Gracilaria chorda attenuates obesity‐related muscle wasting through activation of SIRT1/PGC1α in skeletal muscle of mice

Abstract Gracilaria chorda (GC) is a red algal species that is primarily consumed in Asia. Here, we investigated the effect of GC on obesity‐related skeletal muscle wasting. Furthermore, elucidating its impact on the activation of sirtuin 1 (SIRT1)/peroxisome proliferator‐activated receptor gamma coactivator 1α (PGC1α) constituted a critical aspect in understanding the underlying mechanism of action. In this study, 6‐week‐old male C57BL/6 mice were fed a high‐fat diet (HFD) for 8 weeks to induce obesity, then continued on the HFD for another 8 weeks while orally administered GC. GC decreased ectopic fat accumulation in skeletal muscle and increased muscle weight, size, and function in obese mice. Furthermore, GC reduced skeletal muscle atrophy and increased hypertrophy in mice. We hypothesized that the activation of SIRT1/PGC1α by GC regulates skeletal muscle atrophy and hypertrophy. We observed that GC increased the expression of SIRT1 and PGC1α in skeletal muscle of mice and in C2C12 cells, which increased mitochondrial function and biogenesis. In addition, when C2C12 cells were treated with the SIRT1‐specific inhibitor EX‐527, no changes were observed in the protein levels of SIRT1 and PGC1α in the GC‐treated C2C12 cells. Therefore, GC attenuated obesity‐related muscle wasting by improving mitochondrial function and biogenesis through the activation of SIRT1/PGC1α in the skeletal muscle of mice.

amidst skeletal muscle tissue, as observed in aging and obesity, is associated with an accelerated progression of muscle atrophy (Zhu et al., 2019).
The accumulation of lipids in skeletal muscle leads to impaired mitochondrial function and upregulation of pro-inflammatory cytokines secretion, consequently activating cellular stress signaling and leading to subsequent muscle atrophy (Rubio-Ruiz et al., 2019).
The compromised mitochondrial function additionally influences the expression of genes implicated in oxidative phosphorylation, modulated by peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1 alpha (PGC1α) regulation.Consequently, PGC1α plays a central role as the master regulator of mitochondrial biogenesis, wherein its activity is modulated through phosphorylation by AMP-activated protein kinase (AMPK) and subsequent deacetylation by sirtuin 1 (SIRT1) (Devarshi et al., 2017).In addition, SIRT1 deacetylation of PGC1α has a suppressive effect on fat accumulation by modulating genes associated with mitochondrial and lipid metabolism (Finck & Kelly, 2006).Therefore, activation of the SIRT1-PGC1 axis may be a major regulatory protein in obesityrelated muscle wasting.
Gracilaria chorda (GC) is a red alga, a type of seaweed, known to thrive in the coastal regions of several countries, such as Korea, Japan, and Sakhalin Island of Russia.It is widely used as a food ingredient because it is rich in fiber, minerals, and vitamins and is used for agar production (Armisen, 1995).It is known to have effects such as excretion of heavy metals through the smooth activity of the intestine and suppression of cholesterol deposition in blood vessels (Hong et al., 2016).Previous studies also demonstrated that GC has anticancer, anti-inflammatory, antioxidative, antiadipogenic, insulin-sensitive, neuritogenic and neuroprotective effects (Dang et al., 2008;Faten & Emad, 2009;Hong et al., 2016;Mohibbullah et al., 2015Mohibbullah et al., , 2016Mohibbullah et al., , 2018;;Woo et al., 2013).In addition, sea grapes, which are a type of seaweed, have been shown to possess antihyperglycemic, antihypercholesterolemic, oxidative stress-reducing, metabolic disorders-addressing, and cardiometabolic syndrome-addressing effects (Kuswari et al., 2021;Manoppo et al., 2022;Nurkolis et al., 2023).However, the precise effects of GC on obesity-induced skeletal muscle wasting in skeletal mus- cle have yet to be fully elucidated.Therefore, this investigation aimed to assess whether GC protects against obesity-related muscle wasting in C57BL/6 mice.Furthermore, our study aimed to examine the influence of GC on obesity-induced mitochondrial dysfunction by assessing the activation of SIRT1/PGC1α in the skeletal muscle of mice.
The dried GC (0.5 kg) was subjected to extraction using 10 L of 50% ethanol at 80°C for a duration of 3 h.The obtained extract was subjected to filtration utilizing No. 2 filter paper (Toyo Roshi Kaisha, Tokyo, Japan) to remove solid matter.Subsequent to this, the extract was concentrated at 37°C under vacuum conditions to reduce solvent content, and freeze-drying was employed to attain a desiccated state.The extraction yield was 0.95 g/100 g GC.

| Ultra-high performance liquid chromatography (UPLC)-quadrupole time-of-flight (QTOF)-mass spectrometry (MS) analysis
To perform the sample analysis, a coupling of an Acquity UPLC system (Waters Corporation, Milford, MA, USA) with a Waters SYNAPT G2-Si mass spectrometer (Waters Corporation) was employed.Using an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm), the chromatographic separation was performed with a mobile phase containing 0.1% formic acid in water (solvent A) and acetonitrile (solvent B).The specific conditions for the optimization of the elution gradient were as reported in the previous study (Yoo, Kim, et al., 2022).
The acquired raw data were subsequently subjected to processing using the Progenesis QI software (Nonlinear Dynamics, Newcastle upon Tyne, UK).

| Animals
All animal experiments adhered to the approved guidelines set by the Institutional Animal Care and Use Committee (IACUC) of the Korea Food Research Institute (KFRI-M-19032).Five-week-old male C57BL/6 mice were kept under standardized conditions, maintaining a controlled environment with consistent ambient temperature (21-25°C) and humidity (50%-60%).A 12 h light/12 h dark cycle was maintained, and the mice had ad libitum access to both food and water.Following a 1-week acclimation period, the mice were exposed to an 8-week exposure to a high-fat diet (HFD; Research Diets, New Brunswick, NJ, USA) containing 45% of calories from fat to induce obesity.Subsequently, the HFD-induced obese mice were divided into two groups and received either a continued HFD with oral administration of distilled water (HFD, n = 8) or GC (100 mg/kg dissolved in distilled water, GC, n = 8) for an additional 8-week period.An age-matched control group of mice was kept on a chow diet (chow, n = 8) for the same duration.The composition of the experimental diets is shown in Supplementary Table S1.During the experimental period, weekly evaluations of body weight and food intake were carried out.At the end of the 8-week intervention, the mice were euthanized, and blood samples were collected, followed by the immediate excision and measurement of adipose and muscle tissues.

| Treadmill exercise and grip strength assessments
Prior to the running test, all mice underwent a treadmill acclimation process using a treadmill (Ugo Basile, Gemonio, ltaly) for a duration of 2 days.Detailed experimental conditions were implemented with reference to the previous study (Yoo, Ahn, et al., 2022).Grip strength measurements were obtained using a grip strength meter (model GS3,Bioseb,Vitrolles,France).The recorded values were standardized based on body weight, and the average value was determined, with exclusion of the maximum and minimum values.

| Hematoxylin and eosin (H&E) staining
Following dissection, the gastrocnemius muscle was immediately fixed in 4% formaldehyde and subsequently embedded in paraffin to obtain 4 μm thick sections.H&E staining was then carried out on the gastrocnemius sections.Microscopic images were obtained using the Olympus BX51 microscope, and the cross-sectional area (CSA) was measured using IMT iSolution DT 9.2 software.

| Cell culture
C2C12 myoblast cells (ATCC, Manassas, VA, USA) were cultured in DMEM containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin.The cells were maintained in a humidified environment at 37°C with 5% CO 2 .To induce differentiation, C2C12 cells were seeded at a density of 2 × 10 5 cells per well in 6-well plates.Two days later, approximately 100% confluent C2C12 cells were transferred to DMEM supplemented with 2% horse serum, 100 U/mL penicillin, and 100 μg/mL streptomycin.On the initiation of differentiation (day 0), the cells were subjected to treatment either with or without the SIRT1-specific inhibitor, 10 μM EX-527 (Sigma-Aldrich, St. Louis, MO, USA) for a period of 1 day, followed by treatment with GC and/or EX-527 for an additional 2 days.

| Western blot analysis
Protein extraction from the quadriceps muscle and C2C12 cells was performed using RIPA buffer.The protein concentration of 20 μg in the supernatant was determined using a Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) with bovine serum albumin as the standard, following established procedures (He, 2011).Next, total protein was separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA).
The membranes were subjected to blocking using 5% skimmed milk and 0.1% Tween 20 (Junsei, Tokyo, Japan) in Tris-buffered saline for a duration of 1 h at 25°C.Primary antibodies were allowed to incubate overnight at 4°C with the membranes, followed by thorough washing and subsequent 1 h incubation at 25°C with a horseradish peroxidase-conjugated secondary antibody.Immunodetection was performed utilizing an ECL detection reagent (Bio-Rad), and the subsequent quantification of band density was carried out through analysis with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

| Quantitative real-time PCR
The extraction of total RNA from the quadriceps muscle was achieved through the utilization of the Qiagen RNeasy Fibrous Tissue Mini Kit (Qiagen Inc., Hilden, Germany).Following RNA extraction (30 ng/μL), the synthesis of cDNA was performed using the ReverTra Ace® quantitative RT-PCR master kit (Toyobo Co., Ltd., Osaka, Japan).Quantitative PCR was conducted using SYBR Green real-time PCR Master Mix (Toyobo Co., Ltd.) along with the ViiA7 PCR system (Applied Biosystems, Foster City, CA, USA).The amplification reaction, comprising 20 μL, included cDNA as the template.
The amplification protocol commenced with an initial step at 95°C for 5 min, succeeded by 40 amplification cycles of 95°C for 5 s, 55°C for 10 s, and 72°C for 15 sec.

| Statistical analysis
The data are reported as the mean ± SEM.Statistical analyses were performed using GraphPad Prism software, version 8.3.1 (San Diego, CA, USA).To assess differences between two groups, an unpaired t-test was employed.For comparisons involving more than two groups, one-way analysis of variance (ANOVA) was utilized, followed by Dunnett's multiple comparison test.

| GC decreased ectopic fat accumulation in skeletal muscle and increased skeletal muscle weight, size, grip strength, and treadmill running in HFD-fed mice
To evaluate the impact of GC on obesity-associated muscle wasting, we initially recorded body weight and quantified epididymal fat mass.In addition, the TG content within skeletal muscle was measured.HFD-fed mice showed elevated body (p < .001)and epididymal fat (p < .001)weights in comparison to those fed a chow diet.In the GC group, there was no change in body weight (Figure 1a); however, it was observed that the GC group had decreased epididymal fat weights (p < .05)(Figure 1b) compared with those of the HFD group.Moreover, the GC group exhibited a notable decrease in HFD-induced TG accumulation within the skeletal muscle (p < .05)(Figure 1c).In comparison to the chow-fed group, the HFD group exhibited decreased muscle weight (TA, p < .05,Quad, p < .05),while the GC group showed elevated skeletal muscle weight relative to the HFD-fed mice (TA, p < .001,EDL, p < .05,Gastroc, p < .05,Quad, p < .05,Triceps, p < .01)(Figure 1d).Furthermore, the HFD-feeding resulted in a reduction in the CSA of skeletal muscle fibers compared to that observed in chow-fed mice, whereas GC administration led to an augmentation of muscle fiber size relative to the HFD group (Figure 1e).These findings suggest that GC mitigated obesity-induced fat accumulation in obese mice, leading to an increase in both skeletal muscle weight and size.Furthermore, grip strength and treadmill running were assessed to investigate muscle function.The HFD group showed decreased grip strength (p < .001),running distance (p < .001),and time to exhaustion (p < .001)compared with those of the chow group.However, GC significantly enhanced grip strength (p < .01),running distance (p < .01),and time to exhaustion (p < .01) in a noteworthy manner (Figure 1f).These findings suggest that GC administration led to improved skeletal muscle performance, as evidenced by enhanced grip strength and treadmill running capacity in mice.(p < .01)(Figure 2b).Phosphorylation of smad2/3 in skeletal muscle mediates the activation of skeletal muscle atrophy through the ubiquitin-proteasome system (UPS) (Sartori et al., 2014).We observed increased protein levels of phosphorylated Smad2 (p < .05)and Smad3 (p < .05) in HFD-fed mice.However, in the GC group, phosphor-Smad2 and Smad3 (p < .05)expression decreased while the total Smad2 and Smad3 protein expression remained unaffected compared to the HFD group (Figure 2c).Taken together, these findings suggest that GC ameliorates obesity-induced inflammation and skeletal muscle atrophy through its regulatory effect on Smad 2/3 in mice.

| GC ameliorated mitochondrial biogenesis and function via activation of SIRT1/PGC1α, in vivo and in vitro
To examine the impact of GC on mitochondrial biogenesis and function in obese mice, we evaluated the expression of PGC1α, a central regulator of mitochondrial biogenesis and respiratory activity, as well as proteins under the control of PGC1α.Activation of Ca 2+ /calmodulin-dependent protein kinase (CaMkk) α, β, SIRT1, and AMPK increases PGC1α expression in skeletal muscles (Iwabu et al., 2010).As depicted in Figure 4a, the HFD group exhibited  4d).To investigate whether GC increases mitochondrial biogenesis and function via SIRT1, we treated C2C12 cells with or without the SIRT1-specific inhibitor 10 μM EX-527 on day 0 of differentiation for 1 d, and subsequently with GC and/or EX-527 for 2 days.In the absence of EX527 treatment, GC led to heightened protein levels of SIRT1 and PGC1α; however, no change was noted in EX527-treated C2C12 cells (Figure 4e).

| Twenty-three tentative compounds were identified in GC
The composition of GC was determined by conducting nontargeted analysis using UPLC-QTOF MS to identify compounds in the GC extract.As shown in Figure 5, 23 tentative compounds are based on accurate mass, retention time, and comparison of MS/ MS fragments with standard compounds.In the negative mode, we confirmed the presence of diphlorethol/difucol, dalbergin, r-linolenic acid, linoleic acid, acacetin, luteolin, diosmetin, eicosapentaenoic acid (EPA), cinnamoyl glucose, carnosic acid, hydroxyeckol, and fucofuroeckol A. In the positive mode, we confirmed the presence of phloroglucinol, 4-hydroxybenzoic acid-glucoside, alginate oligosaccharides, eckol, dimethylmatairesinol, fucosterol, deoxyschisandrin, hydroxyfucosterol, alginate oligosaccharides, β-carotene, and fucoxanthin.

| DISCUSS ION
In the present study, GC increased skeletal muscle weight while re- and obesity-induced increases in pro-inflammatory cytokines and lipid metabolites lead to an imbalance between muscle hypertrophy and atrophy (Kalinkovich & Livshits, 2017).Therefore, these findings propose that the impact of GC on obesity-related muscle wasting is due to a decrease in lipid accumulation in skeletal muscle, especially intramuscular lipids.In addition, it has been reported that accumulated muscular lipids induce a muscle lipotoxic effect, reducing muscle strength and power in the elderly (Tumova et al., 2016).As we observed that GC improved skeletal muscle performance, it is expected to be due to a decrease in intramuscular lipids.
In skeletal muscle, the interplay between muscle hypertrophy and atrophy is primarily controlled by the insulin-like growth factor 1 (IGF-1)-AKT-mTOR signaling pathway (Glass, 2010), which exerts a favorable influence on muscle growth, and the myostatin-Smad2/3 pathway, which serves as a negative regulator (Schiaffino et al., 2013).GC supplementation reduced the increase in pro-inflammatory cytokines and ameliorated muscle atrophy by regulating Smad2/3, and activated the inhibited AKT signaling, which was suppressed by obesity.It has been reported that obesity suppresses IGF-1/AKT signaling owing to increased pro-inflammatory cytokines and toxic lipid metabolites and increases muscle atrophy by allowing phosphorylated Smads to enter the nucleus and regulate MuRF1 and Atrogin1 transcription and active UPS (Glass, 2010;Ji et al., 2022;Mahfouz et al., 2014).Our results suggested that GC improves obesity-related muscle wasting by activating AKT signaling and suppressing Smad2/3 signaling.
Moreover, GC supplementation significantly increased the expression of SIRT1 and PGC1α, both in vivo and in vitro.SIRT1, an NAD + -dependent protein deacetylase, plays a role in the posttranslational modification of PGC1α through deacetylation.Activated PGC1α, through SIRT1, increases mitochondrial function and biogenesis as crucial genes governing mitochondrial DNA replication, oxidative phosphorylation proteins, and fatty acid oxidation are activated (e.g., PPARδ, NRF1, NRF2, TFAM, ERRα) (Finck & Kelly, 2006;Kelly & Scarpulla, 2004).Next, to investigate whether the increased expression of PGC1α by GC supplementation was mediated by SIRT1, we treated C2C12 cells with GC in the presence of the SIRT1-specific inhibitor EX-527.We observed no changes in SIRT1 and PGC1α protein levels in GC-treated C2C12 cells.These results suggested that SIRT1 mediates the advantageous effects of GC in increasing the expression of genes related to mitochondrial function and biogenesis.In addition, SIRT1 can control muscle atrophy, as SIRT1-regulated factors contribute to inducing atrophy, and PGC1α can block this process (Sandri et al., 2006) and contribute to muscle growth by increasing AKT (Koltai et al., 2017).This suggested that the activation of SIRT1/PGC1α by GC in skeletal muscle affected mitochondrial function, biogenesis, and skeletal hypertrophy and atrophy.Activation of PGC1α has been shown to induce muscle fiber-type transformation in type 1 fibers (Lin et al., 2002).This is because type 1 muscle fibers have a dark red color, a higher mitochondrial density, and more oxidative enzymes than type 2 fibers (Scott et al., 2001).
Although the present study did not conduct muscle fiber-type conversion, it was observed that MHC1 was reduced in HFD-fed mice.
GC increased MHC1 expression.However, it was observed that MHC1, total MHC, MHC2A, and 2B were decreased in HFD-fed mice.This appears to be due to the reduction in skeletal muscle weight and size, and further studies are required to measure muscle fiber-type conversion.
We identified 23 tentative compounds in GC.Among them, linoleic acid, luteolin, EPA, deoxyschisandrin, β-carotene, and fucoxanthin have been reported to prevent muscle atrophy in a muscle atrophy model (Hedya et al., 2019;Lee et al., 2022;Ogawa et al., 2013;Yeon et al., 2020;Yoshikawa et al., 2021) Therefore, several bioactive compounds in GC may exert synergistic effects against obesity-related muscle wasting.As the bioactive compounds of GC are unknown, future studies are needed to identify the physiologically active compounds of GC through component analysis and to investigate whether these compounds affect obesity-related muscle wasting.
In conclusion, our findings indicate that GC attenuates obesityrelated muscle wasting by improving mitochondrial function and biogenesis via the activation of SIRT1/PGC1α in the skeletal muscle of mice.Taken together, these findings suggested that GC may be a useful food resource for both preventing and treating obese sarcopenia.
ducing epididymal fat weight and TG content in skeletal muscle, without a change in body weight in obese mice.Obesity causes increased body fat mass and ectopic fat accumulation in the skeletal muscle, F I G U R E 4 Effect of GC on mitochondrial biogenesis and function through activation of SIRT1/PGC1α in vivo and in vitro.(a) The protein levels of CaMkkα, β, SIRT1, p-AMPK, AMPK, and GAPDH in the quadriceps muscle were assessed using western blotting.(b) qRT-PCR was employed to quantify the mRNA expression levels of Nampt, SIRT1, PGC1α, NRF1, and TFAM in the quadriceps muscle.(c) Western blotting was utilized to measure the protein levels of PGC1α, PPARδ, NRF1, NRF2, ERRα, γ, CytoC, and GAPDH in the quadriceps muscle.(d) Western blotting was performed to determine the protein levels of SIRT1, p-AMPK, AMPK, PGC1α, PPARδ, NRF1, NRF2, ERRα, γ, and β-Actin in C2C12 cells.(e) Western blotting was used to examine the protein levels of SIRT1 and PGC1α in C2C12 cells treated with GC and/or EX527.Results are expressed as mean ± SEM. *p < .05,**p < .01,***p < .001versus the Chow group or the control C2C12 cells.#p < .05,##p < .01,###p < .001versus the HFD-fed group.

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I G U R E 5 UPLC-QTOF-MS/MS analysis of GC extracts.Total ion chromatograms of GC extract in (a) negative mode and (b) positive mode by UPLC-QTOF-MS/MS.| 5085 YOO et al.