Abstract
MOTS-c, a peptide encoded by the mitochondrial 12S rRNA gene, emerges as a significant player in the body's response to stress and exercise. It translocates to the nucleus, influencing the expression of genes related to stress adaptation and antioxidant responses. Acting primarily through the Folate-AICAR-AMPK pathway, MOTS-c impacts energy metabolism, insulin resistance, inflammation, exercise, aging, and associated pathologies. Its potential to maintain energy and stress homeostasis, particularly relevant in the context of an aging global population, necessitates a comprehensive review of relevant studies.
Introduction
Mitochondria, traditionally recognized as cellular powerhouses, are also integral to stress responses, information transfer, cell death, and aging. Dysfunction in mitochondria can trigger oxidative stress, metabolic imbalances, inflammation, neurodegenerative diseases, and accelerated aging, posing a substantial socioeconomic burden. Understanding the mechanisms by which mitochondria influence these diseases is crucial for improving the quality of life for the elderly and promoting healthy aging.
In 2015, the MOTS-c peptide, encoded by mitochondrial DNA, was discovered. Activated by stress and exercise, MOTS-c expression declines with age. Initial findings highlighted its role in regulating glucose uptake, lipid metabolism, insulin resistance, and aging-related physiological changes. Recent research suggests its involvement in obesity, inflammation, neuroprotection, and age-related hypokinesia, underscoring its potential contribution to healthy aging.
This review summarizes MOTS-c signaling, its role in various physiological and pathological processes, and the underlying molecular mechanisms.
Mitochondrial DNA: The Source of MOTS-c
Mitochondria, originating from engulfed alpha-proteobacteria, produce ATP via oxidative phosphorylation (OXPHOS), synthesize biosynthetic intermediates, and participate in cellular stress responses. Human mitochondrial DNA (mtDNA), a 16.6 kb double-stranded molecule, encodes 11 mRNAs, 2 rRNAs (12S and 16S rRNA), and 22 tRNAs. These mtDNA-encoded proteins, while a minority of OXPHOS subunits, are essential.
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The regulation of mtDNA expression involves intricate control of various processes, including mtDNA maintenance and transcription, RNA processing, mRNA stability, tRNA modification, and translation. Furthermore, the existence of numerous unannotated short open reading frames (sORFs) within the genome, transcriptome, and proteome suggests the potential translation of these sORFs into peptides and proteins. Some mRNAs contain multiple ORFs, with shorter upstream ORFs (uORFs) in the 5'UTR of longer downstream ORFs. While initially considered non-coding, studies have revealed that some uORFs are translated into peptides, playing a crucial role in downstream ORF translation. Certain sORFs have demonstrated biological activities in metabolism, apoptosis, and development.
Mitochondria-Derived Peptides (MDPs)
Mitochondrial DNA utilizes a unique genetic code, with ATA and ATT as start codons and AGA and AGG as stop codons. Based on standard and special genetic codes, mitochondrial DNA can be classified into four categories of sORFs of 9-40 amino acids. To date, identified MDPs, including Humanin, SHLP1-6, and MOTS-c, are encoded by the standard genetic code.
Humanin
Humanin, the first discovered biologically active MDP, exhibits anti-apoptotic and neuroprotective effects. Discovered in 2001, it is encoded by a 75 bp ORF, translating into 21 or 24 amino acid peptides. Humanin interacts with intracellular molecules and cell membrane receptors, enhancing cellular resistance to Alzheimer's disease-associated toxins, improving insulin sensitivity, preventing oxidative stress damage, and increasing resistance to apoptosis.
SHLP1-6
Six sequences encoding 20-38 amino-acid-peptides, named SHLP1-6, were identified in 16S rRNA. SHLP2 and SHLP3 enhanced cell viability and reduced apoptosis in mouse β-cells and human prostate cancer cells. SHLP2 and SHLP4 promoted cell proliferation, while SHLP6 significantly increased apoptosis. SHLP2 treatment in age-related macular degeneration (AMD) restored normal levels of OXPHOS complex protein subunits, increased mitochondrial DNA copy number, attenuated amyloid beta-induced toxicity, induced anti-apoptotic effects, and prevented cell and mitochondrial loss.
MOTS-c
In 1983, cDNAs corresponding to the 12S rRNA region were cloned. Subsequent electronic searches revealed a 51 base pair sequence translated into a 16-amino acid peptide named MOTS-c. Basic local alignment search tool (BLAST) searches indicated a mitochondrial DNA origin for MOTS-c, ruling out a nuclear origin. MOTS-c translation likely occurs in the cytoplasm.
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MOTS-c is mainly present in skeletal muscle and blood, with concentrations decreasing with age. It significantly impacts stress responses, cellular metabolism, sports ability, and inflammation through altered expression of nuclear genes.
MOTS-c: Mediating Communication Between Mitochondrial and Nuclear Genes
Mitochondria communicate extensively with the nucleus, primarily through nuclear-mitochondrial anterograde signals. Retrograde signaling is mainly activated by OXPHOS and mitochondrial DNA defects, activating AMPK or NF-κB pathways to alter nuclear gene expression. MOTS-c, however, can translocate to the nucleus in response to metabolic stress and regulate adaptive nuclear gene expression, complementing the understanding of mutual signaling communication between mitochondrial and nuclear genes.
In a resting state, MOTS-c is predominantly extranuclear. However, metabolic stress induces rapid translocation to the nucleus, returning to its extranuclear state within 24 hours. This translocation is AMPK-dependent, suggesting that inhibiting AMPK activity may prevent stress-induced nuclear translocation of MOTS-c. Metformin and AICAR, AMPK activators, mimic stress-like cellular responses, leading to MOTS-c translocation to the nucleus. ROS may also influence translocation, as N-acetylcysteine treatment inhibits oxidant-induced nuclear translocation. Nuclear translocation may require interaction with other proteins, as substitution of a hydrophobic core residue prevents its entry into the nucleus.
Once in the nucleus, MOTS-c interacts with stress response transcription factors, including NFE2L2/NRF2 and ATF1/ATF7. It can bind to the promoter regions of NRF2 target genes with antioxidant response element (ARE) sequences, regulating drug metabolism, antioxidant defenses, and oxidative signaling. Additionally, some transcription factor binding motifs are enriched in the promoters of genes regulated by MOTS-c genes, such as ATF1/ATF7 and JUND. Through retrograde signaling affecting nuclear gene expression, MOTS-c plays roles in obesity, insulin resistance, exercise, inflammation, and lifespan.
The Pathways Regulated by MOTS-c
AMPK
AMPK, a highly conserved heterotrimeric kinase complex, is activated under energy stress conditions. Lee and colleagues identified the folate-methionine cycle and the de novo purine biosynthesis pathway as targets of MOTS-c. MOTS-c treatment decreased levels of 5-methyltetrahydrofolate (5Me THF) and methionine, while homocysteine levels increased, ultimately leading to increased AICAR levels. AICAR increases AMP levels, activating AMPK by phosphorylation. Activated AMPK stimulates glucose uptake and fatty acid oxidation, increasing ATP production and maintaining energy homeostasis. AMPK also regulates SIRT1 and PGC-1α, producing anti-inflammatory effects. Furthermore, AMPK promotes nuclear translocation of MOTS-c, forming a feedback loop.
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Folate Cycle and Purine Biosynthesis
MOTS-c modulates the folate cycle and purine biosynthesis, impacting AICAR levels and subsequently activating AMPK. This pathway is crucial for maintaining metabolic homeostasis and responding to energy stress.
MOTS-c and Bone Metabolism
Overview of Bone Tissue Physiology
Bone tissue provides support, protection, hematopoiesis, and mineral balance. Bone development involves bone growth and thickening (modeling) and bone unit renewal (remodeling). Modeling ceases in adulthood, while remodeling continues throughout life. Osteoblasts, differentiating from bone marrow mesenchymal stem cells (BMSCs), promote bone formation. Osteoclasts, originating from hematopoietic stem cells, are responsible for bone resorption. The dynamic balance between bone formation and bone resorption is crucial for healthy bones. Mitochondria and mitochondria-derived peptides, including MOTS-c, play a role in bone metabolism.
Potential Mechanisms of MOTS-c Involvement in Bone Metabolism in Osteoblasts
Role of MOTS-c in TGF-β-Mediated Osteoblasts
MOTS-c promotes osteoblast differentiation, increasing the expression of alkaline phosphatase (ALP), bone gamma-carboxyglutamic-acid-containing proteins (BGLAP), and Runx2. Local injection of MOTS-c rescued bone loss in a polymer compound model. TGF-β, a multifunctional protein, plays a key role in cell proliferation, differentiation, apoptosis, and tissue development. MOTS-c increases the expression of TGF-β1 and TGF-β2 during osteogenic differentiation of BMSCs. Silencing TGF-β1 significantly inhibited osteogenic differentiation of BMSCs, suggesting a close relationship between MOTS-c and TGF-β.
Role of MOTS-c in Osteoblasts Mediated via the TGF-β/Smad Pathway
TGF-β and Smad act synergistically to regulate osteoblasts, promoting osteoblast differentiation, inhibiting bone resorption, and promoting bone formation. Smad, a key intracellular effector of TGF-β, mediates osteoblast TGF-β and osteoclast bone morphogenetic protein signaling pathways. Smad-dependent and non-dependent pathways greatly promote osteoblast differentiation and bone formation and can regulate mesenchymal differentiation by mediating the Runx2 gene. MOTS-c promotes bone marrow MSC differentiation toward osteogenesis via the TGF-β/SMAD pathway.
Role of MOTS-c in the Synthesis of Type I Collagen by Osteoblasts
MOTS-c promotes the expression of type I collagen secreted by osteoblasts. Type I collagen, the main bone matrix protein, consists of two α1 chains and one α2 chain. TGF-β is the most direct cytokine affecting the synthesis and breakdown of type I collagen. MOTS-c promotes the protein synthesis of TGF-β and Smad7 in osteoblasts and promotes the expression of type I collagen-related genes COL1A1 and COL1A2.
Regulatory Role of MOTS-c in Bone Metabolism and Related Mechanisms
Recent studies have demonstrated a direct hormonal link between bone remodeling, food intake, and glucose metabolism, and the involvement of MOTS-c in bone metabolism. These studies demonstrated that MOTS-c promotes osteoblast proliferation, differentiation, and mineralization. Furthermore, it inhibits the osteoclastic differentiation involved in the regulation of bone metabolism and remodeling and possesses anti-osteoporotic effects. Therefore, exploring the potential application of MOTS-c as a valuable anti-osteoporosis treatment strategy to promote bone health is promising.
MOTS-c and Exercise
MOTS-c as an Exercise-Induced Regulator
MOTS-c functions as an exercise-induced regulator of metabolic homeostasis, dynamically translocating to the nucleus in response to metabolic stress to regulate gene expression. Exercise interventions show promise as effective adjunct strategies to prevent and/or attenuate chemotherapy-associated toxicity in patients with early-stage breast cancer.
Metformin and MOTS-c
Metformin, a putative exercise mimetic, shares many mechanistic features with MOTS-c, including insulin sensitization, enhanced glucose utilization, suppression of mitochondrial respiration, and targeting the folate-AICAR-AMPK pathway. However, studies have shown that metformin does not operate as an exercise mimetic to augment the circulating levels of MOTS-c in patients with breast cancer treated with neoadjuvant therapy. This may be due to the different target tissues of metformin and MOTS-c.
MOTS-c and Physical Activity
MOTS-c increases in skeletal muscle following long-term physical activity and improves acute exercise performance after a single dose. Exercise stimulates the expression of MOTS-c, which is encoded by mitochondria, in humans. Those with limited movement can benefit tremendously from kinesimatology, which also accelerates metabolism. MOTS-c, as a new type of mitochondrial signal molecule, may stimulate exercise-mediated physiological responses to increase endurance.
MOTS-c and Disease Treatment
MOTS-c in Aging
Adaptation of cellular responses to changing internal and external environments is necessary for the health of an organism. Studies have shown that the interaction of MOTS-c/NRF2 can improve the expression of mitochondrial protective genes. The aging process could lead to a decrease in MOTS-c levels. Studies have shown that blood MOTS-c levels in young people are higher than those in middle- and old-aged people. Moreover, MOTS-c may improve diabetes by inhibiting insulin resistance and diet-induced obesity.
MOTS-c in Cardiovascular Disease
Obesity is the main culprit of cardiovascular problems. Recent studies have shown a protective effect of MOTS-c against cardiac dysfunction and pathological remodeling. MOTS-c prevented the development of heart failure via the activation of the AMPK pathway and improved angiogenesis, inflammation and apoptosis in terms of cell function. The addition of exogenous MOTS-c increases the level of myocardial MOTS-c, which activates AMPK. This study reveals a new pathway for MOTS-c to protect against cardiovascular disease.
MOTS-c in Insulin Resistance
Insulin resistance can lead to a decrease in the number and the abnormal morphology of mitochondria in tissue cells, which in turn hinders the synthesis of ATP. MOTS-c enhances insulin sensitivity throughout the body through muscles. Previous studies have revealed that MOTS-c can enhance the insulin sensitivity of skeletal muscle and improve the utilization of glucose. Aging leads to increased insulin resistance, which reduces MOTS-c levels in skeletal muscle and blood of mice. It has been reported that MOTS-c improves age-related insulin resistance in male mice by increasing glucose intake in soleus muscles.
MOTS-c in Obesity
MOTS-c treatment prevented obesity in mice fed a high-fat diet, but did not affect the weight of mice fed a normal diet. In addition, MOTS-c can improve blood glucose balance and prevent hyperinsulinemia caused by high-fat diet. A major benefit of MOTS-c is that it promotes metabolic homeostasis and reduces obesity and insulin resistance.
MOTS-c's Impact on Cancer
The METTEN Trial
The METTEN trial, a phase 2 study, investigated neoadjuvant metformin in combination with trastuzumab and chemotherapy in women with early HER2-positive breast cancer. The trial's design involved random assignment of patients to receive either metformin combined with neoadjuvant chemotherapy and trastuzumab or an equivalent regimen without metformin. The results indicated that metformin does not operate as an exercise mimetic to augment the circulating levels of MOTS-c in patients with breast cancer treated with neoadjuvant therapy.
Limitations and Future Directions
Several limitations exist in current MOTS-c research. Endogenous levels of circulating MOTS-c have been shown to vary significantly depending on the assay method used. The mechanisms of MOTS-c production, secretion, distribution, and metabolism in the human body remain to be fully elucidated. Likewise, the extent of involvement of various tissue targets and/or the effects of metformin on skeletal muscle metabolism and how they determine the pharmacodynamics and endogenous serum levels of MOTS-c in patients with BC await evaluation in future studies. Further research is needed to address these limitations and fully understand the therapeutic potential of MOTS-c.