Glucose, glutamine, lactic acid and &alpha;‑ketoglutarate restore metabolic disturbances and atrophic changes&nbsp;<br />in energy‑deprived muscle cells

Ikeda,Miu; Matsumoto,Moe; Tamura,Miki; Kobayashi,Masaki; Iida,Kaoruko

doi:10.3892/mmr.2025.13562

Glucose, glutamine, lactic acid and α‑ketoglutarate restore metabolic disturbances and atrophic changes
in energy‑deprived muscle cells

Authors:
- Miu Ikeda
- Moe Matsumoto
- Miki Tamura
- Masaki Kobayashi
- Kaoruko Iida
View Affiliations

Affiliations: Department of Food and Nutritional Sciences, Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo 1128610, Japan
Published online on: May 12, 2025 https://doi.org/10.3892/mmr.2025.13562
Article Number: 197
Copyright: © Ikeda et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Skeletal muscle atrophy is often triggered by catabolic conditions such as fasting, malnutrition and chronic diseases; however, the efficacy of nutritional supplementation in maintaining muscle mass and preventing muscle atrophy remains controversial. The present study aimed to compare the inhibitory effects of various nutritional substrates on starvation‑induced catabolic changes and muscle cell atrophy. C2C12 muscle cells were starved for up to 24 h in medium lacking serum and main nutrients (glucose, glutamine and pyruvate). To assess the effects of exogenous substrates, the cells were incubated in starvation medium and individually supplemented with each of the following nutrients: Glucose, amino acids, fatty acids, lactate or ketone bodies. The expression of each gene and protein was analyzed by reverse transcription‑quantitative PCR and western blotting, respectively. Mitochondrial activity was determined by MTT assay and cell morphology was observed by immunofluorescence staining. The results revealed that starvation for >3 h suppressed mitochondrial activity, and after 5 h of starvation, the expression levels of several metabolic genes were increased; however, the levels of most, with the exception of Scot and Cpt‑1b, were suppressed after 24 h. Protein degradation and a decrease in protein synthesis were observed after 5 h of starvation, followed by autophagy with morphological atrophy at 24 h. Supplementation with specific substrates, with the exception of leucine, such as glucose, glutamine, lactic acid or α‑ketoglutarate, attenuated the suppression of mitochondrial activity, and altered gene expression, protein degradation and myotube atrophy in starved myotubes. Furthermore, the decrease in intracellular ATP production after 24 h of starvation was reversed by restoring glycolysis in glucose‑treated cells, and via an increase in mitochondrial respiration in cells treated with glutamine, lactic acid or α‑ketoglutarate. In conclusion, increasing the availability of glucose, glutamine, lactic acid or α‑ketoglutarate may be beneficial for countering muscle atrophy associated with inadequate nutrient intake.

View Figures

View References

1	Frontera WR and Ochala J: Skeletal muscle: A brief review of structure and function. Calcif Tissue Int. 96:183–195. 2015. View Article : Google Scholar : PubMed/NCBI
2	Sieber CC: Malnutrition and sarcopenia. Aging Clin Exp Res. 31:793–798. 2019. View Article : Google Scholar : PubMed/NCBI
3	Yuan S and Larsson SC: Epidemiology of sarcopenia: Prevalence, risk factors, and consequences. Metabolism. 144:1555332023. View Article : Google Scholar : PubMed/NCBI
4	Owens DJ: Nutritional support to counteract muscle atrophy. Adv Exp Med Biol. 1088:483–495. 2018. View Article : Google Scholar : PubMed/NCBI
5	Wang Y, Liu Q, Quan H, Kang SG, Huang K and Tong T: Nutraceuticals in the prevention and treatment of the muscle atrophy. Nutrients. 13:19142021. View Article : Google Scholar : PubMed/NCBI
6	Hoffman EP and Nader GA: Balancing muscle hypertrophy and atrophy. Nat Med. 10:584–585. 2004. View Article : Google Scholar : PubMed/NCBI
7	Sartori R, Romanello V and Sandri M: Mechanisms of muscle atrophy and hypertrophy: Implications in health and disease. Nat Commun. 12:3302021. View Article : Google Scholar : PubMed/NCBI
8	Carbone JW, Margolis LM, McClung JP, Cao JJ, Murphy NE, Sauter ER, Combs GF Jr, Young AJ and Pasiakos SM: Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis. FASEB J. 27:5104–5111. 2013. View Article : Google Scholar : PubMed/NCBI
9	Pasiakos SM, Margolis LM and Orr JS: Optimized dietary strategies to protect skeletal muscle mass during periods of unavoidable energy deficit. FASEB J. 29:1136–1142. 2015. View Article : Google Scholar : PubMed/NCBI
10	Gielen E, Beckwée D, Delaere A, De Breucker S, Vandewoude M and Bautmans I; Sarcopenia Guidelines Development Group of the Belgian Society of Gerontology Geriatrics (BSGG), : Nutritional interventions to improve muscle mass, muscle strength, and physical performance in older people: An umbrella review of systematic reviews and meta-analyses. Nutr Rev. 79:121–147. 2021. View Article : Google Scholar : PubMed/NCBI
11	Bertero E and Maack C: Metabolic remodelling in heart failure. Nat Rev Cardiol. 15:457–470. 2018. View Article : Google Scholar : PubMed/NCBI
12	Lopaschuk GD, Karwi QG, Tian R, Wende AR and Abel ED: Cardiac energy metabolism in heart failure. Circ Res. 128:1487–1513. 2021. View Article : Google Scholar : PubMed/NCBI
13	Olson B, Marks DL and Grossberg AJ: Diverging metabolic programmes and behaviours during states of starvation, protein malnutrition, and cachexia. J Cachexia Sarcopenia Muscle. 11:1429–1446. 2020. View Article : Google Scholar : PubMed/NCBI
14	Mengeste AM, Rustan AC and Lund J: Skeletal muscle energy metabolism in obesity. Obesity (Silver Spring). 29:1582–1595. 2021. View Article : Google Scholar : PubMed/NCBI
15	Boya P, Reggiori F and Codogno P: Emerging regulation and functions of autophagy. Nat Cell Biol. 15:713–720. 2013. View Article : Google Scholar : PubMed/NCBI
16	Deshmukh AS, Murgia M, Nagaraj N, Treebak JT, Cox J and Mann M: Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors. Mol Cell Proteomics. 14:841–853. 2015. View Article : Google Scholar : PubMed/NCBI
17	Pavlovic K, Krako Jakovljevic N, Isakovic AM, Ivanovic T, Markovic I and Lalic NM: Therapeutic vs suprapharmacological metformin concentrations: Different effects on energy metabolism and mitochondrial function in skeletal muscle cells in vitro. Front Pharmacol. 13:9303082022. View Article : Google Scholar : PubMed/NCBI
18	Akhtar J, Han Y, Han S, Lin W, Cao C, Ge R, Babarinde IA, Jia Q, Yuan Y, Chen G, et al: Bistable insulin response: The win-win solution for glycemic control. iScience. 25:1055612022. View Article : Google Scholar : PubMed/NCBI
19	Yang B, Liu Y and Steinacker JM: α-Ketoglutarate stimulates cell growth through the improvement of glucose and glutamine metabolism in C2C12 cell culture. Front Nutr. 10:11452362023. View Article : Google Scholar : PubMed/NCBI
20	Desgeorges MM, Freyssenet D, Chanon S, Castells J, Pugnière P, Béchet D, Peinnequin A, Devillard X and Defour A: Post-transcriptional regulation of autophagy in C2C12 myotubes following starvation and nutrient restoration. Int J Biochem Cell Biol. 54:208–216. 2014. View Article : Google Scholar : PubMed/NCBI
21	Li F, Li X, Peng X, Sun L, Jia S, Wang P, Ma S, Zhao H, Yu Q and Huo H: Ginsenoside Rg1 prevents starvation-induced muscle protein degradation via regulation of AKT/mTOR/FoxO signaling in C2C12 myotubes. Exp Ther Med. 14:1241–1247. 2017. View Article : Google Scholar : PubMed/NCBI
22	Matsuba I, Fujita R and Iida K: Palmitic acid inhibits myogenic activity and expression of myosin heavy chain MHC IIb in muscle cells through phosphorylation-dependent MyoD inactivation. Int J Mol Sci. 24:58472023. View Article : Google Scholar : PubMed/NCBI
23	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
24	Musser DA and Oseroff AR: The use of tetrazolium salts to determine sites of damage to the mitochondrial electron transport chain in intact cells following in vitro photodynamic therapy with photofrin II. Photochem Photobiol. 59:621–626. 1994. View Article : Google Scholar : PubMed/NCBI
25	Dice JF: Selective degradation of cytosolic proteins by lysosomes. Ann N Y Acad Sci. 674:58–64. 1992. View Article : Google Scholar : PubMed/NCBI
26	Dice JF: Molecular determinants of protein half-lives in eukaryotic cells. FASEB J. 1:349–357. 1987. View Article : Google Scholar : PubMed/NCBI
27	Duan Y, Li F, Guo Q, Wang W, Zhang L, Wen C, Chen X and Yin Y: β-Hydroxy-β-methyl butyrate is more potent than leucine in inhibiting starvation-induced protein degradation in C2C12 myotubes. J Agric Food Chem. 66:170–176. 2018. View Article : Google Scholar : PubMed/NCBI
28	Caldow MK, Ham DJ, Trieu J, Chung JD, Lynch GS and Koopman R: Glycine protects muscle cells from wasting in vitro via mTORC1 signaling. Front Nutr. 6:1722019. View Article : Google Scholar : PubMed/NCBI
29	Zhang H, Wang F, Pang X, Zhou Y, Li S, Li W, Zhang P and Chen X: Decreased expression of H19/miR-675 ameliorates muscle atrophy by regulating the IGF1R/Akt/FoxO signaling pathway. Mol Med. 29:782023. View Article : Google Scholar : PubMed/NCBI
30	Nakai N, Kitai S, Iida N, Inoue S and Higashida K: Autophagy under glucose starvation enhances protein translation initiation in response to re-addition of glucose in C2C12 myotubes. FEBS Open Bio. 10:2149–2156. 2020. View Article : Google Scholar : PubMed/NCBI
31	Zeidler JD, Fernandes-Siqueira LO, Carvalho AS, Cararo-Lopes E, Dias MH, Ketzer LA, Galina A and Da Poian AT: Short-term starvation is a strategy to unravel the cellular capacity of oxidizing specific exogenous/endogenous substrates in mitochondria. J Biol Chem. 292:14176–14187. 2017. View Article : Google Scholar : PubMed/NCBI
32	de Lange P, Moreno M, Silvestri E, Lombardi A, Goglia F and Lanni A: Fuel economy in food-deprived skeletal muscle: Signaling pathways and regulatory mechanisms. FASEB J. 21:3431–3441. 2007. View Article : Google Scholar : PubMed/NCBI
33	Samec S, Seydoux J, Russell AP, Montani JP and Dulloo AG: Skeletal muscle heterogeneity in fasting-induced upregulation of genes encoding UCP2, UCP3, PPARgamma and key enzymes of lipid oxidation. Pflugers Arch. 445:80–86. 2002. View Article : Google Scholar : PubMed/NCBI
34	Frier BC, Jacobs RL and Wright DC: Interactions between the consumption of a high-fat diet and fasting in the regulation of fatty acid oxidation enzyme gene expression: An evaluation of potential mechanisms. Am J Physiol Regul Integr Comp Physiol. 300:R212–R221. 2011. View Article : Google Scholar : PubMed/NCBI
35	Saneyasu T, Kimura S, Kitashiro A, Tsuchii N, Tsuchihashi T, Inui M, Honda K and Kamisoyama H: Differential regulation of the expression of lipid metabolism-related genes with skeletal muscle type in growing chickens. Comp Biochem Physiol B Biochem Mol Biol. 189:1–5. 2015. View Article : Google Scholar : PubMed/NCBI
36	Jagoe RT, Lecker SH, Gomes M and Goldberg AL: Patterns of gene expression in atrophying skeletal muscles: Response to food deprivation. FASEB J. 16:1697–1712. 2002. View Article : Google Scholar : PubMed/NCBI
37	Lecker SH, Jagoe RT, Gilbert A, Gomes M, Baracos V, Bailey J, Price SR, Mitch WE and Goldberg AL: Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression. FASEB J. 18:39–51. 2004. View Article : Google Scholar : PubMed/NCBI
38	Harvald EB, Sprenger RR, Dall KB, Ejsing CS, Nielsen R, Mandrup S, Murillo AB, Larance M, Gartner A, Lamond AI and Færgeman NJ: Multi-omics analyses of starvation responses reveal a central role for lipoprotein metabolism in acute starvation survival in C. elegans. Cell Syst. 5:38–52.e4. 2017. View Article : Google Scholar : PubMed/NCBI
39	Dall KB, Havelund JF, Harvald EB, Witting M and Faergeman NJ: HLH-30-dependent rewiring of metabolism during starvation in C. elegans. Aging Cell. 20:e133422021. View Article : Google Scholar : PubMed/NCBI
40	Skiba-Cassy S, Collin A, Chartrin P, Médale F, Simon J, Duclos MJ and Tesseraud S: Chicken liver and muscle carnitine palmitoyltransferase 1: Nutritional regulation of messengers. Comp Biochem Physiol B Biochem Mol Biol. 147:278–287. 2007. View Article : Google Scholar : PubMed/NCBI
41	Benjamin DI, Both P, Benjamin JS, Nutter CW, Tan JH, Kang J, Machado LA, Klein JDD, de Morree A, Kim S, et al: Fasting induces a highly resilient deep quiescent state in muscle stem cells via ketone body signaling. Cell Metab. 34:902–918.e6. 2022. View Article : Google Scholar : PubMed/NCBI
42	Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, Turk BE and Shaw RJ: AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell. 30:214–226. 2008. View Article : Google Scholar : PubMed/NCBI
43	Tavares MR, Pavan IC, Amaral CL, Meneguello L, Luchessi AD and Simabuco FM: The S6K protein family in health and disease. Life Sci. 131:1–10. 2015. View Article : Google Scholar : PubMed/NCBI
44	Diaz-Troya S, Pérez-Pérez ME, Florencio FJ and Crespo JL: The role of TOR in autophagy regulation from yeast to plants and mammals. Autophagy. 4:851–865. 2008. View Article : Google Scholar : PubMed/NCBI
45	Mugume Y, Kazibwe Z and Bassham DC: Target of rapamycin in control of autophagy: Puppet master and signal integrator. Int J Mol Sci. 21:82592020. View Article : Google Scholar : PubMed/NCBI
46	Durán RV, Oppliger W, Robitaille AM, Heiserich L, Skendaj R, Gottlieb E and Hall MN: Glutaminolysis activates Rag-mTORC1 signaling. Mol Cell. 47:349–358. 2012. View Article : Google Scholar : PubMed/NCBI
47	Dodd KM and Tee AR: Leucine and mTORC1: A complex relationship. Am J Physiol Endocrinol Metab. 302:E1329–E1342. 2012. View Article : Google Scholar : PubMed/NCBI
48	Han JM, Jeong SJ, Park MC, Kim G, Kwon NH, Kim HK, Ha SH, Ryu SH and Kim S: Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1-signaling pathway. Cell. 149:410–424. 2012. View Article : Google Scholar : PubMed/NCBI
49	Leenders M and van Loon LJ: Leucine as a pharmaconutrient to prevent and treat sarcopenia and type 2 diabetes. Nutr Rev. 69:675–689. 2011. View Article : Google Scholar : PubMed/NCBI
50	Ham DJ, Caldow MK, Lynch GS and Koopman R: Leucine as a treatment for muscle wasting: A critical review. Clin Nutr. 33:937–945. 2014. View Article : Google Scholar : PubMed/NCBI
51	Mobley CB, Fox CD, Ferguson BS, Amin RH, Dalbo VJ, Baier S, Rathmacher JA, Wilson JM and Roberts MD: L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy. J Int Soc Sports Nutr. 11:382014. View Article : Google Scholar : PubMed/NCBI
52	Oelkrug C, Horn K, Makert GR and Schubert A: Novel in vitro platform to investigate myotube atrophy. Anticancer Res. 35:2085–2091. 2015.PubMed/NCBI
53	Wang L, Yi D, Hou Y, Ding B, Li K, Li B, Zhu H, Liu Y and Wu G: Dietary supplementation with α-ketoglutarate activates mTOR signaling and enhances energy status in skeletal muscle of lipopolysaccharide-challenged piglets. J Nutr. 146:1514–1520. 2016. View Article : Google Scholar : PubMed/NCBI
54	Cai X, Yuan Y, Liao Z, Xing K, Zhu C, Xu Y, Yu L, Wang L, Wang S, Zhu X, et al: α-Ketoglutarate prevents skeletal muscle protein degradation and muscle atrophy through PHD3/ADRB2 pathway. FASEB J. 32:488–499. 2018. View Article : Google Scholar : PubMed/NCBI
55	Tsukamoto S, Shibasaki A, Naka A, Saito H and Iida K: Lactate promotes myoblast differentiation and myotube hypertrophy via a pathway involving MyoD in vitro and enhances muscle regeneration in vivo. Int J Mol Sci. 19:36492018. View Article : Google Scholar : PubMed/NCBI
56	Ohno Y, Oyama A, Kaneko H, Egawa T, Yokoyama S, Sugiura T, Ohira Y, Yoshioka T and Goto K: Lactate increases myotube diameter via activation of MEK/ERK pathway in C2C12 cells. Acta Physiol (Oxf). 223:e130422018. View Article : Google Scholar : PubMed/NCBI
57	Ohno Y, Nakatani M, Ito T, Matsui Y, Ando K, Suda Y, Ohashi K, Yokoyama S and Goto K: Activation of lactate receptor positively regulates skeletal muscle mass in mice. Physiol Res. 72:465–473. 2023. View Article : Google Scholar : PubMed/NCBI
58	Goodpaster BH and Sparks LM: Metabolic flexibility in health and disease. Cell Metab. 25:1027–1036. 2017. View Article : Google Scholar : PubMed/NCBI
59	Hui S, Ghergurovich JM, Morscher RJ, Jang C, Teng X, Lu W, Esparza LA, Reya T, Zhan L, Yanxiang Guo J, et al: Glucose feeds the TCA cycle via circulating lactate. Nature. 551:115–118. 2017. View Article : Google Scholar : PubMed/NCBI
60	Li M, Wang Y, Wei X, Cai WF, Wu J, Zhu M, Wang Y, Liu YH, Xiong J, Qu Q, et al: AMPK targets PDZD8 to trigger carbon source shift from glucose to glutamine. Cell Res. 34:683–706. 2024. View Article : Google Scholar : PubMed/NCBI