Mitochondrial-derived peptide MOTS-c activates metabolic signaling but blunts reparative function in human mesenchymal stromal cells

Abstract

Background

Mesenchymal stromal cells (MSCs) possess therapeutic potential largely reliant on intact mitochondrial function to maintain reparative function. However, obesity compromises MSC metabolism and reparative capacity. MOTS-c, a mitochondria-derived peptide, is known to regulate cellular metabolism, but its role in human MSC biology remains unclear. We hypothesized that restoring MOTS-c signaling rescues the impaired functionality of adipose-derived MSCs from individuals with obesity.

Methods

MSCs isolated from abdominal fat of patients with obesity (BMI ≥ 30 kg/m²) and lean donors (BMI < 30 kg/m²) (n = 6/group) were assessed in vitro for changes in proliferation, senescence (p16, p21) TNF-α, and antioxidant gene expression following MOTS-c co-incubation. In vivo, the effects of MOTS-c pre-treatment on the reparative capacity of obese MSC were tested in stenotic mouse kidneys.

Results

Basal MOTS-c expression was lower in obese vs. lean MSCs. Nevertheless, although exogenous MOTS-c restored intracellular levels and activated AMPK signaling in obese MSCs, it reduced proliferation, increased expression of senescence-associated genes (p16, p21), and upregulated TNF-α. In vivo, in a murine model of renal artery stenosis, MOTS-c-pretreated MSCs failed to improve renal perfusion, fibrosis, or tubular injury, while pretreatment also blunted the reparative efficacy of lean MSCs. These findings reveal that restoration of mitochondrial metabolic signaling is insufficient to reverse obesity-induced MSC dysfunction and may paradoxically exacerbate senescence and inflammation.

Conclusion

These results suggest a dissociation between metabolic activation and functional stemness, underscoring context-dependent effects of mitochondrial-derived peptides in MSC biology.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s41232-026-00431-7.

Introduction

Mesenchymal stromal cells (MSCs) are an endogenous cell repair system endowed with anti-inflammatory and pro-angiogenic properties [1]. Harvesting and exogenous delivery of MSCs is a promising strategy for application in regenerative medicine. Compared with other stem cell types, MSCs are present in most adult human tissues in niches like perivascular regions, can be sampled without ethical concerns, retain high differentiation potential, and present minimal oncogenic risk [2]. However, the function of endogenous MSCs may be impaired in patients with certain pathologies, limiting their reparative capacity in situ as well as their utility for autologous therapy. Understanding the mechanisms underlying the impairment of both endogenous and exogenous MSCs could be valuable in order to restore their functionality.

Obesity represents a major health burden, which is associated with a 1.5-fold increased risk of mortality [3] and amplifies the risks associated with cardiometabolic conditions. Previous studies have demonstrated that compared to lean individuals, MSCs derived from adipose tissue of patients with obesity show elevated expression of senescence and inflammatory genes like p16, p21, and IL-6, which correlates with body mass index (BMI) [4]. Congruently, obese human MSCs fail to restore the angiogenic potential of injured human umbilical vein endothelial cells in vitro [5] and of stenotic murine kidneys in vivo [6], indicating compromised repair capabilities. Similarly, extracellular vesicles (EVs) derived from obese pigs MSCs contain pro-inflammatory components and fewer reparative proteins compared to non-obese EVs, inducing inflammation in co-cultured renal tubular cells [7].

Mitochondria regulate the viability and functionality of MSCs, partly by encoding mitochondrial-derived peptides (MDPs). Importantly, MSCs from both pigs [4] and human subjects [8] with obesity exhibit altered expression and activation of genes associated with mitochondrial dysfunction and inflammation. Mitochondrial open-reading-frame of the 12S rRNA type-c (MOTS-c) is an important bioactive MDP that was identified in 2015 [9], is encoded by the mitochondrial genome, and is prominently involved in regulating metabolic homeostasis [9]. MOTS-c is present in the plasma of rodents and humans, but its levels may fall in conditions associated with metabolic dysregulation like diabetes [10] and endothelial dysfunction [11]. In animal studies, systemic administration of MOTS-c has conferred metabolic benefits. It prevents high-fat diet-induced obesity by enhancing energy expenditure, improving glucose utilization, and increasing insulin sensitivity [9]; alleviates hyperglycemia and insulin resistance in gestational diabetes patients [12]; regulates mitochondrial activation in brown adipose tissue [13, 14]; and exhibits anti-inflammatory and immunomodulatory functions [15–17]. MOTS-c also modulates age-related physical decline and muscle homeostasis [18] and improves cardiovascular function [19, 20]. Skeletal muscle expression of MOTS-c is also reduced in patients with chronic kidney disease [21], but its relevance for kidney repair is unknown.

MOTS-c has been shown to stabilize the internal environment of aging placenta-derived MSCs through reduced oxygen consumption and reactive oxygen species (ROS) production, thereby enhancing mitochondrial homeostasis [22]. However, its involvement in modulating the function of MSCs in the deranged metabolic environment associated with obesity remains unclear. We hypothesized that MOTS-c regulates MSC function in an obese state and employed an exogenous supplementation strategy to examine whether restoring MOTS-c levels can rescue obesity-induced MSC function, and elucidate its potential mechanism through in vitro and in vivo experiments.

Methods

We isolated adipose-derived MSCs from individuals with or without obesity and quantified MOTS-c levels. Their function was studied in vitro, as were the effects of exogenous MOTS-c supplementation in the culture medium on the proliferation, adhesion, paracrine function, and senescence of MSCs, and the AMPK-related signaling pathway. Subsequently, MSCs pre-treated with MOTS-c to raise its intracellular levels were injected in vivo in mice with renal artery stenosis (RAS) to test their reparative function including stenotic kidney perfusion, oxygenation, tubular injury, and fibrosis.

This animal study complies with the ARRIVE guidelines for the reporting of animal experiments and was approved by the Mayo Clinic Institutional Animal Care and Use Committee. The human research protocol was approved by the Institutional Review Board of Mayo Clinic (protocol #18–005076), and informed consent was obtained from all participants. Cell and vehicle infusions, as well as data analysis, were conducted in a blinded manner. The unblinding procedure was performed after all data had been collected and analyzed.

Participant recruitment

Individuals with or without (lean) obesity (n = 6 each), aged 18–80, were recruited as MSC donors at the Mayo Clinic in Rochester between February 2018 and January 2023. Obesity was defined as a BMI ≥ 30, and non-obesity (‘lean’) as BMI < 30 kg/m². Exclusion criteria included pregnancy, chronic inflammatory diseases, active malignancies, recent stroke or myocardial infarction, solid organ transplant recipients, and those receiving immunosuppressive or anticoagulation therapy.

MSC harvesting, cultivation, and characterization

Subcutaneous abdominal adipose tissue samples (0.5–2.0 g) were collected during weight reduction surgery (in patients with obesity) or donor kidney removal (in non-obese patients) and processed according to standard protocols [5]. Briefly, the tissue was minced, digested, and filtered. The cells were centrifuged, and the pellets were resuspended in advanced MEM medium supplemented with 5% platelet lysate. MSCs were expanded and cultured for three passages prior to experimentation. Our previous studies [5, 23, 24] have demonstrated that our laboratory protocol yields human MSC that align with the standards set by the International Society for Cellular Therapy [25]. These include expression of CD90, CD73, and CD105, but negative for the hematopoietic markers CD45 and CD34, as well as tri-lineage differentiation into chondrocytes, adipocytes, and osteoblasts.

Subsequently, MSCs were cultured in a medium containing 10 μM MOTS-c (GenScript, Lot No.:P19781308) or Phosphate buffered solution (PBS) (Life Technologies) for 48 h before experiments, based on previous research [18]. The dose was based on a prior study showing that MOTS-c improved skeletal muscle viability and adaptation to metabolic stress [18]. Previous studies confirmed that a 24–48 h-treatment with MOTS-c suffices to modulate cellular function [18, 26, 27].

Cell proliferation

MSC proliferation was assessed using a Cell Imaging Multimode Reader (Cytation-5, BioTek, Santa Clara, CA). Cells were seeded in a 24-well plate (3 × 10⁴/well) and kept at 37 °C with 5% CO₂. Cell confluence was captured hourly for 72 h, and data analyzed using Gen5 software (BioTek), as previously published [28].

Cell adhesion

MSCs were incubated in a 24-well plate (3 × 10⁴/well) at 37 °C with 5% CO2. After 2 h the wells were washed, followed by the addition of fresh culture medium and incubation with MTS/PMS solution (CellTiter-96® AQueous Non-Radioactive Cell Proliferation Assay, Promega, Madison, WI, Cat.#G5421) for 3 additional hours [29]. MSC adhesion was assessed by absorbance recorded at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

MSC protein expression

Protein extracts from non-obese and obese MSCs were used for standard protein immunoblotting using specific antibodies against human MOTS-c (ThermoFisher, Cat #MOTSC-101AP), p-AMPKα (Cell signaling, Cat.#2531), AMPKα (Cell signaling, Cat.#2532), mammalian target of Rapamycin (mTOR) (Cell signaling, Cat.#2983), p-mTOR (Cell signaling, Cat.# 5536), extracellular signal-regulated kinases (ERK)1/2 (Cell signaling, Cat.#4695), p-ERK1/2 (Cell signaling, Cat.# 4370), protein kinase B (AKT) (Cell signaling, Cat.# 9272), p-AKT (Cell signaling, Cat.# 9271), p38 mitogen-activated protein kinase (p38MAPK) (Cell signaling, Cat.# 9212), p-p38MAPK (Cell signaling, Cat.# 4511) and GAPDH for loading control (Abcam, Cat#ab8245). Protein concentrations were measured using a BCA assay. Lean and obese MSC samples were run on the same membrane, exposed together, and band intensities were read using ImageJ software (NIH).

Given the small molecular weight of MOTS-c, we used a Trans-Blot Turbo system (Bio RAD) for transfer in our experiments, during which we employed the machine's built-in low molecular weight mode and a double PVDF membrane to prevent the protein from crossing the membrane. Simultaneously, we incubated the antibody and exposed the membrane to both layers to ensure that the protein was not over-transferred.

MSC gene expression

For quantitative reverse-transcription polymerase chain reaction in the human MSCs, total RNA was isolated (Cat.#AM1556, Life Technologies) and the SuperScript VILO cDNA synthesis kit (Cat.#11754-050) was applied to get cDNA. The probes used (all from ThermoFisher) were p16 (Hs00923894), p21 (Hs00355782), p53 (Hs01034249), SOD1 (Hs00533490), TNF-α (Hs00174128), GPX (Hs00829989), CAT (Hs00156308) and GAPDH (Hs02786624). The relative mRNA expression levels were calculated using the 2^−ΔΔCT method after normalization with GAPDH.

Mouse studies

Eleven-week-old 129-S1 mice (Jackson Lab, Bar Harbor, ME) were housed in an accredited facility and provided with free access to water. Mice were randomly divided into 6 groups based on treatments: sham + vehicle, RAS + vehicle, RAS + lean MSC, RAS + obese MSC, RAS + lean MSC + MOTS-c, and RAS + obese MSC + MOTS-c (n = 6 mice/group) using a simple randomization method [30].

RAS surgery performed by experienced animal technologists involved surgical implantation of a 0.15-mm diameter plastic cuff around the right renal artery, while the sham surgery involved the same procedure without cuff placement [31, 32]. Two weeks after surgery, via a left internal carotid artery cannulation, a small plastic tube was inserted caudally and secured with surgical silk for subsequent cell or vehicle injections. Obese or lean MSCs (5 × 10⁵ in 200 µL PBS [32]), pre-treated or untreated with MOTS-c (10 μM, 48 h), or PBS alone, were slowly injected into the aorta, and the mice were allowed to recover. Anesthesia was induced using 1.5–2.0% isoflurane inhalation, and mice received buprenorphine SR-LAB (1.0 mg/kg) post-operatively for pain management. Two weeks after infusion, mice underwent MRI scans followed by euthanasia via exsanguination, during which blood was collected. Kidneys were harvested for ex vivo analysis. Blood pressure (BP) measurements (tail-cuff) and body weight were recorded at baseline, 2 weeks (prior to infusion), and 4 weeks (prior to euthanasia).

Imaging studies

Two weeks after MSC injection, murine renal perfusion was assessed using arterial spinning label (ASL), and oxygenation with blood oxygen-dependent (BOLD)-MRI in both the cortex and medulla, as described [33]. All images were analyzed and quantified using MATLAB R2015-a (MathWorks, INC). Kidney volume was assessed using Analyze® software (version 12.0; Bio-medical Imaging Resource, Mayo Clinic, MN) [33].

Histological studies

Five-µm kidney cross-sections were used for histological studies. Tubular injury (scale 0–5 where 5 is the worst) was assessed in H&E-stained slides [34] and interstitial fibrosis by Masson trichrome (MT) with the fibrosis area quantified using MATLAB in 10 random 20X fields.

Statistical analysis

Statistical analysis was performed using Prism (GraphPad). Levene’s test was used to test variance homogeneity, and the Shapiro–Wilk test for normality. Accordingly, statistical differences were analyzed using the Kruskal–Wallis test and one-way ANOVA or Welch one-way ANOVA test. Normally distributed data are presented as mean ± standard deviation, and non-normal data as the median and interquartile range (in box-plot format). Statistical significance was accepted for p ≤ 0.05.

Results

Patients with obesity had higher BMI, systolic BP, and proteinuria compared to the non-obese patients (Table 1), but similar sex distribution, age, glucose levels, and kidney function.

Table 1.

Demographics of obese and non-obese human MSC donors

Characteristics	Lean	Obese	P-value
n	6	6
Sex (F:M)	3:3	3:3	1.00
Age (years)	58.33 ± 10.69	56 ± 9.01	0.69
Blood pressure (mm/Hg)
Systolic	115.67 ± 10.783	134.17 ± 9.2826	0.01
Diastolic	76.33 ± 12.04	78.67 ± 17.37	0.79
Mean Arterial Pressure	89.44 ± 7.69	97.17 ± 12.07	0.22
Body mass index(Kg/m2)	25.92 ± 1.81	49.92 ± 6.04	< 0.001
Serum Creatinine(mg/dl)	0.92 ± 0.235	0.87 ± 0.15	0.69
Estimated GFR (eGFR)	75.5 ± 10.56	81 ± 9.17	0.36
Serum Glucose(mg/dl)	92 ± 15.17	103.2 ± 15.6	0.26
Serum Albumin(g/dl)	4.45 ± 0.25	4.22 ± 0.42	0.29
Urinary Protein(mg/dl)	5.83 ± 1.94	98.67 ± 10.50	0.004

Results are mean ± SD or median(IQR). GFR: Glomerular Filtration Rate

MOTS-c reduces obese MSCs proliferation rate and increases senescence and inflammatory factors

There was no difference in the proliferation rate between lean and obese MSCs under baseline conditions. MOTS-c increased the proliferation rate of the non-obese (lean) MSCs but reduced it in obese MSCs (Fig. 1A), without affecting MSC adhesion capacity (Fig. 1B). Senescence gene expression was higher in obese than in lean MSCs under basal conditions, and MOTS-c further increased p16 and p21 gene expression only in obese MSCs (Fig. 1C). MOTS-c also upregulated gene expression of the antioxidant superoxide dismutase 1 (SOD1) (Fig. 1D), while the expression of CAT and GPX genes remained unaffected (Supplemental figure B). Furthermore, MOTS-c upregulated TNF-α expression only in obese MSCs (Fig. 1D).

Fig. 1.

The effects of MOTS-c on the function and gene expression in human adipose tissue-derived MSC in vitro. A–B Obese and non-obese (“lean”) MSCs showed similar basal proliferation rates and adhesion. MOTS-c reduced the proliferation rate of obese MSCs but increased it in non-obese MSCs, without affecting their adhesion. C Compared to lean MSCs, the expression of the senescence-related genes p16, p21, and p53 was higher in obese MSCs. MOTS-c further upregulated the mRNA expression of p16 and p21 in obese MSCs. Basal TNF-α mRNA and SOD1 expression were similar between the groups, but MOTS-c upregulated both in obese MSCs (n = 6/group). #p < 0.05 vs. lean MSCs, ￥p < 0.05 vs. MOTS-c-untreated counterpart

MOTS-c protein affects obese MSC through AMPK pathways in vitro

Under resting conditions, MOTS-c expression was significantly lower in obese vs. lean MSCs (Fig. 2A). Coincubation with MOTS-c increased and restored its intracellular levels only in obese MSCs. Furthermore, in obese MSCs MOTS-c activated the key regulator of cellular homeostasis AMP-activated protein kinase (AMPKα), reflected in the p-AMPKα/AMPKα ratio. MOTS-c increased AMPKα total protein expression in non-obese MSCs group, but did not activate it. On the other hand, MOTS-c slightly downregulated in obese MSCs both the total protein expression of mTOR, a major protein complex involved in aging, and its phosphorylation. Contrarily, MOTS-c slightly decreased the total protein expression of ERK1/2 [35] only in obese MSCs compared to untreated obese MSCs, but not its phosphorylation (activation) (Fig. 2B). Moreover, there were no differences in the total protein content or phosphorylation ratios (activation) of AKT and p38 MAPK (Supplemental figure A).

Fig. 2.

MOTS-c alters metabolic pathways in MSCs. A Obese MSCs showed lower MOTS-c expression than lean MSCs, which significantly increased only in obese MSCs after MOTS-c treatment, indicating efficient uptake. B Baseline protein expression of mTOR, ERK1/2, and AMPKα were similar between lean and obese MSCs. MOTS-c upregulated total protein expression of AMPKα in lean MSCs and decreased that of mTOR and ERK1/2 in obese MSCs. However, MOTS-c activated AMPKα (increased p-AMPKα/AMPKα ratio) and decreased p-mTOR/mTOR ratio only in obese MSCs (n = 6/group). #p < 0.05 vs. lean MSCs, ￥p < 0.05 vs. MOTS-c-untreated counterpart

MOTS-c does not impact the hemodynamic effect of obese MSCs in RAS mice

Body weight was similar among the mouse groups (Fig. 3A). At 4 weeks, all RAS groups showed significantly elevated systolic BP compared to sham, except for RAS + lean MSCs that showed blunted hypertension. Therefore, obesity abolished the antihypertensive effect of MSCs in RAS mice, which MOTS-c did not improve (Fig. 3B). Compared to sham, stenotic kidney cortical perfusion was significantly reduced in all RAS-operated groups, except for RAS + lean MSCs that was higher than RAS + vehicle. MOTS-c did not affect cortical perfusion in RAS + obese MSCs + MOTS-c, but blunted the improvement in cortical perfusion achieved by untreated lean MSCs, so that RAS + lean MSCs + MOTS-c remained lower than sham. Medullary perfusion showed a similar pattern (Fig. 3C).

Fig. 3.

MOTS-c did not improve the effect of obese MSCs on stenotic kidney (STK) function. A Body weight was similar among the groups. B–C Compared to sham, systolic blood pressure (SBP) rose and STK cortical and medullary perfusion was decreased in RAS + vehicle. Only RAS + lean MSC blunted the rise in SBP from baseline to 4 weeks and improved STK perfusion compared to RAS + vehicle (n = 6/group). &p < 0.05 vs. sham + vehicle, ¢p < 0.05 vs. RAS + vehicle, #p < 0.05 vs. lean MSCs

MOTS-c boosts the effect of obese MSCs on medullary oxygenation

Compared to RAS + Vehicle, MSC treatment improved cortical oxygenation in all groups regardless of MOTS-c, and lean MSC treatment also improved medullary oxygenation. Contrarily, RAS + obese MSCs showed no significant difference in medullary oxygenation from either RAS + vehicle or sham, but this was slightly improved in RAS + obese MSCs + MOTS-c in which medullary hypoxia became lower than RAS + vehicle (Fig. 4A). MSCs significantly increased stenotic kidney volume similarly in all groups compared to RAS + vehicle (Fig. 4B).

Fig. 4.

MOTS-c pretreatment improved the effect of obese MSCs on medullary oxygenation in the stenotic kidney (STK). RAS + vehicle showed a significant increase in STK cortical and medullary T2* (i.e., decreased oxygenation) (A) and a decrease in volume (B). Compared to RAS, cortical oxygenation and STK volume were improved similarly in all the MSC-treated RAS mice. Contrarily, medullary oxygenation became significantly higher than RAS + vehicle only after MOTS-c pre-treatment of obese MSCs (n = 6/group). &p < 0.05 vs. sham + vehicle, ¢p < 0.05 vs. RAS + vehicle, #p < 0.05 vs. lean MSCs

MOTS-c blunts the protective effects of obese MSCs in the stenotic kidney

Tubular injury scores in the stenotic kidney were markedly higher in all RAS groups vs. sham. Although it remained higher than in sham, all MSC-treated groups except for RAS + obese MSCs + MOTS-c had lower tubular injury than RAS + vehicle. RAS + lean MSCs induced a greater fall in tubular injury scores than RAS + obese MSCs, but MOTS-c blunted this effect (Fig. 5A). Stenotic kidney fibrosis also increased in all RAS groups vs. sham and was reduced only by lean MSCs vs. RAS + vehicle. Yet, MOTS-c significantly attenuated the anti-fibrotic efficacy of both obese and lean MSCs in the stenotic kidney compared to their PBS-treated counterparts (Fig. 5B).

Fig. 5.

MOTS-c pretreatment of obese or non-obese (lean) MSCs does not improve tubular injury in the murine stenotic kidney (STK) and worsens fibrosis. RAS + vehicle showed a significant increase in STK tubular injury score (A) and fibrosis (B). Compared to RAS + vehicle, only RAS + lean MSC both improved STK tubular injury scores and decreased fibrosis, while RAS + obese MSCs only improved STK tubular injury scores. Furthermore, compared to their counterparts, MOTS-c pretreatment significantly increased STK tubular injury scores and fibrosis in RAS groups treated with lean MSC (n = 6/group). &p < 0.05 vs. sham + vehicle, ¢p < 0.05 vs. RAS + vehicle, #p < 0.05 vs. lean MSCs

Discussion

This study demonstrates that replenishment of MOTS-c in human obese MSCs confers limited functional benefit. Although coincubation of obese MSCs with MOTS-c activated AMPKα and downregulated mTOR expression and activation levels, it reduced MSC proliferation, increased senescence, and attenuated their reparative function in vivo. Thus, while MOTS-c enhanced antioxidant responses, like upregulating SOD1, its concomitant effects on cell cycle regulators and pro-inflammatory gene expression may undermine MSC regenerative efficacy. Despite mitochondrial dysfunction being a hallmark of obesity-impaired MSCs, our findings demonstrate that restoration of the mitochondrial-derived peptide MOTS-c does not rejuvenate obese MSCs. These observations suggest that MOTS-c operates within a narrow physiological range in MSCs and that perturbation of this balance may impair cellular function in the context of kidney repair. Importantly, our data suggest a metabolic–stemness uncoupling, whereby MOTS-c–linked metabolic adaptation can occur without recovery of the paracrine reparative program that underlies MSC efficacy.

Obesity compromises the therapeutic efficacy of MSCs, promoting cellular senescence and impairing the regenerative capacity of MSCs [6] and their EVs [36]. Systemic application of MOTS-c has been shown to prevent insulin resistance and diet-induced obesity in aging and high-fat conditions [9] and to regulate age-related physical decline and muscle homeostasis [18]. However, while MOTS-c improves the mitochondrial homeostasis of aging placenta-derived MSCs [22], its effects on the reparative function of MSCs and specifically on obese adipose tissue-derived MSCs were unknown. Interestingly, our study shows that although its levels are decreased in obese MSCs, co-incubation with MOTS-c did not effectively reverse obesity-induced functional deficits in MSCs for kidney repair and further reduced their anti-fibrotic and tubule-protective capacity.

An important consideration is that exogenous supplementation of 10 µM MOTS-c does not fully recapitulate physiological conditions. Comparable doses are frequently used to produce robust signaling effects [18, 37, 38] and achieve AMPK activation, nuclear translocation, and transcriptional effects, while preserving cell viability. However, given that this concentration is much higher than plasma MOTS-c concentrations in healthy individuals [39], it should generally be viewed as pharmacological rather than an endogenous physiological exposure. Obesity may lead to decreased circulating MOTS-c levels [9, 40, 41], so that replenishing its levels in MSCs does not necessarily mimic the prevailing baseline milieu, but rather stress adaptation. Indeed, our experimental design was not intended to reproduce the obesity microenvironment itself, but rather to determine whether supplementation with mitochondrial-derived peptides could rescue obesity-induced MSC dysfunction.

Consistent with prior findings [6], we observed that obesity upregulates in MSCs the senescence-associated genes p21, p16, and p53, and we now show that this is accompanied by a marked reduction in intracellular MOTS-c levels compared to lean MSCs. Nevertheless, while co-incubation with MOTS-c restores its intracellular levels, it concurrently upregulated p16 and p21 gene expression, consistent with sustained cellular senescence. Our observations align with a previous report suggesting that MOTS-c exposure may enhance the senescence-associated secretory phenotype in aged cells [42].

Post-metabolic stress MOTS-c relocates to the nucleus via an AMPK-dependent mechanism, modulating nuclear gene expression [37], and in turn activates AMPK [43]. This energy sensor sustains cellular metabolic homeostasis, and is activated by MDPs like humanin [44] and MOTS-c [37]. AMPK is a heterotrimer composed of several subunits, of which α is catalytic [45]. In obese MSCs, we observed that MOTS-c significantly increased AMPK phosphorylation (p-AMPKα/AMPKα levels), indicating AMPK pathway activation, which in turn may improve metabolism and inhibit protein synthesis, cell growth, and proliferation [46]. An important regulatory mechanism of inhibiting protein synthesis is AMPK’s inhibition of mTOR [47, 48], which coordinates cell growth and metabolism, aligning with our observation that mTOR protein expression as well as phosphorylation-dependent activation in obese MSCs was also reduced by MOTS-c. Given the central role of mTOR in regulating cellular metabolism and growth, this finding suggests that MOTS-c preferentially modulates metabolic signaling pathways. Interestingly, in our study, AMPK activation and mTOR inhibition were associated with increased senescence. Possibly, in obese MSCs, AMPK pathway activation by MOTS-c may function as a stress-adaptive but senescence-permissive signal, prioritizing metabolic survival over proliferative or reparative capacity. Indeed, AMPK activation or mTOR inhibition may reverse or exacerbate senescence-associated phenotypes, depending on the context [46]. For example, in cancer mTORC1 may be necessary for the survival of senescent cells [49]. Thus, the extent to which mTORC1 activity promotes or reverses cellular senescence varies [50]. Furthermore, other activated pro-senescence factors might offset the effects of mTOR inhibition.

Moreover, MOTS-c treatment slightly downregulated in obese MSCs the total protein expression of ERK1/2, which regulates cell cycle progression, proliferation, senescence, death, migration, and adhesion [51], as well as survival signaling of growth factors [52], and its inhibition increases ischemic myocardial cell damage [53]. However, its phosphorylation was not significantly altered following MOTS-c treatment, indicating that ERK1/2 activation remained largely unchangedf implying that MOTS-c may regulate ERK1/2 protein abundance, turnover, or feedback control but not activation. On the other hand, while the AKT and p38MAPK pathways showed no significant changes in total protein levels, they were both activated in obese MSCs vs. lean MSCs, consistent with a baseline stress-activated state. However, neither was modulated by MOTS-c (Supplemental Fig. A). Hence, while the proposed role of AMPK-mTOR-ERK1/2 in the MOTS-c pathway is compelling, our data suggest that ERK1/2, AKT, and p38MAPK are not primary mediators of MOTS-c effects in this context, and the causality in our study remains speculative.

Overall, MOTS-c may improve MSC metabolism by increasing SOD1 expression and activating AMPK. Some studies suggest that MOTS-c promotes the stabilization of the internal environment in aging MSCs by reducing oxygen consumption and ROS production, thereby enhancing mitochondrial homeostasis [22], which is consistent with the increased expression of SOD1 observed in MOTS-c-treated obese MSCs. However, unchanged CAT and GPX expression suggests that this represents a selective rather than comprehensive antioxidant response, possibly favoring superoxide detoxification without broadly activating downstream peroxide-scavenging systems. Yet, upregulation of p16 [54] in MOTS-c-treated obese MSCs may have downregulated total ERK1/2 protein expression, which can reduce their stemness and reparative potency [55]. Upregulation of cyclin-dependent kinases leading to cell cycle arrest may also inhibit cell growth, which may account for the reduced proliferation rate of obese MSCs following MOTS-c treatment in vitro. Moreover, the elevated expression of TNF-α in MOTS-c-treated obese MSCs suggests development of a pro-inflammatory phenotype, which promotes the aggregation and proliferation of immune cells [56], thereby preventing improvement in kidney perfusion and fibrosis in ischemic mouse kidneys. Compared to the renal cortex, the renal medulla has lower perfusion and partial oxygen pressure [57]. By upregulating SOD1 expression in obese MSCs, MOTS-c might enhance their survival in the hypoxic environment, allowing improvement in medullary oxygenation in ischemic murine kidneys likely through reduction of superoxide burden rather than global redox restoration. Nonetheless, this did not translate into protection from fibrosis or tubular injury, highlighting a potential disconnect between metabolic effects and functional outcomes. Alternatively, this may also be attributable to metabolic alterations that prolong the pro-inflammatory phenotype of MSCs. Likely, the increased senescence phenotype may explain the negative effect of MOTS-c on obese MSCs and might explain their decreased proliferation in vitro.

Notably, the effects of MOTS-c on lean MSCs were unexpected. The divergent response of lean MSCs to MOTS-c suggests a context-dependent regulation of peptide uptake and downstream signaling. The absence of intracellular MOTS-c accumulation despite increased extracellular availability indicates that lean MSCs may actively restrict its uptake under non-metabolic stress conditions. Although AMPK-α protein abundance was increased following MOTS-c exposure, the lack of canonical AMPK pathway activation implies that MOTS-c may modulate AMPK expression independently of its activation state, potentially through compensatory or feedback regulatory mechanisms rather than direct energetic sensing. Importantly, the observed attenuation of therapeutic efficacy in vivo raises the possibility that excess, unutilized MOTS-c in the culture environment may induce maladaptive signaling that compromises tissue repair. Our data therefore suggest that MOTS-c operates within a narrow physiological window, where excess signaling, particularly in metabolically intact MSCs, disrupts homeostatic repair programs.

These findings have direct implications for MSC preconditioning strategies. While metabolic enhancement is often pursued to improve cell survival, our data indicate that indiscriminate activation of metabolic stress pathways may compromise MSC stemness and paracrine function. Effective preconditioning may therefore require simultaneous modulation of senescence and inflammatory programs, rather than isolated metabolic interventions.

Our study has certain limitations. Donor homogeneity (Caucasian) does not account for potential ethnic variations. Moreover, the small sample size might have reduced the ability to detect functional differences between Lean-MSCs and obese MSCs in vivo, which were instead inferred indirectly through comparisons with the corresponding control groups. MOTS-c supplemented in culture may also not mimic changes induced by its intracellular upregulation, although it might reflect cellular alterations that occur following systemic administration of MOTS-c. Our use of extracellular MOTS-c was intended to test whether increasing peptide availability is sufficient to alter MSC behavior. Indeed, the experimental design of this study likely simulates scenarios involving pharmacological intervention or states of stress adaptation, rather than basal physiological conditions. In addition, the study was not designed to test potential effects of systemic MOTS-c on effector or accessory cells that interact with MSCs rather than the MSCs themselves. Finally, additional studies are needed to explore the mechanisms by which MOTS-c affects MSC cellular function and explore alternative dosing regimens, time course, and combination approaches that might optimize the metabolic benefits of MOTS-c while avoiding detrimental effects on proliferation or inflammation.

Together, these findings demonstrate that mitochondrial signal restoration alone cannot overcome obesity-induced MSC senescence and may paradoxically impair reparative efficacy, emphasizing the need for integrated strategies that address both metabolic dysfunction and cellular aging. MOTS-c application is likely complex, context-dependent, and finely tuned. Furthermore, these findings suggest that metabolic interventions targeting MSCs may require stratification by donor metabolic state and baseline senescence burden, and that uniform preconditioning strategies may inadvertently worsen outcomes in otherwise competent cells. Overall, the distinct regulatory mechanisms of MOTS-c in obese MSCs warrant further study, potentially aiding in the identification of specific obesity-related MSC targets and informing MOTS-c applications. Furthermore, studies are needed to determine if MOTS-c modulation could offer advantages in different disease states under different delivery schemes.

Supplementary Information

41232_2026_431_MOESM1_ESM.jpg ^{(604.8KB, jpg)}

Supplementary Material 1: Supplemental Figure: (A) Baseline total protein expression of AKT and p38MAPK were similar between Lean and Obese-MSCs, while their activation (p-AKT/AKT and p-p38MAPK/p38MAPK ratios) were higher in Obese-MSCs. MOTS-c did not affect either AKT or p38MAPK expression or activation (n = 6/group). (B) Basal CAT mRNA was similar among the groups, while GPX expression was higher in obese groups, but MOTS-c did not affect either in Obese-MSCs. #p < 0.05 vs. lean-MSCs, ￥p < 0.05 vs. MOTS-c-untreated counterpart.

Abbreviations

MSCs

Mesenchymal stromal cells

EVs

Extracellular vesicles

BMI

Body mass index

MDPs

Mitochondrial-derived peptides

MOTS-c

Mitochondrial open-reading-frame of the 12S rRNA type-c

ROS

Reactive oxygen species

RAS

Renal artery stenosis

BP

Blood pressure

BOLD

Blood oxygen-dependent

ASL

Arterial spinning label

MT

Masson trichrome

mTOR

Mammalian target of Rapamycin

SOD1

Superoxide dismutase 1

AMPK

AMP-activated protein kinase

ERK

Extracellular signal-regulated kinases

STK

Stenotic kidney

CAT

Catalase

GPX

Glutathione peroxidase

AKT

Protein kinase B

p38MAPK

p38 mitogen-activated protein kinase

Authors’ contributions

LX: Conception, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing- Original Draft, Writing- Review & Editing. XZ: Data Curation, Formal Analysis, Investigation, Methodology, Validation, Writing- Review & Editing. LB: Data Curation, Formal Analysis, Investigation, Methodology, Software. MAS: Data Curation, Resources, Investigation, Methodology, Software, Visualization, Writing- Review & Editing. AL: Funding Acquisition, Investigation, Writing- Review & Editing. AE: Data Curation, Project Administration, Resources, Supervision, Writing- Review & Editing. PC: Data Curation, Project Administration, Resources, Supervision, Writing- Review & Editing. LOL: Conception, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Writing- Review & Editing. All authors read and approved the final manuscript.

Funding

This study was partly supported by NIH grant numbers: DK120292, DK122734, HL158691, and AG062104.

Data availability

The data supporting this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Title of the approved human study: “Obesity-induced mesenchymal stem cell senescence”.

Name of the approving ethics committee: Mayo Clinic Institutional Review Board.

Ethics approval reference number: 18-005076. Date approved: August 17, 2018.

Consent for publication

Not applicable.

Competing interests

Dr. Lerman is an advisor to CureSpec, LiveKidney.bio, and Cellergy. The authors declare no conflict.