of leukocytes, such as lymphocytes, to increase. Unlike skeletal muscle, white blood cells do not have glutamine synthase and cannot synthesize glutamine. In addition, macrophages, lymphocytes, and other inflammatory cells will consume glutamine as a substrate. Therefore, to meet the demand for glutamine anabolism under inflammatory conditions, skeletal muscle cells increase their glutamine production, which becomes released into the blood, causing an increase in plasma glutamine concentration. Indeed, Ilaiwy et al. observed that in vitro muscle cell atrophy is related to an increase in glutamine concentration in culture media (44). Moreover, considering that the DS group was older than the DM group and aging is defined as a disease of anabolic resistance to nutrients (such as amino acids), these patients may have exhibited impaired protein synthesis of glutamine, resulting in reduced muscle protein synthesis and development of sarcopenia. Based on these collective study results, we postulate that glutamine concentration is elevated in the DS group and may serve as an effective metabolic marker of DS.

In addition, we found that IX levels were lower in DS patients than in the DM group. IX is a prenyl flavonoid that inhibits the production of secondary bile acids. Secondary bile acids are cytotoxic DNA-damaging metabolites converted from primary bile acids by 7-α-dehydroxylase (45) that interfere with proper metabolism of sugars and lipids, causing lipids to accumulate in muscle tissues, induction of oxidative stress and the inflammatory response, mitochondrial dysfunction, insulin resistance, and damage to skeletal muscle cells, thereby increasing the risk of developing DS (46). Moreover, xanthohumol, a precursor of IX, has been shown to have anti-obesity effects in animal studies (47). Therefore, IX might improve glucose and lipid metabolism to a certain extent, thereby improving lipid accumulation in diabetes and reducing the possibility of DS.

We also found that 3-methylxanthine was highly expressed in the DS group. 3-Methylxanthine is nephrotoxic in that it impacts kidney function, causing kidney damage and increasing the risk of end-stage renal disease (48). Decreased renal function, as an independent risk factor for sarcopenia, suggests that 3-methylxanthine appears to be a predictive metabolite for diabetic myopathy (49).

The level of L-(+)-arginine was also significantly higher in the DS group than in the DM control group. L-(+)-arginine is an inducer of mTOR, which induces muscle protein synthesis primarily through the mTOR pathway (50). However, studies have shown that when sarcopenia occurs, the L-(+)-arginine concentration in muscles begins to decrease (51). Hence, we postulate that our findings may have resulted from protein decomposition in plasma and a subsequent compensatory increase in L-(+)-arginine concentration, reflecting metabolic changes secondary to sarcopenia.

We also observed a significant increase in D-gluconic acid levels within the DS group. D-Gluconic acid is involved in the antioxidative stress response, thereby upregulating glycolysis and downregulating the TCA cycle, leading to mitochondrial dysfunction and muscle ATP deficiency (52, 53). However, due to the lack of further evidence related to sarcopenia, more studies are needed to verify its applicability as a metabolic marker for DS.

We further identified metabolic pathways associated with DS using KEGG enrichment analysis. Among the enriched pathways were ABC transporter superfamily, mTOR signaling pathway, Chagas disease, amoebiasis, amyotrophic lateral sclerosis, pyrimidine metabolism, D-arginine and D-ornithine metabolism, as well as seven other metabolic pathways. Of these pathways, ABC transporters and the mTOR signaling pathway are the most influential and may represent potential target pathways for DS. ABC transporters, one of the largest families of membrane proteins in most organisms, are involved in myriad key physiological processes and serve as pathogenic factors in many diseases. In fact, mutations in ABC-related genes cause neurological disorders and defects in cholesterol and bile transport (54). Cholesterol transport defects affect lipid metabolism and lead to fat deposition in skeletal muscle, thus affecting the activity of skeletal muscle cells and causing skeletal muscle atrophy. Meanwhile, neurological diseases directly affect the activity of innervated muscles, thus impacting communication between muscles and nerves, as well as the types of muscle fibers and expression of myosin (55). This causes muscle weakness and atrophy, which increases the risk of developing sarcopenia. The mTOR signaling pathway plays a key regulatory role in mitochondrial autophagy in skeletal muscles. Mitochondrial quality in skeletal muscle cells is closely associated with sarcopenia; reduced quality deteriorates the muscle microenvironment, leading to the progression of sarcopenia in naturally or rapidly aging mice (56). In addition, L-(+)-arginine also has an important role in the mTOR signaling pathway. Hence, we purport that ABC transporters and the mTOR signaling pathway may contribute to DS development and, therefore, may represent target pathways for the prevention and treatment of DS.

Certain limitations were noted in this study. First, the relatively small sample size limited the statistical ability to definitively identify metabolites associated with DS. Second, due to the cross-sectional design, we were unable to predict DS prognosis or determine the causal relationship between serum differential metabolites and DS. Further studies are warranted to elucidate the clinical correlation between these metabolites and DS, as well as the related mechanisms. Nevertheless, the advantage of our study lies in the application of metabolomics analysis to identify potential metabolites related to the development and progression of DS. These findings may inform the development of effective preventative and therapeutic modalities for DS.

In conclusion, this study explored potential biomarkers of DS using a metabolomic strategy. Pentadecanoic acid, 5’-MTA, ADMA, and glutamine were identified as potential biomarkers of DS, while ABC transporters and the mTOR signaling pathway likely make significant contributions toward DS development. The potential relationships between differential metabolites and DS provide novel insights into the mechanisms underlying DS and potential strategies for future treatments.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Medical Ethics Committee of the Qilu Hospital of Shandong University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

YT Conceptualization, Methodology, Resource, Investigation, Formal analysis, Data curation, Visualization, Writing - Original draft and Writing - Review & Editing. XL Data curation and Formal analysis. YY Data curation and Formal analysis. BL Conceptualization, Validation and Writing - Review & Editing. YF Data curation and Formal analysis. WZ Data curation. CF Data curation. XY Data curation. ZH Formal analysis and Funding acquisition. MC Supervision, Funding acquisition, Resources and Project administration. All authors contributed to the article and approved the submitted version.