Stem cell secretome treatment improves whole‐body metabolism, reduces adiposity, and promotes skeletal muscle function in aged mice

Abstract Aging coincides with the progressive loss of muscle mass and strength, increased adiposity, and diminished physical function. Accordingly, interventions aimed at improving muscle, metabolic, and/or physical health are of interest to mitigate the adverse effects of aging. In this study, we tested a stem cell secretome product, which contains extracellular vesicles and growth, cytoskeletal remodeling, and immunomodulatory factors. We examined the effects of 4 weeks of 2×/week unilateral intramuscular secretome injections (quadriceps) in ambulatory aged male C57BL/6 mice (22–24 months) compared to saline‐injected aged‐matched controls. Secretome delivery substantially increased whole‐body lean mass and decreased fat mass, corresponding to higher myofiber cross‐sectional area and smaller adipocyte size, respectively. Secretome‐treated mice also had greater whole‐body physical function (grip strength and rotarod performance) and had higher energy expenditure and physical activity levels compared to control mice. Furthermore, secretome‐treated mice had greater skeletal muscle Pax7+ cell abundance, capillary density, collagen IV turnover, reduced intramuscular lipids, and greater Akt and hormone sensitive lipase phosphorylation in adipose tissue. Finally, secretome treatment in vitro directly enhanced muscle cell growth and IL‐6 production, and in adipocytes, it reduced lipid content and improved insulin sensitivity. Moreover, indirect treatment with secretome‐treated myotube culture media also enhanced muscle cell growth and adipocyte size reduction. Together, these data suggest that intramuscular treatment with a stem cell secretome improves whole‐body metabolism, physical function, and remodels skeletal muscle and adipose tissue in aged mice.


| INTRODUC TI ON
Aging is accompanied by the progressive development of frailty, chronic diseases, and impaired quality of life.While multifaceted, the severity of the aging phenotype is largely controlled by musculoskeletal and metabolic health and associated cellular and molecular underpinnings (Fried et al., 2021;Kennedy et al., 2014).Skeletal mass and function as well as metabolic health are markedly impaired in older adults and are associated with numerous negative health effects under the umbrella of aging (Larsson et al., 2019;Pontzer et al., 2021).Aged skeletal muscle is characterized by structural disorganization (Wood et al., 2014), fibrosis, neuromuscular degeneration (Liu et al., 2017), blunted growth, and impaired regenerative capacity (Tidball et al., 2021).Similarly, adipose tissues exhibit metabolic dysregulation (Goodpaster et al., 2005), adverse redistribution (Goodpaster et al., 2005), and inflammatory profiles in aged individuals (Wang et al., 2022).Moreover, aginginduced muscle and adipose dysfunction, either individually or combined (termed sarcopenic obesity) (Trouwborst et al., 2018), diminish the effectiveness of exercise and dietary interventions (Peterson et al., 2011;Phillips et al., 2017) and lead to generalized frailty, increased disease morbidity, and ultimately, pre-mature mortality (Fried et al., 2021;He et al., 2021;Kennedy et al., 2014).Therefore, there is great interest in the development of therapeutic strategies that target age-related deficits in musculoskeletal and adipose tissue health that result in improved physical activity and physical function levels (Chakhtoura et al., 2023;Kwak & Kwon, 2019).
Stem cell therapies show promise in regenerative medicine, yet when delivered directly may induce adverse responses.However, secondary approaches have arisen including the enrichment of culture media with stem cell secretory factors (Fix et al., 2021;Kim et al., 2016;Sandonà et al., 2021;Sanz-Ros et al., 2022;Wu et al., 2022).These secretomes include numerous soluble and encapsulated (extracellular vesicles) signaling molecules (cytokines, growth factors) that exert cellular and tissue adaptive effects (Daneshmandi et al., 2020;Hsieh et al., 2013;Sandonà et al., 2021).Suitably, secretome products from various stem cell origins have been utilized to enhance muscular outcomes and combat the effects of aging in mice (Sanz-Ros et al., 2022), including use of these products in a regenerative capacity following muscular atrophy or damage (Amankwaah et al., 2019;Fix et al., 2021;Wu et al., 2022).For example, we previously demonstrated that intramuscular secretome treatment reduces the loss of muscle mass and strength associated with disuse-atrophy and accelerates recovery from hindlimb unloading-associated atrophy (Fix et al., 2021).Similarly, others have shown that intravenous treatment with extracellular vesicles from adipocytederived stem cells improves muscular strength and function and decreases frailty in old mice (Sanz-Ros et al., 2022).Ostensibly, stem cell secretome treatments can combat the effects of aging in skeletal muscle, yet the secondary whole-body effects including changes in adipose tissue and body composition, as well as metabolic function are unknown.
Therefore, the purpose of this study was to investigate the effects of twice weekly intramuscular treatment for 4 weeks with a pluripotent stem cell secretome product in aged mice on wholebody metabolism (energy expenditure, tissue composition, activity levels), physical function, as well as skeletal muscle and adipose tissue remodeling.Furthermore, we examined the acute skeletal muscle transcriptional response to the secretome treatment as well as the autocrine and paracrine effects of the secretome product on muscle cells and adipocytes in-vitro.We hypothesized that chronic intramuscular secretome treatment would ameliorate muscle aging (decrease muscle fibrosis, increase capillarity, stem cells, and myofiber hypertrophy) and adiposity while improving whole-body energy metabolism and physical function capacity in aged mice.Moreover, we hypothesized that acute secretome treatment would directly and indirectly impact C2C12 muscle cell growth and reduce lipid accumulation in 3T3-L1 adipocytes.
Cultured media collected from these cells was pooled, sterile filtered, concentrated, and prepared as a USP-grade cell-free stem cell-based secretome product (IMMUNA; Immunis, Inc., Irvine, CA).Fifty microliters of secretome or saline (0.9% USP) were delivered to the right quadricep muscle via intramuscular injection under sterile conditions twice per week for 4 weeks (8 total injections/mouse).Secretome treatment was injected at a 0.4% concentration in saline based on pilot data and our previous work (Fix et al., 2021).
As previously described by our lab (Fix et al., 2021), the secretome product contains a host of soluble signaling molecules with prominent proteins regulating cellular growth, remodeling, and immunomodulation (Table 1).Extracellular vesicles from the secretome product were examined via nanoparticle tracking followed by microRNA isolation and next-generation sequencing by an independent party (Creative Biolabs, Inc.).Extracellular vesicles had an average particle size of 118 (nm) and concentration 2.8 12 (particles/ mL), while microRNA concentration was 23 ng/μL with a quality score of 30 (99.9%).The top 10 identified microRNAs and their validated target genes are also presented in Table 1.

| Body tissue composition and physical function testing
A nuclear magnetic resonance (NMR) instrument (Bruker Minispec MQ20 NMR analyzer, Rheinstetten, Germany) was used to assess whole body tissue composition.In addition, whole-body strength, plus balance and coordination, were assessed by grip strength and rotarod instrument, respectively, as we have conducted previously (Petrocelli et al., 2021).NMR and grip strength were assessed weekly, while rotarod was tested before treatment and then repeated after the 4-week intervention.Whole-body grip strength was assessed using a grip strength meter with a mesh wire attachment (Columbus Instruments, Columbus, OH, USA).After acclimation testing a week prior, mice were placed on the mesh wire and pulled by the base of their tail, parallel to the mesh wire.Peak force was recorded, and an average of three trials was recorded.Balance and coordination were assessed using rotarod testing on a Rotamex-5 (Columbus Instruments, Columbus, OH).The speed began at 0.1 rpm and increased by 0.3 rpm/s increments with the final recorded time when mice fell off the rotating bar.Each mouse performed the test three times, and an average time was recorded.Mice were acclimated on the rotarod 2 days prior to testing.All physical function tests were conducted by the same research personnel.(RER) was calculated from V ̇CO 2 production and V ̇O2 consumption, and energy expenditure was calculated by dividing heat production (kcal/hr) by body weight.Ambulatory activity was calculated by summing ambulatory beam breaks in the x, y, and z directions.Food intake was calculated from a food scale inside the CLAMS unit.

| Metabolic measurements
Prior to tissue collection following the 4-week experiments, separate mice from above performed a 120-min glucose tolerance test (10% glucose solution injected at 1 g/kg) with tail blood sampling including assessment of fasting and 30 min fed insulin levels via Ultra-Sensitive Mouse ELISA kit (Crystal Chem, Elk Grove Village, Il, USA) per manufacturer recommendations.

| Fat and liver histology
After dissection, a portion of each fat pad and the left lobe of the liver were placed in 4% paraformaldehyde for 24 h, after which they were stored in 70% ethanol until analysis.Samples were submitted to Associated Regional and University Pathologists (ARUP) laboratories at the University of Utah and the Department of Pathology for hematoxylin and eosin (H&E) (fat pads) while livers were stained for H&E and Masson's trichome.Briefly, samples were embedded in paraffin, sectioned at 5μm thickness then H&E stained to visualize lipid droplets, and trichrome stained to visualize tissue fibrosis.
Slides were imaged on a Zeiss Slide Scanner Axio Scan.Z1 (Carl Zeiss Inc.) with a 10× (fat pads, liver trichrome) or 20× (liver H&E) objective lens.Fat pad images were analyzed using a Fiji plugin Adiposoft as described by others (Galarraga et al., 2012) to determine average adipocyte diameter for each sample across three randomly selected fields.Liver lipid accumulation was assessed from H&E images in Fiji, briefly, by thresholding to identify lipid droplets, analyzing particles, and removing erroneous areas from analysis.Liver fibrosis was analyzed using the Automated Fibrosis Analysis Toolkit plugin for Fiji as described elsewhere (Gratz et al., 2020).

| RNA sequencing and Western blotting
In a separate experiment to determine the acute effects of the secretome treatment on muscle, we delivered a single intramuscular treatment of secretome or saline to C57BL/6 mice (26-28 months old) in a fasted (4 h) state.Three hours after injection, mice were euthanized, and injected quadriceps were collected for bulk RNA sequencing, qPCR, and immunoblotting.Hallmark, KEGG, and REACTOME pathways were identified using the fast gene set enrichment analysis in MSigDB using a 5% FDR.Data can be found on the Gene Expression Omnibus (GSE242211).
Injected quadriceps muscle from the acute and 4-week experiments as well as adipose pads from the 4-week investigation were additionally used to isolate RNA and/or protein for downstream real-time PCR and western blotting for anabolic, catabolic, and metabolic targets.Additional details on methodology can be found in the Supplemental Methods.

| Lipidomics
A portion of injected quadricep muscle (~15 mg) from the 4-week experimental study was used to extract lipids and prepare samples for liquid chromatography mass spectrometry (LC-MS) metabolomic analysis as described elsewhere (de Hart et al., 2023).Processed lipid metabolites including triglycerides, ceramides, and diacylglycerides with relative standard deviation less than 30% of quality controls were examined individually and as population totals.Detailed methods for all analyses can be found in the Supplemental Methods.

| Adipocyte and muscle cell culture experiments
C2C12 myoblasts were grown to confluence then differentiated for ~4 days under standard conditions in 6-well dishes.Media was replaced every 48 h during growth and differentiation.Cultured myotubes were treated with 4% secretome product replacement for 24 h based on previous experiments (Fix et al., 2021) and analyzed for changes to myotube area and fusion index (n = 6).Separate groups of control and treated (4% secretome) myotubes were used to produce culture media for 3 h (n = 6).Culture media was then placed on fully differentiated myotubes for 24 h to assess autocrine and paracrine effects of cultured media followed by the same measurements described above (n = 4).Cultured media replicates (n = 7) and undiluted secretome product (n = 3) were additionally tested for IL-6 concentration using a Mouse Quantikine ELISA Kit per manufacturer recommendations (R&D Sytems, Minneapolis, MN, USA).Detailed methodologies and reagents can be found in the Supplemental Methods.
3T3-L1 preadipocytes were prepared according to manufacturer recommendations then differentiated into adipocytes in 8well chamber slides for 5-7 days until confluent, followed by 3 days of differentiation, and finally cellular maintenance and treatment (Montanari et al., 2019).Media was replaced every 48-72 h during growth and differentiation.Replicate wells of cultured adipocytes (n = 7) were treated via 5% or 20% secretome product media replacement for 24 h and compared to untreated controls.Adipocytes were stained and assessed for content as described elsewhere (Montanari et al., 2019) and in the Supplemental Methods.Separate groups (n = 6) of control and treated adipocytes (20% secretome) with and without insulin treatment (100 nM) were then analyzed for insulin sensitivity via western blot (Akt phosphorylation, Ser473).Finally, groups of differentiated 3T3-L1 adipocytes (n = 4) were treated with 20% media replacement using the C2C12 culture mediums above and assessed as previously described.
To assess persistence of the described whole-body tissue composition and physical function effects, in a separate group of mice, we ceased the secretome treatment for 2 weeks (withdrawal) following the 4-week treatment period and compared this response to the 4-week treatment timepoint for whole body tissue composition outcomes, grip strength, rotarod performance, and tissue weights (Figure S1).We found that following 2 weeks of withdrawal in previously secretome-treated mice, that the prior gains in body mass (Figure S1E) and lean mass (Figure S1F) were lost.However, secretome-mediated adaptations in fat mass, % body fat, grip strength, and rotarod performance (Figure S1G-J) were maintained.
Representative histochemical images of fiber types are shown in between groups suggesting a transient activation following secretome treatment (Figure S2G).

| Secretome treatment altered muscle and adipocyte cells in-vitro through direct and indirect mechanisms
To explore the direct and indirect effects of the secretome product on muscle and adipose tissue, we performed a series of experiments using myotubes and 3T3-L1 preadipocytes in-vitro (Figure 6a).
Based on our previous publication (Fix et al., 2021), we first confirmed that 4% media replacement with secretome product for 24 h increased myotube area (31.8 ± 3.8 vs. 19.2± 3.5%, p = 0.01) and myonuclear fusion index (0.46 ± 0.04 vs. 0.32 ± 0.09 au, p = 0.03) compared to untreated controls (Figure 6b,c).To examine the indirect effects of muscle secretome treatment, we treated a separate group of differentiated myotubes with 4% secretome replacement for 24 h, then allowed them to produce fresh culture media for 3 h (Secretome CM).This cultured media was tested for IL-6 content, and then the Secretome CM was used to treat both fully differentiated myotubes and adipocytes for 24 h followed by the same analyses as described previously (Figure 6a).IL-6 was present in control culture media (Control CM) (132.08 ± 7.5 pg/mL) but was over three times as concentrated in Secretome CM (424.02 ± 29.29 pg/mL, p < 0.01) (Figure 6d).We additionally tested the undiluted secretome product (n = 3) and found considerably lower IL-6 concentrations

| DISCUSS ION
Here we examined the effects of biweekly intramuscular treatment in aged mice for 4 weeks with a stem cell-derived secretome We also report increased whole-body lean mass as well as greater quadriceps mass and myofiber size, with an emphasis on type II fiber growth, following 4-weeks of unilateral intramuscular secretome treatment.Additionally, grip strength progressively increased over time and persisted with two weeks of withdrawal independent of lean mass maintenance.These responses were underpinned by robust muscular remodeling including greater collagen IV turnover, capillarization, and muscle stem (satellite) cell content, which are known to enhance muscular function, regeneration, and recovery from disuse atrophy (Fix et al., 2021;Sambasivan et al., 2011).
Furthermore, the adaptations to muscle mass and fiber type with secretome treatment are important for maintaining body composition, metabolism (Akasaki et al., 2014), and muscle function with age (Korhonen et al., 2006), and perhaps lends some evidence to the observed improvements to whole-body metabolic and grip strength.
Cumulatively, the rapid increase of muscle size, strength, and remodeling following secretome-based treatment could be relevant for atrisk aging populations including those affected by health conditions including metabolic dysfunction and sarcopenia (Fried et al., 2021;Kennedy et al., 2014;Larsson et al., 2019;Pontzer et al., 2021).
Consistent with reduced adiposity and enhanced leanness, we observed lower muscle triglycerides and ceramides following 4-weeks of secretome treatment.The accumulation of skeletal muscle lipids including triglycerides and ceramides is associated to impaired metabolic health and the development of metabolic diseases (Blachnio-Zabielska et al., 2016;Turpin-Nolan et al., 2019).
Accordingly, increased intramuscular triglyceride turnover and reductions in storage have positive effects on the health of humans and animals (Bergman et al., 2018;Ko et al., 2018).Improved triglyceride handling is further associated with decreases in muscle ceramides (Bergman et al., 2018) proteostasis in aged mice (Lima et al., 2023).Therefore, reductions in muscle lipids, specifically muscle ceramides, may be a possible mechanism for the improved muscle and physical function following secretome treatment.
The secretome product also enhanced skeletal muscle remodeling in aging noted by higher levels collagen IV turnover, capillarization, and muscle stem cell (satellite cell) content.Interestingly, excessive collagen deposition not only impairs contractile function, but also the ability for satellite cells to proliferate and infiltrate the extracellular muscle environment (Fry et al., 2017;Lacraz et al., 2015).Similarly, greater muscle capillarization, and thereby perfusion, enhances satellite cell dynamics and promotes muscular recovery following damage (Christov et al., 2007), which is diminished yet reversable in aging (Snijders et al., 2017).Accordingly, the robust increase in muscle satellite cell expansion observed here and what we have reported previously (Fix et al., 2021) could be partially driven by enhanced cell migration triggered by enhance perfusion and increased collagen turnover.Noting that satellite cell content and function are limited in aging, an enhanced satellite cell pool would be beneficial to promote muscle regrowth following disuse atrophy (Fix et al., 2021) and/or muscle regeneration following injury (Sambasivan et al., 2011).
The simultaneous adaptations observed for whole-body and site-specific lean and fat tissues, as well as whole-body metabolic rate in the current study are encouraging and warrant discussion.
Lean mass is the primary determinant of metabolic rate and is the main site of glucose uptake (Hulett et al., 2022) Appropriately, the secretome product contains a host of bioactive signaling factors (Fix et al., 2021) capable of stimulating growth, metabolism, and remodeling in both muscle and adipose tissue (Hoier & Hellsten, 2014;Zhang et al., 2021).Moreover, we report the presence of extracellular vesicles containing microRNAs within the secretome product, which can have pronounced effects on skeletal muscle, immune cells, and adipose tissues (Kim et al., 2016;Sanz-Ros et al., 2022;Wu et al., 2022).Though we are not yet able to identify the specific factors driving the direct effects of secretome product treatment, the responses in skeletal muscle and adipose tissue have application to metabolic and musculoskeletal diseases and conditions.Furthermore, while it is unknown if the product directly reaches tissues secondary to the site of injection, we have identified direct and indirect treatment effects in muscle cells and adipocytes with muscle-derived IL-6 as a possible mediator for the secondary actions.Both skeletal muscle (Watanabe et al., 2022;Whitham et al., 2018) and adipose tissue (Luo & Liu, 2016;Sjøberg et al., 2017) release extracellular vesicles, myokines, and adipokines upon adequate stimulation, such as exercise.Here, we have shown that prior secretome treatment enhances the production of IL-6 in myotube culture media, which subsequently stimulates myotube growth and lipid reduction in adipocytes.In agreement, muscle-derived IL-6 can mediate adaptations including hypertrophy (Serrano et al., 2008) and lipolysis (Wolsk et al., 2010) in skeletal muscle as well as lipolysis (van Hall et al., 2003) and metabolic reprograming (Li et al., 2021) Male C57BL/6 mice (obtained from the National Institute of Aging rodent colony) began experiments at 22-24 months and finished at 23-25 months.Mice were maintained in a temperature-controlled (22-23°C) facility on a 12:12-h light/dark cycle and housed with ad libitum food and water access.After ≥1 week of acclimating to their cages, mice were assigned to either secretome treatment or saline control groups (n = 16/group) in a matched fashion (body, fat, and lean mass).After the intervention, a subset of mice (n = 6/group) underwent metabolic chamber measurements (detailed below).These same mice were utilized to examine the effects of secretome/ saline treatment and withdrawal for 2 weeks after the final injections.Most of the remaining mice (n = 8/group) were fasted for ~4 h and euthanized under isoflurane followed by cervical dislocation.Tissues (quadriceps, gastrocnemius, inguinal: I-WAT and epididymal: these data suggest that intramuscular treatment with a stem cell secretome improves whole-body metabolism, physical function, and remodels skeletal muscle and adipose tissue in aged mice.K E Y W O R D S adipogenesis, fibrosis, lipids, metabolic rate, sarcopenia, stem cells E-WAT fat pads, heart, liver) were dissected, weighed, and placed in paraformaldehyde, frozen in liquid nitrogen, or frozen in optimal cutting temperature compound (OCT) (Fisher Scientific 23-730-571, Waltham, MA, USA) in isopentane, and stored at −80°C based on the specific analysis.All animal procedures were conducted in agreement with standards set by the University of Utah Institutional Animal Care and Use Committee.

3. 2 |
Secretome treatment increased lean mass and physical function and reduced whole body fat mass in aged miceTo gain insight into the whole-body metabolic effects of the secretome treatment, we analyzed the weekly effects of the secretome treatment on whole-body tissue content via NMR spectroscopy, the impact on physical function (grip strength and rotarod performance) at baseline and at the end of the 4-week treatment, and localized effects on specific tissues (quadriceps, I-WAT, E-WAT).Weekly assessment revealed that the secretome treatment progressively increased body mass and decreased body fat percentage over baseline levels, which were driven by consistent increases in lean mass and decreases in fat mass (Figure2a-d).Conversely, aged mice treated with saline showed a decrease in body mass compared to baseline (Figure2d), likely as result of modest decrements in lean mass (Figure2a) by week 4. Group comparison after 4 weeks treatment demonstrated greater lean mass (24.8 ± 1.3 vs. 23.6 ± 1.1 g, p = 0.02), lower fat mass (2.9 ± 1.3 vs. 4.3 ± 1.4 g, p = 0.02), and lower body fat percentage (12.5 ± 3.7 vs. 8.4 ± 3.7%, p < 0.01) in secretome-treated mice relative to control mice (Figure2a-c).Assessment of strength, balance, and coordination demonstrated that (1) whole-body grip strength was increased over baseline strength at 3 and 4 weeks in secretome-treated mice;

F
Skeletal Muscle Morphology.Average quadriceps fiber cross sectional area μm 2 (a), minimum feret diameter μm 2 (b), and fiber type proportion (c; MyHC -IIa/IIb %) for control (n = 8) and secretome treated (n = 8) mice.Size distribution (%) for total (e), IIa (f) and IIb (g) fiber types across 500 μm 2 increments.Representative histochemical image of fiber type including cell border with laminin in blue, MyHC IIa in green, MyHC IIb in red, and scale bar of 50 μm (d).All data presented as mean ± SD, white circles and bars represent controls while blue squares and bars represent secretome-treated mice.n = 8 for each group.Analyzed via t-tests and mixed effects models with Holm-Bonferroni comparisons.* indicates significant difference between groups at indicated category with * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
Figure 6c).Representative images for myotubes are presented in Figure6h.Next, we performed 24 h direct secretome treatment in 3T3-L1 cells using a low (5%) and high (20%) proportion of media replacement.20% secretome replacement (but not 5% replacement) robustly decreased the amount of area occupied by lipids corrected for the area of DAPI (Figure6e; 0.24 ± 0.13 fold change, p < 0.01) compared to untreated controls.The reported effects were the same

F
Adipose Morphology and Muscle Lipid Content.Average cellular diameter μm of I-WAT (a) and E-WAT (b) depots.Size distribution (%) of I-WAT (c) and E-WAT (d) cells across 5 μm increments.Average liver lipid droplet (e) and fibrosis (f; trichrome staining) area (%) as assessed with H&E.Representative image of I-WAT and E-WAT depots as well as liver H&E and trichrome staining with scale bar 50 μm (g).Protein phosphorylation status for protein kinase B (Akt) and hormone sensitive lipase (HSL) for I-WAT and E-WAT depots and representative western blot image (h).Muscle lipid content including total triglycerides (TAGs) diglycerides (DAGs), ceramides (Cer), and C18:0 ceramide (C18:0 Cer).All data presented as mean ± SD, white circles and bars represent controls while blue squares and bars represent secretome treated groups.(a-g) n = 8 for each group, (h-i) n = 7 for control, n = 6 for secretome, (i) n = 6 for control, n = 8 for secretome.Analyzed via t-tests (a, b, e, f, h, i) or two-way ANOVA with Holm-Bonferroni comparison (c, d).* indicates significant difference between groups at indicated category with * = p < 0.05, ** = p < 0.01.product that is enriched with extracellular vesicles and matrix, immunomodulatory, and growth factors.We found that, in 4 weeks, secretome treatment increased lean mass and enhanced local muscle cellular remodeling including reductions in intramuscular lipid content.Remarkably, the secretome treatment enhanced wholebody energy expenditure, physical activity, and physical function while reducing whole-body and local adiposity.Moreover, direct secretome treatment in-vitro robustly enhanced muscle cell growth and reduced lipid droplet content.Finally, cultured media from secretome-treated muscle cells displayed autocrine paracrine functions stimulating muscle cell growth and lipid droplet reduction in naive cells.Together, 4-weeks administration of a stem cell secretome ameliorated several hallmarks of aging in mice resulting in improvements in physical function, metabolic rate, adiposity, and muscle cellular remodeling.A major finding of this current investigation was that intramuscular secretome treatment in aged mice decreased whole-body adiposity, reduced fat depot mass and adipocyte size, increased insulin sensitivity and lipolysis (Akt and HSL phosphorylation) in adipose tissues, and improved whole-body metabolic rate.As adverse body fat accumulates with age (Goodpaster et al., 2005; Wang et al., 2022), the reduction of fat mass following the secretome treatment and the maintenance of this loss after treatment cessation are impactful.Excessive adiposity with age contributes to reduced capacity to perform or adapt to physical activity (Peterson et al., 2011; Phillips et al., 2017; Pontzer et al., 2021), F I G U R E 6 Direct and Indirect Cellular Experiments.Cell culture design for media replacement and cultured media (CM) experiments (a).Average myotube area (%) (b) and myonuclear fusion index (au) (c) for control, secretome treated, control CM, and secretome CM conditions in differentiated C2C12 myotubes.Interleukin 6 (IL-6) content in the culture media collected from secretome treated (4%) and control C2C12 myotubes (Fold change) (d).Average lipid droplet area corrected to DAPI area (Fold Change) in 3T3-L1 adipocytes for control, 5 and 20% secretome product media replacement (e).Phosphorylated corrected to total Akt protein (fold change) for 20% secretome treated and control 3T3-L1 adipocytes following overnight fast and insulin (100 nM) stimulation (f).Average lipid droplet area corrected to DAPI area (Fold Change) in 3T3-L1 adipocytes for control and 20% media replacement with culture media from control and secretome treated C2C12 cells (g).Representative images of myotubes (h) and adipocytes treated with secretome (i) and culture media (j).(b-g) n = 4-7 per group or replicate.Analyzed via one-way ANOVA with Holm-Bonferroni comparison (b-c, e, g) or t-tests (d, f).* indicates significant difference between groups as indicated with * = p < 0.05, ** = p < 0.01, *** = p < 0.001.whichwas also improved following secretome treatment.Previous research demonstrated that 4-weeks of daily treadmill running modestly increased metabolic rate (VCO 2 and RER) in old mice while decreasing body fat to levels in young counterparts(Yoon et al., 2021).Accordingly, the robust increases in metabolic outcomes (VO 2 , VCO 2 , RER, activity levels) and reductions in wholebody and localized fat depots (I-WAT, E-WAT) with secretome treatment alone across a similar timeframe as exercise are very promising.
, particularly ceramide C18:0, which is linked to insulin resistance and the development of type 2 diabetes(Blachnio-Zabielska et al., 2016;Turpin-Nolan et al., 2019).In fact, reducing C18:0 ceramide content in the skeletal muscle of mice improves whole-body metabolic health(Turpin-Nolan et al., 2019).Moreover, muscle ceramides have been shown to promote muscle atrophy(De Larichaudy et al., 2012;Morigny et al., 2020) while genetic ablation of ceramides enhanced mitochondrial function and , which may partly explain the greater energy expenditure and glucose utilization (indicated by RER) following secretome treatment in aged mice.Under this premise, a subsequent decrease in fat mass (due to increased lipolysis) may be partially explained by increases in lean mass (and metabolic rate) to mobilize the energy demands of muscle tissue.It is possible that increased physical activity levels in the secretometreated mice may further accelerate this cycle.Further, the magnitude and rapidity of effects on body composition observed in the present study are notable and suggestive of direct as well as secondary effects across tissues following secretome treatment.
in adipose tissue.Accordingly, we postulate that secretome treatment may have autocrine and paracrine effects on muscle and fat tissue through the release of IL-6.However, we cannot rule out other bioactive factors that may be produced by muscle or other cell types following secretome treatment that could be contributing to the phenotype observed in adipose tissue.Future investigations including exploration of tissue-secreted factors and crosstalk following secretome treatment are justified.In summary, the results from this experiment show that 4-weeks of bi-weekly intramuscular treatment with a stem cell-derived secretome product enhanced metabolic rate while reducing whole-body and site-specific adiposity in aged mice.Moreover, the secretome treatment promoted skeletal muscle hypertrophy, cellular remodeling, and greater physical function in aged mice.Finally, secretome treatment in-vitro demonstrates both direct and indirect effects on myotube growth and adipocyte lipolysis.Together, these results suggest that as little as 4 weeks of secretome treatment ameliorated many hallmarks of aging in mice.While currently unknown, the translatability of these results to humans is a topic of interest and is under investigation in a phase 1/2a clinical trial (NCT05211986) currently underway.Considering the beneficial pre-clinical effects reported here and elsewhere, human investigations using stem cellderived secretome products are warranted.AUTH O R CO NTR I B UTI O N SExperiments and data analysis were conducted by Z.J.F, P.B., N.M.D., J.J.P., E.M.Y, and A.K. Experimental design was conducted by M.J.D. The manuscript was prepared by Z.J.F. and M.J.D.All authors contributed to editing and the approval of the final draft of the manuscript.