MOTS-c: Comprehensive Research Monograph and Technical Review

Database ID: BIOLOGIX-2024-MOTS-019

Mitochondrial-Derived Peptide Research Metabolic Regulation and Longevity Peptide

Executive Summary

MOTS-c (Mitochondrial Open Reading Frame of the 12S rRNA-c) represents a groundbreaking discovery in mitochondrial biology—a bioactive peptide encoded directly by the mitochondrial genome that functions as a systemic metabolic regulator and exercise mimetic. First identified in 2015, this 16-amino acid peptide has emerged as a critical mediator of mitochondrial-nuclear communication, cellular energy metabolism, and age-related physiological decline. MOTS-c demonstrates remarkable capacity to enhance insulin sensitivity, promote metabolic homeostasis, improve physical performance, and counteract age-dependent functional deterioration across multiple organ systems. This comprehensive monograph examines MOTS-c's molecular characteristics, biosynthetic origins, mechanistic foundations, extensive preclinical evidence base, emerging clinical data, and therapeutic potential in metabolic disorders, aging-related pathologies, and performance enhancement.

Key Research Findings

First peptide discovered to be encoded by mitochondrial 12S rRNA with systemic regulatory functions
Demonstrates potent insulin-sensitizing effects and prevents diet-induced obesity in preclinical models
Functions as an exercise mimetic, improving physical capacity and extending healthspan in aged animals
Translocates to the nucleus under metabolic stress to regulate gene expression via AMPK-dependent pathways
Clinical trials of MOTS-c analogs demonstrate safety and potential efficacy in metabolic disorders
Age-dependent decline in circulating levels correlates with metabolic dysfunction and physical decline

1. Molecular Characterization and Structure

1.1 Chemical Structure and Genomic Origin

MOTS-c is a 16-amino acid peptide with the sequence Met-Arg-Trp-Gln-Glu-Met-Gly-Tyr-Ile-Phe-Tyr-Pro-Arg-Lys-Leu-Arg (MRWQEMGYIFYPRKLR), encoded by a small open reading frame (sORF) within the mitochondrial 12S ribosomal RNA gene. This discovery, first reported by Lee and colleagues in 2015, revolutionized understanding of mitochondrial genome function by demonstrating that mtDNA encodes bioactive peptides beyond the traditional 13 proteins involved in oxidative phosphorylation [Lee et al., Cell Metab 2015; PMID: 25738459]. The peptide is translated in the cytoplasm using the standard genetic code rather than the mitochondrial genetic code, representing a unique biosynthetic mechanism.

The MOTS-c coding sequence spans nucleotide positions 1,382-1,432 of the mitochondrial 12S rRNA gene, a region previously considered non-coding. The first 11 amino acids of MOTS-c are highly conserved across 14 mammalian species, suggesting evolutionary preservation of critical functional domains. This conservation pattern indicates that MOTS-c represents an ancient regulatory mechanism that has been maintained throughout mammalian evolution, underscoring its physiological importance.

Table 1: Molecular Specifications of MOTS-c
Parameter	Value	Notes
Amino Acid Sequence	Met-Arg-Trp-Gln-Glu-Met-Gly-Tyr-Ile-Phe-Tyr-Pro-Arg-Lys-Leu-Arg	16-mer peptide (MRWQEMGYIFYPRKLR)
Molecular Formula	C₁₀₁H₁₅₂N₂₈O₂₂S₂	Contains 2 methionine residues
Molecular Weight	2174.62 Da	Monoisotopic mass
CAS Number	1627580-64-6	Chemical registry identifier
Genomic Location	mtDNA 12S rRNA (nt 1382-1432)	Mitochondrial genome position
Isoelectric Point (pI)	~10.7	Theoretical calculation; highly basic
Net Charge at pH 7.4	+4.0	Physiological pH; highly cationic
Hydrophobicity	-0.456 (GRAVY score)	Moderately hydrophilic character
Extinction Coefficient	11,460 M^-1cm^-1 at 280nm	Based on Trp and Tyr content

1.2 Structural Features and Functional Domains

MOTS-c exhibits several distinctive structural features that contribute to its biological function and stability. The peptide contains two methionine residues (positions 1 and 6), two tyrosine residues (positions 8 and 11), a single tryptophan (position 3), and is highly enriched in basic amino acids (arginine and lysine), conferring a strong positive charge at physiological pH. This cationic character facilitates cellular uptake and nuclear translocation, critical aspects of MOTS-c's mechanism of action.

Secondary structure predictions suggest that MOTS-c adopts predominantly random coil and turn conformations in aqueous solution, with potential for α-helical structure formation under certain conditions or upon receptor binding. The presence of proline at position 12 introduces a structural kink that may be important for molecular recognition. The aromatic residues (Trp3, Tyr8, Tyr11, Phe10) likely contribute to hydrophobic interactions and may form a functional cluster important for biological activity. Studies examining structure-activity relationships have confirmed that the N-terminal region (residues 1-11) is critical for metabolic regulatory functions.

1.3 Genetic Polymorphism: The K14Q Variant

A significant discovery in MOTS-c biology is the identification of the m.1382A>C polymorphism (rs111033358), which results in substitution of lysine with glutamine at position 14 (K14Q variant). This East Asian-specific polymorphism, with allele frequencies of approximately 5-6.5% in Korean and Japanese populations, produces a biologically distinct peptide with altered functional properties [Fuku et al., Aging Cell 2015; PMID: 26289118]. The K14Q variant exhibits reduced AMPK activation capacity and altered metabolic effects compared to the wild-type peptide.

Interestingly, the K14Q polymorphism associates with distinct physiological phenotypes in human populations. Male carriers of the variant allele demonstrate higher percentages of fast-twitch (type IIx) muscle fibers, greater muscular strength, and enrichment among sprint/power athletes compared to endurance athletes. Conversely, in older populations, the polymorphism shows sex-specific associations with sarcopenia risk, with protective effects observed in men but not women. This genetic variation provides natural evidence for MOTS-c's role in muscle function and metabolic regulation in humans.

1.4 Biosynthesis and Cellular Distribution

MOTS-c biosynthesis follows an unusual pathway reflecting its mitochondrial genomic origin. The mitochondrial 12S rRNA containing the MOTS-c coding sequence is transcribed and subsequently polyadenylated, a modification typically associated with mRNA. The polyadenylated transcript is then exported from mitochondria to the cytoplasm, where it serves as an mRNA template for translation using the standard genetic code and cytoplasmic ribosomes. This mechanism differs fundamentally from the 13 conventional mitochondrial genome-encoded proteins, which are translated within mitochondria using the mitochondrial genetic code.

Following synthesis, MOTS-c localizes to multiple cellular compartments depending on physiological conditions. Under basal conditions, MOTS-c is present in cytoplasm and demonstrates significant expression in metabolically active tissues including skeletal muscle, heart, liver, and kidney. Upon metabolic stress or exercise stimulation, MOTS-c undergoes nuclear translocation, where it regulates gene expression programs involved in cellular stress response and metabolic adaptation [Kim et al., Cell Metab 2018; PMID: 29983246]. This dynamic subcellular localization is central to MOTS-c's function as a metabolic stress sensor and adaptive regulator.

2. Synthesis and Manufacturing

2.1 Solid-Phase Peptide Synthesis

MOTS-c is manufactured using standard solid-phase peptide synthesis (SPPS) methodology, typically employing Fmoc (9-fluorenylmethoxycarbonyl) chemistry for orthogonal protection and sequential amino acid coupling. The synthesis proceeds from the C-terminal arginine to the N-terminal methionine on a solid resin support, with careful attention to coupling efficiency given the peptide's 16-amino acid length and presence of challenging sequences including the Ile-Phe-Tyr-Pro tetrapeptide segment.

Critical synthesis considerations include the incorporation of two methionine residues, which are susceptible to oxidation during synthesis and storage. Protected methionine derivatives or post-synthetic reduction protocols may be employed to prevent Met oxidation to methionine sulfoxide, which significantly reduces biological activity. The presence of tryptophan at position 3 requires protection strategies to prevent side reactions during acidic cleavage conditions. HBTU (O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate) or HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) coupling reagents with DIEA (N,N-diisopropylethylamine) base typically achieve >99% coupling efficiency per amino acid addition.

Following complete assembly, the peptide is cleaved from the resin using trifluoroacetic acid (TFA) cocktails containing scavengers such as triisopropylsilane (TIS), water, and ethanedithiol (EDT) to prevent oxidation and side reactions. The crude peptide undergoes initial precipitation in cold ether to remove organic contaminants, followed by dissolution and extensive purification.

2.2 Purification and Quality Control

Purification of synthetic MOTS-c employs preparative reverse-phase high-performance liquid chromatography (RP-HPLC) using C18 silica columns with acetonitrile-water gradient systems. Due to MOTS-c's moderately hydrophobic character (GRAVY score -0.456) and presence of aromatic residues, the peptide typically elutes at 25-35% acetonitrile concentrations. Multiple purification passes may be necessary to achieve pharmaceutical-grade purity exceeding 98%, with removal of deletion sequences, truncation products, and oxidized variants being primary purification objectives.

Table 2: Manufacturing Quality Specifications for MOTS-c
Quality Parameter	Specification	Analytical Method
Purity (HPLC)	≥98.0%	RP-HPLC at 220 nm
Peptide Content	≥95.0%	Amino acid analysis or UV spectroscopy
Sequence Verification	100% match to target	MS/MS peptide sequencing
Molecular Weight	2174.62 ± 1.0 Da	ESI-MS or MALDI-TOF MS
Methionine Oxidation	≤2.0%	RP-HPLC with MS detection
Water Content	≤8.0%	Karl Fischer titration
TFA Content	≤0.1%	Ion chromatography or ¹⁹F NMR
Acetate Content	≤15.0%	Ion chromatography
Bacterial Endotoxins	≤5 EU/mg	LAL assay (USP <85>)
Sterility	Sterile	USP <71> sterility test

Mass spectrometric analysis provides definitive molecular weight confirmation and detects oxidation products, which appear as +16 Da species for each oxidized methionine. High-resolution mass spectrometry enables discrimination between correctly synthesized MOTS-c and closely related impurities. Amino acid analysis following complete acid hydrolysis confirms the amino acid composition, with particular attention to methionine recovery, which may be underestimated due to oxidation during hydrolysis.

2.3 Formulation and Lyophilization

MOTS-c is typically formulated and supplied as a sterile lyophilized powder, most commonly as an acetate or trifluoroacetate salt depending on purification conditions. The lyophilization process involves careful optimization of freezing protocols, primary drying, and secondary drying to produce an elegant, readily reconstitutable cake with optimal stability characteristics. Excipients such as mannitol or glycine may be incorporated as bulking agents to improve cake structure and facilitate reconstitution.

For enhanced stability during storage, some formulations incorporate antioxidants such as methionine or ascorbic acid to prevent oxidation of the two methionine residues in the MOTS-c sequence. The presence of these sulfur-containing amino acids represents the primary stability challenge for long-term storage. Lyophilized MOTS-c demonstrates excellent stability when stored at -20°C to -80°C with protection from moisture, light, and atmospheric oxygen. Stability studies indicate minimal degradation over 24-36 months under proper storage conditions.

3. Mechanism of Action

3.1 The Folate-AICAR-AMPK Pathway

The primary mechanism underlying MOTS-c's metabolic regulatory effects involves modulation of the folate cycle and subsequent activation of AMP-activated protein kinase (AMPK), the master cellular energy sensor. MOTS-c inhibits the enzyme methylenetetrahydrofolate dehydrogenase (MTHFD), a key enzyme in the folate cycle that is coupled to de novo purine biosynthesis [Lee et al., Cell Metab 2015; PMID: 25738459]. This inhibition redirects metabolic flux, leading to accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an intermediate in purine biosynthesis.

AICAR accumulation triggers AMPK activation through multiple mechanisms. AICAR is converted by adenosine kinase to ZMP (AICAR monophosphate), an AMP mimetic that directly binds to AMPK's γ-subunit, mimicking the allosteric activation normally triggered by elevated AMP:ATP ratios during energy stress. Studies in MOTS-c-overexpressing cells demonstrate >20-fold increases in AICAR levels, sufficient to robustly activate AMPK. This creates a metabolic state resembling exercise or caloric restriction, even under nutrient-replete conditions.

AMPK activation by MOTS-c initiates a cascade of downstream metabolic effects including: enhanced glucose uptake via GLUT4 translocation in skeletal muscle, increased fatty acid oxidation through phosphorylation and inactivation of acetyl-CoA carboxylase (ACC), stimulation of mitochondrial biogenesis via PGC-1α activation, and suppression of anabolic processes including lipogenesis and protein synthesis. These coordinated metabolic shifts favor catabolic pathways and energy production, contributing to improved metabolic homeostasis and insulin sensitivity.

3.2 Nuclear Translocation and Transcriptional Regulation

A paradigm-shifting discovery in MOTS-c biology revealed that the peptide translocates to the nucleus in response to metabolic stress, glucose restriction, or oxidative stress, where it directly regulates gene expression [Kim et al., Cell Metab 2018; PMID: 29983246]. This nuclear translocation is AMPK-dependent, representing a feed-forward mechanism whereby AMPK activation by MOTS-c facilitates its own nuclear import. The cationic character of MOTS-c (net charge +4 at physiological pH) likely facilitates interaction with nuclear import machinery.

Within the nucleus, MOTS-c binds to specific DNA regulatory regions, particularly antioxidant response elements (ARE), where it interacts with transcription factors including Nuclear Factor Erythroid 2-Related Factor 2 (NFE2L2/NRF2). This interaction modulates expression of genes involved in cellular stress defense, antioxidant responses, and metabolic adaptation. Genome-wide transcriptional profiling reveals that nuclear MOTS-c regulates hundreds of genes involved in oxidative stress response, glucose metabolism, mitochondrial function, and inflammatory pathways.

The nuclear regulatory function of MOTS-c represents a novel paradigm in mitochondrial-nuclear communication. Rather than solely functioning through classical retrograde signaling pathways, MOTS-c directly enters the nucleus to reprogram gene expression in response to metabolic challenges. This mechanism enables rapid adaptive responses to metabolic stress and may contribute to cellular resilience during aging and disease states.

3.3 Exercise Mimetic Effects and Physical Performance

MOTS-c functions as a bona fide exercise mimetic, recapitulating many metabolic and performance benefits of physical exercise at the molecular level. Exercise induces robust MOTS-c expression in skeletal muscle (11.9-fold increase) and elevates circulating MOTS-c levels (1.6-fold during exercise, 1.5-fold post-exercise), suggesting that MOTS-c mediates some beneficial effects of physical activity [Reynolds et al., Nat Commun 2021; PMID: 33206854]. Conversely, exogenous MOTS-c administration replicates exercise-like adaptations even in sedentary animals.

Studies in aged mice (22 months, equivalent to ~70 human years) demonstrate that two weeks of MOTS-c treatment doubles treadmill running capacity, an effect comparable to exercise training interventions. The peptide improves multiple determinants of physical performance including mitochondrial respiration, muscle glucose uptake, lactate clearance, and resistance to fatigue. Importantly, MOTS-c enhances physical capacity across the lifespan—in young (2 months), middle-aged (12 months), and old (22 months) mice—though the magnitude of effect is most pronounced in aged animals experiencing age-related functional decline.

The molecular mechanisms underlying MOTS-c's exercise mimetic effects involve activation of skeletal muscle AMPK, enhanced expression of PGC-1α (the master regulator of mitochondrial biogenesis), increased mitochondrial oxidative capacity, and favorable shifts in substrate utilization favoring fat oxidation. These adaptations closely mirror those induced by endurance exercise training, providing a molecular basis for MOTS-c's performance-enhancing effects. For researchers interested in related performance-enhancing peptides, Thymosin Beta-4 offers complementary mechanisms in tissue repair and muscle function.

3.4 Insulin Sensitization and Glucose Metabolism

MOTS-c demonstrates potent insulin-sensitizing effects through multiple complementary mechanisms. In skeletal muscle, the primary site of insulin-stimulated glucose disposal, MOTS-c enhances insulin signaling by promoting Akt phosphorylation and subsequent GLUT4 translocation to the plasma membrane. This increases insulin-stimulated glucose uptake by 25-40% in cultured myocytes and muscle tissue from MOTS-c-treated animals.

Hyperinsulinemic-euglycemic clamp studies, the gold standard for assessing insulin sensitivity in vivo, demonstrate that MOTS-c treatment increases the glucose infusion rate by approximately 30% in mice, indicating substantially improved whole-body insulin sensitivity. The peptide restores insulin sensitivity in aged mice to levels comparable to young animals, effectively reversing age-related insulin resistance. Similarly, in diet-induced obesity models, MOTS-c prevents development of insulin resistance despite continued high-fat feeding.

Beyond direct effects on insulin signaling, MOTS-c improves glucose metabolism through AMPK-mediated mechanisms including increased glycolytic flux, enhanced mitochondrial glucose oxidation, and improved metabolic flexibility (the capacity to switch between glucose and fat oxidation based on substrate availability). The peptide also influences hepatic glucose metabolism, reducing hepatic glucose production and improving liver insulin sensitivity, contributing to improved systemic glycemic control.

3.5 Anti-Inflammatory and Immunomodulatory Effects

MOTS-c exerts significant anti-inflammatory effects that contribute to its metabolic and age-protective benefits. Treatment with MOTS-c reduces circulating levels of pro-inflammatory cytokines including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1β (IL-1β) in models of metabolic dysfunction and aging. Conversely, the peptide increases anti-inflammatory mediators and improves the balance between pro- and anti-inflammatory signaling.

These anti-inflammatory effects likely result from multiple mechanisms including: AMPK-mediated suppression of NF-κB signaling (a master regulator of inflammatory gene expression), improved metabolic health reducing metabolic inflammation, enhanced mitochondrial function limiting production of inflammatory reactive oxygen species, and direct effects on immune cell function. Chronic low-grade inflammation ("inflammaging") is a key driver of age-related pathology, and MOTS-c's anti-inflammatory actions may contribute significantly to its age-protective effects.

4. Preclinical Research Evidence

4.1 Metabolic Regulation and Insulin Resistance

The foundational preclinical evidence for MOTS-c established its role as a metabolic regulator capable of preventing and reversing insulin resistance across multiple experimental paradigms. In the seminal 2015 study, mice treated with MOTS-c (0.5 mg/kg/day for 8 weeks) during high-fat diet feeding were completely protected from diet-induced insulin resistance, maintaining insulin sensitivity equivalent to chow-fed controls despite 60% fat intake [Lee et al., Cell Metab 2015; PMID: 25738459]. This protection occurred without reduction in food intake, indicating direct metabolic effects rather than appetite suppression.

Age-associated insulin resistance, a near-universal feature of mammalian aging, is similarly ameliorated by MOTS-c. Aged mice (18-22 months) exhibit severe insulin resistance compared to young animals (2-3 months), but administration of MOTS-c for just 7-14 days restores insulin sensitivity to youthful levels. This rapid reversal of age-related metabolic dysfunction suggests that MOTS-c addresses fundamental mechanisms of metabolic aging rather than requiring prolonged treatment for gradual improvements.

Mechanistically, MOTS-c prevents insulin resistance through multiple pathways: maintaining skeletal muscle insulin signaling capacity, preventing ectopic lipid accumulation in muscle and liver, preserving mitochondrial oxidative function, and reducing inflammatory signaling that interferes with insulin action. The peptide improves both peripheral (muscle, fat) and hepatic insulin sensitivity, contributing to whole-body metabolic homeostasis. Studies employing insulin receptor substrate (IRS) phosphorylation and Akt activation assays confirm enhancement of proximal insulin signaling events in MOTS-c-treated tissues.

4.2 Obesity Prevention and Weight Management

MOTS-c demonstrates significant effects on body composition and obesity prevention in preclinical models. During high-fat diet feeding, MOTS-c-treated mice gain substantially less weight than vehicle-treated controls, with reductions in fat mass accumulation of 30-50% despite equivalent caloric intake. This weight-sparing effect occurs through increased energy expenditure rather than reduced food consumption, as evidenced by indirect calorimetry studies showing elevated oxygen consumption and heat production in MOTS-c-treated animals.

The anti-obesity effects of MOTS-c involve enhanced thermogenesis in brown adipose tissue (BAT) and induction of "beiging" in white adipose tissue, processes that convert energy-storing white fat into energy-expending beige adipocytes. Molecular analysis reveals increased expression of uncoupling protein 1 (UCP1) and other thermogenic markers in adipose tissue from MOTS-c-treated animals. Additionally, MOTS-c enhances whole-body fatty acid oxidation, redirecting lipids toward mitochondrial β-oxidation rather than storage in adipose depots.

Table 3: Summary of Key Preclinical Efficacy Studies
Model/Condition	Intervention	Key Findings	Reference
High-fat diet (mice)	0.5 mg/kg/day, 8 weeks	Complete protection from diet-induced insulin resistance and obesity	Lee 2015 (PMID: 25738459)
Aged mice (22 mo.)	Systemic injection, 2 weeks	Doubled running capacity; restored metabolic function to youthful levels	Reynolds 2021 (PMID: 33206854)
Type 2 diabetes (rats)	MOTS-c + exercise	Synergistic effects on cardiac function; reduced myocardial injury	Multiple studies 2022-2023
Type 1 diabetes (NOD mice)	MOTS-c treatment	Delayed diabetes onset; improved β-cell function and reduced senescence	Recent 2024 studies
Exercise performance	Acute & chronic dosing	11.9-fold increase in muscle expression; enhanced endurance capacity	Reynolds 2021 (PMID: 33206854)
Muscle atrophy	Systemic administration	Prevented immobilization-induced atrophy; reduced lipid infiltration	Recent preclinical data
Gestational diabetes	MOTS-c therapy	Relieved hyperglycemia and insulin resistance in GDM models	Recent studies
Ovarian cancer	MOTS-c treatment	Suppressed progression via LARS1 deubiquitination pathway	2024 Advanced Science

Importantly, the anti-obesity effects of MOTS-c are achieved without activating sympathetic nervous system tone or increasing cardiovascular stress, distinguishing it from traditional thermogenic agents that can cause tachycardia and hypertension. The peptide's mechanism—enhancing metabolic efficiency and substrate oxidation—represents a more physiological approach to weight management.

4.3 Aging, Longevity, and Healthspan Extension

MOTS-c has emerged as a critical mediator of healthy aging, with circulating levels declining progressively with age in both animal models and humans. Plasma MOTS-c concentrations are 11% lower in middle-aged versus young individuals and 21% lower in older versus young populations, correlating with age-related metabolic decline [Reynolds et al., Nat Commun 2021; PMID: 33206854]. This age-dependent reduction suggests that MOTS-c deficiency contributes to metabolic aspects of aging.

Restoration of MOTS-c levels in aged animals produces remarkable anti-aging effects. Late-life intermittent MOTS-c treatment (initiated at 23.5 months, equivalent to ~75 human years) significantly extends healthspan—the period of life spent in good health. Treated aged mice demonstrate improved physical performance, enhanced metabolic function, better maintenance of muscle mass and strength, and improved resistance to age-related pathologies. While effects on maximum lifespan require longer-term studies, the healthspan extension is clear and substantial.

The mechanisms underlying MOTS-c's age-protective effects are multifactorial and include: restoration of metabolic efficiency that declines with age, enhancement of mitochondrial function and biogenesis (countering age-related mitochondrial dysfunction), activation of cellular stress response pathways that promote resilience, reduction of inflammatory signaling that drives age-related pathology, and improvement in stem cell function and regenerative capacity. These pleiotropic effects address multiple hallmarks of aging simultaneously.

4.4 Cardiovascular Protection

Emerging preclinical evidence demonstrates significant cardiovascular protective effects of MOTS-c. In models of diabetic cardiomyopathy, MOTS-c treatment improves cardiac systolic and diastolic function, reduces myocardial ultrastructural damage, and restores mitochondrial respiration in cardiomyocytes. Studies in type 2 diabetic rats show that MOTS-c, with or without concurrent exercise training, significantly improves cardiac output, reduces left ventricular dysfunction, and decreases markers of cardiac stress and damage.

The cardioprotective mechanisms involve direct effects on cardiomyocyte metabolism and survival. MOTS-c enhances cardiac mitochondrial function, improving ATP production efficiency and reducing production of damaging reactive oxygen species. The peptide activates survival signaling pathways including the NRG1-ErbB pathway, which promotes cardiomyocyte survival and contractile function. Additionally, MOTS-c improves cardiac glucose and fatty acid metabolism, addressing the metabolic substrate inflexibility characteristic of diabetic and failing hearts.

4.5 Pancreatic β-Cell Function and Diabetes Prevention

Recent studies have revealed important effects of MOTS-c on pancreatic β-cell function and diabetes prevention. In both type 1 diabetes models (NOD mice) and insulin resistance models (S961-treated mice), MOTS-c treatment prevents or delays diabetes onset. The peptide reduces pancreatic islet cell senescence, a key mechanism of β-cell failure in diabetes, by modulating nuclear gene expression and metabolic pathways involved in cellular aging.

MOTS-c improves glucose-stimulated insulin secretion from pancreatic islets, indicating enhanced β-cell functional capacity. The peptide also provides cytoprotective effects, reducing β-cell apoptosis in the face of metabolic stress, inflammatory cytokines, or autoimmune attack. These dual effects—improving function and enhancing survival of insulin-producing cells—position MOTS-c as a potential disease-modifying therapy for both type 1 and type 2 diabetes rather than merely a symptomatic treatment.

4.6 Neurological and Cognitive Effects

While less extensively studied than metabolic effects, emerging evidence suggests neuroprotective and cognitive benefits of MOTS-c. The peptide crosses the blood-brain barrier and exerts direct effects on brain metabolism and function. In models of neurodegenerative disease and cognitive aging, MOTS-c demonstrates protective effects against neuronal injury, reduces neuroinflammation, and may preserve cognitive function.

The neuroprotective mechanisms likely involve enhancement of neuronal mitochondrial function, activation of antioxidant defenses in brain tissue, modulation of neuroinflammatory responses, and improvement of cerebral metabolic efficiency. Given the critical role of metabolic dysfunction in neurodegenerative diseases including Alzheimer's disease (often termed "type 3 diabetes" due to brain insulin resistance), MOTS-c's metabolic regulatory effects may have particular relevance for brain health and cognitive aging.

4.7 Cancer Biology: A Complex Relationship

The relationship between MOTS-c and cancer biology is complex and context-dependent. Recent studies have identified both potential anti-cancer effects and mechanisms warranting careful consideration. In ovarian cancer models, MOTS-c suppresses tumor progression by interacting with leucyl-tRNA synthetase 1 (LARS1), promoting its ubiquitination and degradation, thereby limiting protein synthesis required for rapid cancer cell proliferation. MOTS-c attenuates the deubiquitinase USP7-mediated stabilization of LARS1, providing a novel mechanism for tumor suppression.

However, the peptide's growth-promoting and metabolic effects raise theoretical concerns about potential effects on tumor growth in other contexts. The metabolic reprogramming induced by MOTS-c—enhanced glucose and lipid metabolism—could potentially provide substrates for proliferating cancer cells. Additionally, the angiogenic and anti-inflammatory effects might, in some contexts, support tumor growth. These considerations highlight the need for careful evaluation of MOTS-c in different cancer types and stages before therapeutic application in oncology settings.

5. Clinical Studies and Human Research

5.1 Published Clinical Evidence

Clinical investigation of MOTS-c remains in early stages, with limited published data from human studies. The most advanced clinical development has focused on CB4211, a MOTS-c analog developed by CohBar Inc. for treatment of nonalcoholic steatohepatitis (NASH) and obesity. Phase 1 clinical trials (NCT03998514) evaluated safety and tolerability in healthy adults, with both single ascending dose (SAD) and multiple ascending dose (MAD) studies completed.

Results from the Phase 1 program demonstrated that CB4211 was safe and well-tolerated following 7 days of dosing in healthy volunteers [Frontiers Endocrinol 2023; PMID: 36761202]. No serious adverse events were reported, and the peptide exhibited acceptable pharmacokinetic properties. Based on these promising safety data, CohBar advanced CB4211 into Phase 1b/2a clinical trials for NASH and obesity, though detailed efficacy results from these studies have not yet been published in peer-reviewed literature.

5.2 Observational Studies in Human Populations

Several observational studies have examined endogenous MOTS-c levels in human populations, providing insights into the peptide's physiological roles. Cross-sectional studies confirm age-related decline in circulating MOTS-c, with plasma levels progressively decreasing from young adulthood through older age. The magnitude of decline correlates with markers of metabolic dysfunction, suggesting that MOTS-c deficiency contributes to age-related metabolic deterioration.

Studies of the m.1382A>C polymorphism (K14Q variant) in human populations provide natural genetic evidence for MOTS-c's physiological functions. Korean and Japanese cohort studies demonstrate associations between the variant allele and muscle fiber type distribution, athletic performance phenotypes, and age-related sarcopenia risk. Male carriers of the C allele (encoding K14Q MOTS-c) show enrichment among sprint/power athletes, higher fast-twitch muscle fiber percentages, and greater muscular strength in young adulthood, but differential sarcopenia risk in older age.

Exercise intervention studies in humans have confirmed that physical activity induces MOTS-c expression and release. Acute exercise increases circulating MOTS-c by 1.5-1.6-fold, with skeletal muscle MOTS-c expression increasing up to 11.9-fold following exercise training. This exercise-induced MOTS-c release suggests the peptide serves as an exercise-responsive myokine, mediating some beneficial effects of physical activity on systemic metabolism.

5.3 Current Clinical Development Status

As of 2024, MOTS-c and its analogs remain investigational compounds without regulatory approval from major health authorities including the FDA or EMA. The most advanced clinical program involves CB4211 for NASH and obesity, currently in Phase 2 development. Additional clinical trials are planned or ongoing for other MOTS-c applications, though public disclosure of these programs remains limited.

Table 4: Clinical Development Summary
Study Phase	Indication	Population	Key Outcomes
Phase 1 (NCT03998514)	Safety/tolerability	Healthy adults (n=~60)	Safe and well-tolerated; acceptable PK profile; no serious AEs
Phase 1b/2a	NASH and obesity	Patients with NASH/obesity	CB4211 well-tolerated; preliminary efficacy signals (full results pending)
Observational	Exercise response	Healthy volunteers	Exercise increases circulating MOTS-c 1.5-1.6-fold; muscle expression 11.9-fold
Genetic association	K14Q polymorphism	Korean/Japanese cohorts	Variant associates with muscle phenotype and athletic performance

The regulatory pathway for MOTS-c development faces several challenges. As a mitochondrial genome-encoded peptide that functions as a metabolic regulator, MOTS-c represents a novel therapeutic modality that may require specialized regulatory considerations. The peptide's pleiotropic effects across multiple organ systems, while promising for addressing complex metabolic diseases, complicate clinical trial design and endpoint selection. Additionally, the optimal patient populations, dosing regimens, and duration of treatment remain to be established through systematic clinical investigation.

5.4 Safety Profile in Human Studies

Available clinical safety data, while limited in scope, suggest favorable tolerability of MOTS-c and its analogs in humans. The Phase 1 studies of CB4211 reported no serious adverse events, dose-limiting toxicities, or clinically significant laboratory abnormalities at doses tested. Minor adverse events were generally mild and transient, primarily consisting of injection site reactions with subcutaneous administration.

Importantly, no evidence of metabolic derangements, cardiovascular effects, or other system toxicities emerged from the Phase 1 program despite MOTS-c's potent effects on cellular metabolism. This safety profile aligns with preclinical toxicology studies showing wide therapeutic margins and absence of target organ toxicity. However, the relatively short duration of human exposure in completed studies (7-14 days maximum) precludes conclusions about long-term safety, rare adverse events, or effects in vulnerable populations including elderly individuals with multiple comorbidities—the target population for many potential MOTS-c applications.

6. Analytical Methods and Quality Assessment

6.1 Identity and Purity Analysis

Comprehensive analytical characterization of MOTS-c requires integration of multiple orthogonal techniques to confirm identity, assess purity, and detect degradation products or manufacturing impurities. Reverse-phase high-performance liquid chromatography (RP-HPLC) serves as the primary method for purity determination, with detection at 220 nm (peptide bond absorbance) and 280 nm (aromatic amino acid absorbance from Trp and Tyr residues). Typical HPLC methods employ C18 columns (150-250 mm length, 4.6 mm diameter, 5 μm particle size) with linear acetonitrile gradients in 0.1% TFA.

Critical method development considerations include optimization of gradient slope to achieve baseline resolution between MOTS-c and closely related impurities including deletion sequences (missing one or more amino acids), oxidized variants (Met+16 Da species), and truncated products. Pharmaceutical-grade MOTS-c should demonstrate ≥98% main peak purity by HPLC, with individual impurities not exceeding 0.5% and total impurities below 2.0%.

Mass spectrometry provides definitive molecular weight confirmation and structural verification. Electrospray ionization mass spectrometry (ESI-MS) in positive ion mode typically produces multiply charged ion species ([M+2H]²⁺, [M+3H]³⁺, [M+4H]⁴⁺) that can be deconvoluted to yield the monoisotopic mass of 2174.62 Da. High-resolution mass spectrometry (HRMS) with mass accuracy <5 ppm enables discrimination between MOTS-c and isobaric impurities. Tandem mass spectrometry (MS/MS) with collision-induced dissociation provides complete sequence verification through systematic fragmentation.

Table 5: Analytical Characterization Methods for MOTS-c
Technique	Purpose	Key Specifications
RP-HPLC (UV detection)	Purity assessment	≥98% main peak at 220 nm; baseline resolution of impurities
ESI-MS or MALDI-TOF MS	Molecular weight confirmation	2174.62 ± 1.0 Da (monoisotopic mass)
MS/MS sequencing	Sequence verification	100% match to MRWQEMGYIFYPRKLR; all b- and y-ions detected
Amino acid analysis	Compositional analysis	All residues within ±10% of theoretical molar ratios
Peptide mapping (LC-MS)	Sequence and modification analysis	Tryptic digest analysis; detection of oxidation or modifications
Karl Fischer titration	Water content	≤8.0% (lyophilized powder)
Ion chromatography	Counter-ion quantification	TFA ≤0.1%; acetate as specified by formulation
UV-Vis spectroscopy	Concentration determination	Using ε₂₈₀ = 11,460 M^-1cm^-1
LAL endotoxin assay	Bacterial endotoxin testing	≤5 EU/mg (for injectable formulations)

6.2 Methionine Oxidation Assessment

The presence of two methionine residues (positions 1 and 6) in MOTS-c makes oxidation monitoring a critical quality control parameter. Methionine oxidation to methionine sulfoxide (+16 Da) or sulfone (+32 Da) significantly reduces biological activity and represents a major degradation pathway. Analytical methods must be capable of detecting and quantifying oxidized species at low levels (typically <2% specification for pharmaceutical-grade material).

RP-HPLC with MS detection provides optimal sensitivity and specificity for oxidation product analysis. Oxidized MOTS-c variants typically elute slightly earlier than the native peptide due to increased polarity. Mass spectrometric detection enables unambiguous identification: mono-oxidized species appear at m/z = 2190.62 Da (+16 Da), while di-oxidized species appear at 2206.62 Da (+32 Da). Quantification is achieved by comparing peak areas of oxidized and native forms, with corrections for potential differences in ionization efficiency.

Forced degradation studies under oxidative stress conditions (hydrogen peroxide treatment, metal ion catalysis, elevated temperature) establish oxidation kinetics and identify the more susceptible methionine residue. Such studies inform formulation development, guide storage condition selection, and establish stability-indicating analytical methods. Protective strategies including addition of antioxidants (methionine, ascorbic acid), oxygen-free packaging, and storage at low temperature minimize oxidation during manufacturing and storage.

6.3 Biological Activity Assays

Chemical and physical characterization methods, while essential, do not directly assess biological function. Biological activity assays provide functional verification that MOTS-c retains its metabolic regulatory properties. Cell-based assays measuring AMPK activation serve as primary potency assays. C2C12 myoblasts or primary skeletal muscle cells are treated with MOTS-c, and phosphorylation of AMPK (Thr172) and downstream substrates (ACC-Ser79) is quantified by Western blotting or ELISA.

Glucose uptake assays in skeletal muscle cells provide functional assessment of MOTS-c's metabolic effects. Cells are treated with MOTS-c, and glucose incorporation is measured using radiolabeled 2-deoxy-D-glucose or fluorescent glucose analogs. Active MOTS-c increases glucose uptake by 25-50% compared to vehicle controls, providing a quantifiable potency metric.

For comprehensive quality control, nuclear translocation assays assess MOTS-c's ability to enter the nucleus under stress conditions. Cells are treated with MOTS-c under metabolic stress (glucose restriction, oxidative stress), and nuclear localization is quantified by immunofluorescence microscopy or subcellular fractionation. These multi-level biological assays—from molecular signaling to cellular metabolism to subcellular localization—provide confidence that analytical-grade MOTS-c retains full biological functionality.

6.4 Quantification in Biological Samples

Measurement of endogenous MOTS-c in plasma, serum, or tissue samples requires sensitive and specific analytical methods. Enzyme-linked immunosorbent assays (ELISA) using anti-MOTS-c antibodies provide high-throughput quantification but may suffer from cross-reactivity or matrix effects. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers superior specificity and accuracy, representing the gold standard for MOTS-c bioanalysis.

LC-MS/MS methods for MOTS-c typically employ immunoaffinity enrichment or solid-phase extraction to concentrate the peptide from complex biological matrices, followed by reversed-phase liquid chromatography separation and MS/MS detection using multiple reaction monitoring (MRM). Stable isotope-labeled MOTS-c internal standard (heavy lysine or arginine incorporation) enables accurate quantification by correcting for matrix effects and recovery variations. Typical lower limits of quantification (LLOQ) are 0.5-5 ng/mL in plasma, sufficient for measuring physiological concentrations.

7. Research Applications and Experimental Uses

7.1 Metabolic Disease Modeling

MOTS-c serves as a valuable tool for investigating fundamental mechanisms of metabolic regulation and disease pathogenesis. Researchers employ MOTS-c in cellular and animal models to dissect the folate-AMPK axis and its role in insulin sensitivity, obesity, and type 2 diabetes. Loss-of-function studies using MOTS-c antagonists or genetic deletion of the MOTS-c coding sequence reveal the physiological importance of endogenous MOTS-c in metabolic homeostasis.

Comparative studies examining wild-type versus K14Q variant MOTS-c provide insights into structure-activity relationships and the molecular determinants of metabolic regulation. The naturally occurring human polymorphism offers a unique opportunity to understand how single amino acid changes affect peptide function, receptor interactions, and physiological outcomes. Such studies inform rational design of MOTS-c analogs with enhanced potency, stability, or tissue selectivity.

7.2 Aging and Longevity Research

MOTS-c has become an important tool in aging biology research, enabling investigation of mitochondrial-nuclear communication in age-related decline. Researchers use MOTS-c to test whether restoring youthful levels of this mitochondrial-derived peptide can reverse or slow aging processes. Studies combining MOTS-c with other longevity interventions (caloric restriction, rapamycin, senolytics) explore potential synergistic effects and common versus distinct mechanisms of lifespan extension.

The peptide's age-dependent decline and restoration of function upon replacement provide support for the "mitochondrial decline" theory of aging. Research examining tissue-specific MOTS-c expression patterns during aging reveals which organs experience the greatest loss of MOTS-c signaling and may benefit most from therapeutic restoration. Longitudinal studies in long-lived versus short-lived animal strains investigate whether natural variation in MOTS-c levels contributes to lifespan differences.

7.3 Exercise Physiology and Performance Research

MOTS-c's exercise mimetic properties make it a valuable research tool for understanding molecular mechanisms of exercise adaptation. Studies examining how exercise induces MOTS-c expression and release elucidate signaling pathways linking muscle contraction to systemic metabolic benefits. Comparative studies in trained versus untrained individuals investigate whether exercise training modulates MOTS-c responsiveness or baseline expression.

Athletic performance research explores MOTS-c's potential as an ergogenic aid, though ethical considerations regarding sports doping apply. Studies in animal models demonstrate that MOTS-c enhances endurance capacity, delays fatigue, and accelerates recovery from exercise. Mechanistic investigations reveal which aspects of exercise adaptation—mitochondrial biogenesis, angiogenesis, substrate utilization, or others—are most strongly influenced by MOTS-c signaling. For researchers interested in related performance peptides, Ipamorelin and Sermorelin offer complementary mechanisms through growth hormone pathways.

7.4 Drug Development and Formulation Studies

MOTS-c serves as a lead compound for developing novel metabolic therapeutics. Structure-activity relationship (SAR) studies systematically modify the MOTS-c sequence to identify residues critical for AMPK activation, nuclear translocation, or metabolic effects. These studies have revealed that the N-terminal region (residues 1-11) is essential for activity, while the C-terminal region tolerates some modifications without complete loss of function.

Formulation research explores delivery strategies to optimize MOTS-c pharmacokinetics and biodistribution. Approaches under investigation include PEGylation to extend circulation half-life, encapsulation in nanoparticles for sustained release, chemical modifications to enhance oral bioavailability, and fusion to cell-penetrating peptides or targeting moieties for tissue-specific delivery. The native peptide's reasonable stability and membrane permeability provide advantages over many other peptide therapeutics, but optimization can enhance clinical utility.

7.5 Mitochondrial Biology and Retrograde Signaling

Beyond specific therapeutic applications, MOTS-c research has fundamentally advanced understanding of mitochondrial biology and mitochondrial-nuclear communication. The discovery that mitochondria encode bioactive peptides beyond the canonical 13 proteins opened a new field investigating the mitochondrial "cryptome"—hidden coding sequences in supposedly non-coding regions of mtDNA. MOTS-c research established that these mitochondrial-derived peptides (MDPs) function as systemic hormones, expanding the biological role of mitochondria from cellular power plants to endocrine organs.

Studies of MOTS-c nuclear translocation and gene regulation have revealed novel mechanisms of retrograde signaling—communication from mitochondria to nucleus that coordinates cellular responses to metabolic stress. This bidirectional communication enables cells to adapt nuclear gene expression in response to mitochondrial status, maintaining cellular homeostasis. Understanding these mechanisms has implications beyond MOTS-c itself, informing research on other MDPs and potentially identifying new therapeutic targets for metabolic and age-related diseases.

8. Dosing Protocols in Research Settings

8.1 Preclinical Dosing Paradigms

Extensive preclinical research has established effective dose ranges and administration protocols for MOTS-c across diverse experimental models. In rodent studies, effective doses typically range from 0.5 mg/kg to 15 mg/kg body weight, with most metabolic and performance studies employing doses of 0.5-5 mg/kg. The broad dose range reflects different experimental objectives, administration routes, and outcome measures, but consistently demonstrates that MOTS-c produces biological effects across this spectrum.

The seminal metabolic studies employed 0.5 mg/kg/day administered intraperitoneally for 8 weeks during high-fat diet feeding, completely preventing diet-induced insulin resistance and obesity. Acute studies examining immediate metabolic effects have used higher doses (5-10 mg/kg) administered as single injections, producing rapid AMPK activation and metabolic shifts within 30-60 minutes. Exercise mimetic studies demonstrating enhanced physical performance typically employ 0.5-5 mg/kg administered systemically for 1-4 weeks.

Table 6: Representative Preclinical Dosing Protocols
Application	Dose Range	Route	Frequency & Duration
Insulin resistance prevention	0.5-5 mg/kg	IP, SC	Once daily for 4-8 weeks
Obesity prevention (HFD)	0.5-5 mg/kg	IP, SC	Once daily during HFD (8-16 weeks)
Physical performance enhancement	0.5-5 mg/kg	SC, IP	Once daily for 1-4 weeks
Age-related decline reversal	5-15 mg/kg	SC, IP	3x/week for 2-8 weeks
Acute metabolic studies	5-10 mg/kg	IP, IV	Single dose; assess 0.5-24h post-injection
Diabetes models (T1D, T2D)	1-10 mg/kg	IP, SC	Once daily or 3x/week for 2-8 weeks
Cardiac protection (diabetic)	5 mg/kg	IP	Once daily for 4-8 weeks
Muscle atrophy prevention	5 mg/kg	SC	Once daily during immobilization

Dose-response studies reveal a generally monotonic relationship between MOTS-c dose and metabolic effects up to approximately 5 mg/kg, with plateau effects observed at higher doses. Unlike some peptides showing bell-shaped dose-response curves, MOTS-c demonstrates consistent efficacy increases with dose escalation within the tested range, without evidence of reduced efficacy at high doses. This favorable dose-response profile suggests a wide therapeutic window, though determination of optimal human doses requires clinical trials.

8.2 Routes of Administration and Pharmacokinetics

MOTS-c has been successfully administered via multiple routes in research settings, each offering distinct pharmacokinetic profiles and practical considerations. Intraperitoneal (IP) injection, the most common route in rodent studies, provides reliable systemic delivery and is practical for repeated dosing. Subcutaneous (SC) injection offers an alternative parenteral route more relevant to potential clinical applications, with slower absorption kinetics compared to IP administration but sustained plasma levels.

Intravenous (IV) administration provides immediate and complete systemic bioavailability, useful for acute pharmacodynamic studies examining immediate metabolic responses. However, most therapeutic applications employ SC or IP routes due to practicality and sustained exposure. Pharmacokinetic studies of subcutaneous MOTS-c reveal peak plasma concentrations at 1-2 hours post-injection, with elimination half-lives of 2-4 hours in rodents. The relatively short half-life necessitates daily dosing for sustained effects, though recent formulation approaches aim to extend duration of action.

An important consideration is that endogenous MOTS-c is released from mitochondria and acts both locally (autocrine/paracrine) and systemically (endocrine). Exogenous administration via systemic injection may not perfectly replicate the tissue-specific patterns of endogenous MOTS-c expression, potentially contributing to differences between physiological MOTS-c signaling and pharmacological administration. Understanding these differences informs interpretation of preclinical data and design of clinical dosing regimens.

8.3 Timing Considerations and Treatment Duration

Research protocols vary substantially in treatment duration depending on experimental objectives. Acute studies examining immediate signaling events (AMPK phosphorylation, glucose uptake) employ single-dose administration with assessments at 0.5-4 hours post-injection. Short-term studies (1-2 weeks) examine early metabolic adaptations and performance effects, while longer-term protocols (4-16 weeks) assess sustained metabolic improvements and prevention of diet-induced pathology.

Timing of MOTS-c administration relative to other interventions (feeding, exercise, metabolic stress) influences outcomes. Some studies administer MOTS-c immediately before exercise or feeding to capture acute metabolic interactions, while others employ regular dosing schedules independent of behavioral rhythms. Studies in aged animals often employ late-life intervention paradigms, beginning treatment at 18-24 months of age to model therapeutic intervention in older humans, demonstrating efficacy even when initiated after age-related decline is established.

Intermittent dosing regimens (e.g., 3 times per week) have shown efficacy in some aging studies, potentially offering practical advantages over daily dosing while maintaining therapeutic benefits. The intermittent approach may also mimic more physiological patterns, where MOTS-c levels fluctuate in response to metabolic demands and exercise rather than remaining constantly elevated. Optimization of dosing frequency and timing represents an important consideration for clinical translation.

8.4 Combination Therapies and Synergistic Effects

Research increasingly explores MOTS-c in combination with other interventions to identify synergistic effects and optimal therapeutic strategies. Studies combining MOTS-c with exercise training demonstrate additive or synergistic improvements in metabolic parameters and physical performance beyond either intervention alone. The molecular mechanisms involve MOTS-c enhancing exercise-induced AMPK activation, PGC-1α expression, and mitochondrial biogenesis, creating a feed-forward enhancement of exercise adaptations.

Combination studies with metformin, the first-line diabetes medication that also activates AMPK, reveal complementary mechanisms. MOTS-c activates AMPK via the folate-AICAR pathway, while metformin inhibits complex I of the electron transport chain, increasing AMP:ATP ratios. The dual mechanisms produce more robust and sustained AMPK activation compared to either agent alone. Similar synergistic approaches combine MOTS-c with other metabolic modulators, caloric restriction, or senolytics in aging research.

9. Storage and Handling Protocols

9.1 Storage Conditions for Lyophilized Product

Proper storage of MOTS-c is critical for maintaining peptide stability, biological activity, and preventing degradation, particularly oxidation of the two methionine residues. Lyophilized MOTS-c should be stored at -20°C to -80°C for optimal long-term stability, with storage at -80°C recommended for extended periods (>12 months). Under these conditions, properly manufactured MOTS-c maintains >95% purity and full biological activity for 24-36 months.

The lyophilized product must be protected from moisture, as residual water accelerates degradation even in the solid state. Vials should be sealed with appropriate butyl rubber stoppers and aluminum crimp caps to prevent moisture ingress during storage. Storage in desiccated conditions (with desiccant packets in secondary packaging) provides additional protection. Once opened, unused lyophilized material should be re-stored immediately at -20°C or colder with minimal exposure to ambient humidity.

Short-term storage at 2-8°C (refrigerated) is acceptable for unopened vials for periods up to 3-6 months, though freezer storage (-20°C or below) remains preferred for maximum stability. Storage at room temperature should be avoided except during necessary handling periods, as elevated temperatures accelerate oxidative and hydrolytic degradation pathways. Protection from light is also recommended, as photocatalyzed oxidation can affect the tryptophan and tyrosine residues.

Table 7: MOTS-c Storage and Handling Guidelines
Form	Storage Condition	Stability Duration	Special Considerations
Lyophilized powder (unopened)	-80°C (ultra-low freezer)	24-36 months	Optimal long-term storage; protect from moisture
Lyophilized powder (unopened)	-20°C (freezer)	12-24 months	Acceptable long-term storage; desiccated conditions
Lyophilized powder (unopened)	2-8°C (refrigerator)	3-6 months	Short-term storage only; protect from moisture
Reconstituted (sterile water)	2-8°C (refrigerator)	7-14 days	Use within recommended timeframe; protect from light
Reconstituted (bacteriostatic water)	2-8°C (refrigerator)	14-30 days	Extended stability with preservative; multi-use vials
Reconstituted solution aliquots	-20°C (freezer)	1-3 months	Single freeze-thaw only; thaw at 2-8°C before use
Working solutions	2-8°C (refrigerator)	Use within 24-48 hours	Prepare fresh when possible; minimize light exposure

9.2 Reconstitution Procedures

Reconstitution of lyophilized MOTS-c requires careful technique to ensure complete dissolution while minimizing degradation and contamination risks. Sterile water for injection (WFI) or bacteriostatic water for injection (containing 0.9% benzyl alcohol preservative) are the recommended reconstitution vehicles. Bacteriostatic water extends the usable life of reconstituted solutions and is preferred for multi-dose vials, though sterile water is appropriate for single-use applications.

The reconstitution process should be performed under aseptic conditions using sterile technique. Allow the lyophilized vial to reach room temperature (20-25°C) before adding reconstitution vehicle to prevent condensation and ensure uniform dissolution. Add the appropriate volume of reconstitution vehicle slowly to the vial, directing the stream against the vial wall rather than directly onto the lyophilized cake to minimize foaming and shear stress on the peptide.

After adding the solvent, gently swirl or roll the vial to dissolve the peptide—avoid vigorous shaking or vortexing, which can cause aggregation, air bubble formation, and potential degradation. The solution should become clear and colorless to slightly opalescent within 1-2 minutes. If particulates or cloudiness persist, do not use the solution. Typical reconstitution concentrations range from 0.5-5 mg/mL depending on dosing requirements, with 1-2 mg/mL being common for research applications.

9.3 Handling Precautions and Best Practices

Standard laboratory safety practices for handling research chemicals and biological materials should be followed when working with MOTS-c. Although the peptide has demonstrated low toxicity in preclinical studies, appropriate personal protective equipment (PPE) including gloves, lab coat, and eye protection should be worn. Work should be conducted in appropriate laboratory environments (chemical fume hood or biological safety cabinet) following institutional safety protocols.

Minimize exposure of reconstituted MOTS-c to room temperature, light, and atmospheric oxygen. Prepared solutions should be stored refrigerated (2-8°C) when not in active use and protected from light using amber vials or aluminum foil wrapping. The methionine residues are particularly susceptible to oxidation; antioxidants such as dithiothreitol (DTT) or reduced glutathione may be added to working solutions for enhanced stability, though compatibility with downstream applications must be verified.

Avoid repeated freeze-thaw cycles of reconstituted solutions, as this causes cumulative degradation and potential aggregation. If multiple aliquots are needed, divide the reconstituted solution immediately into single-use portions and store frozen (-20°C). Each aliquot should be thawed only once, preferably slowly at 2-8°C rather than room temperature or water bath, to minimize temperature stress. Thawed aliquots should be used within 24-48 hours and not refrozen.

9.4 Stability Monitoring and Quality Maintenance

Recent stability studies have challenged earlier assumptions about MOTS-c degradation rates. Research examining MOTS-c solutions stored at 4°C and 37°C for 30 days found that methionine oxidation remained relatively constant, indicating greater stability than previously assumed. Solutions stored at refrigerated temperatures (2-8°C) demonstrated minimal degradation over 14-30 days, with purity decreasing by less than 0.5% over two weeks.

For critical applications requiring assured potency, periodic stability monitoring is recommended. HPLC analysis of stored solutions can detect degradation products, oxidation species, or aggregates. Solutions showing >2% degradation or development of visible particulates should be discarded. Maintaining detailed records of storage conditions, reconstitution dates, and stability assessments ensures optimal quality and reproducibility in research applications.

10. Safety Profile and Toxicology

10.1 Preclinical Safety Studies

Extensive preclinical safety evaluation of MOTS-c across multiple species and administration routes has established a favorable safety profile with wide therapeutic margins. Acute toxicity studies in rodents have failed to identify a maximum tolerated dose (MTD) or lethal dose, with animals tolerating doses up to 15 mg/kg (30-fold higher than typical effective doses) without mortality or severe adverse effects. This exceptional safety margin substantially exceeds that of most peptide therapeutics and small molecule metabolic drugs.

Chronic toxicity studies involving daily administration for 8-16 weeks at doses up to 5 mg/kg demonstrate no treatment-related adverse effects on survival, body weight (beyond intended anti-obesity effects), food consumption, or general health status. Comprehensive evaluations including clinical chemistry, hematology, urinalysis, and histopathology reveal no abnormalities attributable to MOTS-c treatment. Organ weights and tissue histology of liver, kidney, heart, brain, endocrine organs, and reproductive tissues remain within normal ranges, indicating absence of target organ toxicity.

Importantly, despite MOTS-c's profound metabolic effects—enhancing glucose uptake, fatty acid oxidation, and mitochondrial function—no evidence of metabolic derangements, hypoglycemia, or energy depletion emerges from safety studies. The peptide appears to optimize rather than disrupt metabolic homeostasis, enhancing efficiency without pushing systems beyond physiological limits. This safety profile reflects MOTS-c's role as an endogenous metabolic regulator that evolved to maintain rather than disrupt cellular and systemic function.

10.2 Cardiovascular and Systemic Safety

Cardiovascular safety assessment is particularly important for metabolic therapeutics, as many face safety concerns related to cardiac effects. MOTS-c demonstrates favorable cardiovascular safety profiles in preclinical studies, with no evidence of arrhythmias, blood pressure changes, or cardiac structural abnormalities. Electrocardiographic monitoring in treated animals reveals normal heart rate, QT intervals, and cardiac rhythm. In fact, MOTS-c shows cardioprotective rather than cardiotoxic effects, improving cardiac function in models of diabetic cardiomyopathy.

Comprehensive physiological assessments including cardiovascular function, respiratory parameters, body temperature, and locomotor activity reveal no adverse effects at therapeutic doses. The peptide does not activate sympathetic tone or induce stress responses that often accompany metabolic stimulation by other agents. Blood pressure remains stable, and no signs of cardiovascular stress or decompensation occur even with chronic administration in aged or metabolically compromised animals.

10.3 Genotoxicity and Carcinogenicity Assessment

Standard genotoxicity testing including bacterial reverse mutation assays (Ames test) and mammalian cell mutation assays have not revealed mutagenic potential for MOTS-c. The peptide's mechanism of action—metabolic regulation via AMPK activation—does not involve DNA intercalation, covalent DNA modification, or other genotoxic mechanisms. As an endogenous peptide encoded by the mitochondrial genome and present in all mammalian cells, MOTS-c would not be expected to demonstrate genotoxic properties.

Long-term carcinogenicity studies specifically designed to regulatory standards have not been published, representing a gap in the safety database that would need to be addressed for regulatory approval. However, lifespan studies in rodents treated with MOTS-c for extended periods (up to 12-16 months) have not revealed increased tumor incidence or accelerated tumor growth. The peptide's effects on cellular proliferation are context-dependent, promoting regeneration in injured tissues but not driving uncontrolled growth.

Theoretical concerns about MOTS-c's proliferative and angiogenic effects in cancer contexts require careful consideration. While recent studies demonstrate anti-cancer effects in ovarian cancer models via LARS1 degradation, the complex relationship between MOTS-c and cancer biology warrants caution in oncology settings until more comprehensive data are available. Current evidence does not support carcinogenic risk, but systematic evaluation in diverse cancer models would strengthen the safety profile.

10.4 Reproductive and Developmental Toxicity

Limited data are available regarding reproductive and developmental toxicity of MOTS-c, representing an important gap in the safety profile. Preliminary observations in pregnant animals have not revealed obvious embryotoxic or teratogenic effects, and studies of gestational diabetes models suggest beneficial rather than harmful effects on pregnancy outcomes. However, comprehensive reproductive toxicology studies following regulatory guidelines (fertility assessment, embryo-fetal development studies, pre- and postnatal development studies) have not been published.

The physiological role of MOTS-c in reproduction and development remains incompletely characterized. As an endogenous peptide with age-dependent expression patterns, MOTS-c likely plays normal roles in developmental metabolism and reproductive function. Nevertheless, until systematic developmental toxicity studies are completed, caution is warranted regarding MOTS-c administration during pregnancy, lactation, or in pediatric populations. The precautionary principle dictates avoiding use in these vulnerable populations absent compelling data supporting safety.

10.5 Immunogenicity and Hypersensitivity

As a small peptide with high sequence conservation across mammalian species, MOTS-c would be expected to demonstrate low immunogenic potential. Repeated administration studies in rodents have not revealed anti-MOTS-c antibody formation, hypersensitivity reactions, or evidence of immune-mediated adverse effects. The peptide's endogenous nature and presence as a self-antigen in all individuals should limit adaptive immune responses, though individual variability in immune tolerance cannot be excluded.

Clinical experience with peptide therapeutics indicates that immunogenicity risk increases with peptide size, presence of non-native sequences or modifications, and frequency/duration of administration. MOTS-c's small size (16 amino acids), native sequence, and lack of unusual modifications should minimize immunogenicity risk. However, clinical trials will need to monitor for anti-drug antibodies, especially with chronic administration, as even endogenous peptides can occasionally elicit immune responses when administered exogenously at supraphysiological doses.

10.6 Clinical Safety Considerations

Limited clinical safety data from Phase 1 studies of CB4211 (MOTS-c analog) support the favorable preclinical safety profile. Healthy volunteers tolerated the peptide well, with no serious adverse events, dose-limiting toxicities, or significant laboratory abnormalities. Minor adverse events were predominantly injection site reactions, as expected with subcutaneous peptide administration. No evidence of metabolic derangements, organ toxicity, or systemic adverse effects emerged from the Phase 1 program.

Important clinical considerations include the current investigational status of MOTS-c, meaning it lacks the regulatory oversight, manufacturing controls, and post-market surveillance applied to approved pharmaceuticals. Products marketed as research chemicals or obtained through non-regulated sources may vary significantly in quality, purity, and actual MOTS-c content. Healthcare providers should counsel patients that self-administration of unapproved MOTS-c products carries risks related to product quality uncertainty and lack of clinical safety data in target populations.

Safety Summary

No acute toxicity observed at doses >30-fold higher than effective doses in rodents
No target organ toxicity in chronic administration studies (8-16 weeks)
No cardiovascular adverse effects; cardioprotective in disease models
No genotoxicity in standard assays; no increased tumor incidence in long-term studies
Low predicted immunogenicity as endogenous, conserved mammalian peptide
Phase 1 clinical data support safety and tolerability in healthy adults
Gaps remain: comprehensive reproductive toxicity, long-term human safety, rare adverse events

10.7 Contraindications and Special Populations

While definitive contraindications cannot be established without extensive clinical data, several theoretical considerations warrant caution. Patients with active malignancies should exercise caution given MOTS-c's effects on cellular metabolism and proliferation, despite anti-cancer effects observed in specific models. Until comprehensive oncology safety data are available, conservative approaches are warranted in cancer patients or those with cancer history.

Individuals with severe renal or hepatic impairment may experience altered MOTS-c pharmacokinetics, though the peptide's primary metabolic effects on skeletal muscle suggest these conditions may not substantially impact disposition. Nevertheless, dose adjustments and enhanced monitoring may be prudent in organ dysfunction until pharmacokinetic data in these populations are available. Elderly individuals with multiple comorbidities—a key target population for MOTS-c's age-protective effects—require particular attention to safety given potential for drug-disease and drug-drug interactions.

Pediatric use should be avoided absent specific pediatric safety studies, as children's developing metabolic systems may respond differently to metabolic modulators. Similarly, pregnancy and lactation represent contraindications until comprehensive reproductive safety data are established. Healthcare providers should thoroughly assess risk-benefit ratios when considering MOTS-c for any investigational use, recognizing the limited human safety database and investigational regulatory status. For related peptide therapies with established safety profiles, consider alternatives such as Epithalon or Thymosin Alpha-1.

11. Literature Review and Research Trends

11.1 Historical Development and Discovery

The discovery of MOTS-c in 2015 by Lee and colleagues at the University of Southern California represented a paradigm shift in mitochondrial biology and opened an entirely new field of investigation. Prior to this discovery, the mitochondrial genome was thought to encode only 37 genes: 13 proteins involved in oxidative phosphorylation, 22 transfer RNAs, and 2 ribosomal RNAs. The identification of bioactive peptides encoded within regions previously classified as "non-coding" fundamentally changed understanding of mitochondrial genome function and information content [Lee et al., Cell Metab 2015; PMID: 25738459].

The initial characterization established MOTS-c as a metabolic regulator that prevents insulin resistance and obesity through AMPK activation. This foundational work demonstrated that a mitochondrial-encoded peptide could function as a systemic metabolic regulator, challenging the traditional view of mitochondria as merely cellular power plants. The discovery catalyzed searches for additional mitochondrial-derived peptides (MDPs), leading to identification of SHLP1-6 (small humanin-like peptides) and expansion of the mitochondrial "cryptome"—hidden coding sequences throughout the mitochondrial genome.

11.2 Evolution of Mechanistic Understanding

Following the initial discovery, mechanistic investigations progressively revealed the complexity and sophistication of MOTS-c signaling. The 2018 landmark study by Kim and colleagues demonstrated nuclear translocation of MOTS-c in response to metabolic stress, establishing the peptide as a direct regulator of nuclear gene expression [Kim et al., Cell Metab 2018; PMID: 29983246]. This discovery revealed that MOTS-c functions both in cytoplasm (via AMPK activation) and nucleus (via transcriptional regulation), representing a multi-level regulatory system responsive to cellular metabolic state.

Subsequent research elucidated the folate-AICAR-AMPK pathway as MOTS-c's primary mechanism, demonstrating inhibition of MTHFD and resulting AICAR accumulation. Studies of the K14Q polymorphism provided natural genetic evidence for MOTS-c's physiological functions and revealed structure-activity relationships. The identification of MOTS-c as an exercise-induced myokine established connections between physical activity and mitochondrial signaling, explaining some molecular mechanisms underlying exercise's metabolic benefits.

11.3 Current Research Landscape and Trends

Contemporary MOTS-c research encompasses diverse themes spanning basic biology, translational medicine, and clinical development. Mechanistic studies continue probing receptor identification, signaling pathway components, and tissue-specific effects. The precise membrane receptor(s) mediating MOTS-c cellular uptake and initial signaling events remain incompletely defined, representing an important research priority. Advanced proteomics and receptor binding studies aim to identify these molecular targets.

Aging and longevity research represents a major focus, with studies examining MOTS-c's role in healthspan extension, age-related metabolic decline, and potential lifespan effects. Research in exceptionally long-lived populations investigates whether natural variation in MOTS-c levels or polymorphisms contributes to exceptional longevity. Interventional studies in aged animals explore optimal dosing paradigms for age-protective effects and identify which aspects of aging are most amenable to MOTS-c therapy.

Clinical translation efforts are advancing, with ongoing Phase 2 studies of CB4211 for NASH and obesity. Parallel efforts explore MOTS-c applications in diabetes, cardiovascular disease, sarcopenia, and neurodegenerative disorders. Formulation development focuses on extended-release preparations, oral delivery systems, and tissue-targeted approaches. The convergence of strong preclinical efficacy, favorable safety profiles, and unmet medical needs in metabolic diseases drives continued clinical investment.

11.4 Emerging Applications and Novel Findings

Recent research has expanded MOTS-c applications beyond traditional metabolic diseases. Cancer biology studies reveal context-dependent effects, with anti-tumor properties in ovarian cancer via LARS1 degradation but potential concerns in other malignancies. Membrane repair functions have been discovered, with MOTS-c enhancing TRIM72 trafficking to damaged membranes and improving membrane integrity. Pancreatic β-cell senescence prevention represents a novel mechanism for diabetes prevention, targeting cellular aging processes underlying disease pathogenesis.

Cardiovascular applications have expanded beyond metabolic effects to include direct cardioprotective mechanisms. Studies demonstrate MOTS-c restoration of mitochondrial respiration in diabetic hearts, activation of survival signaling (NRG1-ErbB), and improvement of cardiac mechanical efficiency. Synergistic effects with exercise training suggest combination approaches may optimize therapeutic benefits, relevant for both metabolic disease and performance applications.

11.5 Key Research Groups and Institutions

The University of Southern California, where MOTS-c was discovered, remains a leading center for MDP research. The Cohen laboratory has systematically characterized MOTS-c and related mitochondrial-derived peptides, establishing fundamental mechanisms and therapeutic potential. Collaborative networks have expanded globally, with significant contributions from Korean institutions examining the K14Q polymorphism and athletic phenotypes, Chinese research groups investigating disease applications, and European centers exploring aging and longevity aspects.

Pharmaceutical development has been led primarily by CohBar Inc., which licensed MOTS-c technology from USC and advanced CB4211 through clinical development. Academic-industry partnerships are accelerating translation, combining academic mechanistic expertise with industrial drug development capabilities. International expansion of the research community brings diverse perspectives and experimental approaches, enriching understanding and identifying novel applications.

11.6 Future Directions and Research Priorities

Several critical research priorities will shape MOTS-c's future trajectory. Definitive receptor identification remains the highest priority from a basic science perspective, enabling structure-based drug design and development of more potent or selective analogs. Understanding how MOTS-c crosses cellular membranes and nuclear import mechanisms will inform delivery strategies and formulation development. Tissue-specific effects and differential responsiveness across organs require systematic investigation to optimize therapeutic applications.

Clinical development priorities include well-designed randomized controlled trials in specific indications with clear endpoints. Metabolic diseases—type 2 diabetes, obesity, NASH—represent logical initial targets given strong preclinical evidence. Age-related applications including sarcopenia, frailty, and healthspan extension offer enormous potential but face challenges in trial design and endpoint selection. Combination therapy studies with metformin, exercise, or other metabolic modulators may identify synergistic strategies superior to monotherapy.

Long-term safety evaluation in humans is essential, particularly regarding cancer risk, cardiovascular safety, and effects in vulnerable populations. Pharmacogenetic studies examining how the K14Q polymorphism affects therapeutic responses will enable precision medicine approaches. Understanding sex differences in MOTS-c effects, suggested by polymorphism association studies, may reveal opportunities for sex-specific therapeutic strategies. Finally, investigation of other MDPs and their interactions with MOTS-c may uncover coordinated mitochondrial signaling networks amenable to therapeutic modulation.

Research Milestones

2015: Discovery of MOTS-c as mitochondrial-encoded metabolic regulator (Lee et al., Cell Metab)
2015: Identification of K14Q polymorphism and longevity associations (Fuku et al., Aging Cell)
2018: Discovery of nuclear translocation and transcriptional regulation (Kim et al., Cell Metab)
2021: Identification as exercise-induced myokine and exercise mimetic (Reynolds et al., Nat Commun)
2022-2023: Clinical trials of CB4211 for NASH; cardiovascular protective mechanisms elucidated
2024: β-cell senescence prevention; cancer applications; membrane repair functions discovered

Conclusion

MOTS-c represents a transformative discovery in mitochondrial biology and metabolic medicine—a mitochondrial genome-encoded peptide that functions as a systemic metabolic regulator, exercise mimetic, and mediator of healthy aging. Since its identification in 2015, an extensive body of preclinical research has established MOTS-c's remarkable capacity to enhance insulin sensitivity, prevent obesity, improve physical performance, and counteract age-related metabolic decline. The peptide's mechanisms involve both cytoplasmic AMPK activation through the folate-AICAR pathway and nuclear translocation to directly regulate gene expression, creating a sophisticated multi-level regulatory system responsive to metabolic stress.

Preclinical evidence across diverse animal models demonstrates consistent efficacy in preventing and reversing insulin resistance, obesity, type 2 diabetes, diabetic complications, sarcopenia, and age-related physical decline. The discovery that exercise induces MOTS-c expression and that exogenous MOTS-c replicates exercise-like adaptations establishes the peptide as a bona fide exercise mimetic with potential for individuals unable to engage in physical activity. Age-dependent decline in circulating MOTS-c levels, restoration of which produces anti-aging effects, positions this peptide as a potential therapeutic for extending healthspan and promoting healthy aging.

Safety evaluation has been favorable, with no acute toxicity at high doses, no target organ toxicity in chronic studies, and acceptable tolerability in Phase 1 clinical trials of the CB4211 analog. The peptide's endogenous nature, evolutionary conservation, and physiological role in metabolic homeostasis underlie this favorable safety profile. However, comprehensive long-term safety data in humans, particularly regarding cancer risk, cardiovascular safety in diseased populations, and effects in vulnerable groups, remain to be established through continued clinical investigation.

Clinical development is advancing, with CB4211 currently in Phase 2 trials for NASH and obesity. The convergence of compelling preclinical efficacy, favorable early-stage clinical data, and substantial unmet medical needs in metabolic diseases supports continued investment in clinical translation. However, significant challenges remain, including definitive receptor identification, optimization of formulation and delivery, establishment of optimal dosing regimens, and completion of large-scale efficacy trials with long-term follow-up.

Future research priorities include elucidating precise molecular targets, conducting rigorous clinical trials in metabolic diseases and aging-related indications, evaluating combination therapies, and understanding the broader network of mitochondrial-derived peptides and their coordinated functions. The remarkable discovery that mitochondria encode bioactive peptides beyond the canonical 13 proteins has opened new frontiers in mitochondrial biology, metabolic regulation, and therapeutic development. MOTS-c stands at the forefront of this new field, with potential to become a first-in-class mitochondrial-derived peptide therapeutic for metabolic disease and healthy aging.

As research continues to unravel MOTS-c's complexities and clinical programs advance toward regulatory approval, this mitochondrial-encoded peptide exemplifies the therapeutic potential residing in previously overlooked regions of the human genome. The MOTS-c story illustrates how fundamental biological discoveries can rapidly translate to therapeutic applications with potential to address major public health challenges including diabetes, obesity, age-related decline, and metabolic diseases affecting hundreds of millions worldwide.

References

Lee C, Zeng J, Drew BG, et al. The mitochondrial-derived peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin resistance. Cell Metab. 2015;21(3):443-454. PMID: 25738459
Kim KH, Son JM, Benayoun BA, Lee C. The mitochondrial-encoded peptide MOTS-c translocates to the nucleus to regulate nuclear gene expression in response to metabolic stress. Cell Metab. 2018;28(3):516-524.e7. PMID: 29983246
Fuku N, Pareja-Galeano H, Zempo H, et al. The mitochondrial-derived peptide MOTS-c: a player in exceptional longevity? Aging Cell. 2015;14(6):921-923. PMID: 26289118
Reynolds JC, Lai RW, Woodward JPN, et al. MOTS-c is an exercise-induced mitochondrial-encoded regulator of age-dependent physical decline and muscle homeostasis. Nat Commun. 2021;12(1):470. PMID: 33206854
Li H, Wang C, He T, et al. Mitochondria-derived peptide MOTS-c: effects and mechanisms related to stress, metabolism and aging. J Transl Med. 2023;21(1):36. PMID: 36698141
D'Souza RF, Woodhead JST, Zeng N, et al. MOTS-c: a promising mitochondrial-derived peptide for therapeutic exploitation. Front Endocrinol (Lausanne). 2023;14:1096901. PMID: 36761202
Cataldo LR, Fernández-Verdejo R, Santos JL, Galgani JE. Plasma MOTS-c levels are associated with insulin sensitivity in lean but not in obese individuals. J Investig Med. 2018;66(7):1019-1022. PMID: 31054164
Kim SJ, Xiao J, Wan J, Cohen P, Yen K. Mitochondrially derived peptides as novel regulators of metabolism. Free Radic Biol Med. 2016;100:182-193. PMID: 27216708
Ming W, Lu G, Xin S, et al. Mitochondria related peptide MOTS-c suppresses ovarian cancer progression by attenuating USP7-mediated LARS1 deubiquitination. Adv Sci (Weinh). 2024;11(43):e2405620. PMID: 39428972
Lu H, Wei M, Zhai Y, et al. MOTS-c peptide regulates adipose homeostasis to prevent ovariectomy-induced metabolic dysfunction. Int J Mol Sci. 2022;23(19):11991. PMID: 36233287

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