{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T05:03:43Z","timestamp":1784178223549,"version":"3.55.0"},"reference-count":27,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2026,7,10]],"date-time":"2026-07-10T00:00:00Z","timestamp":1783641600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>\u03b2-hydroxy-\u03b2-methylbutyrate (HMB), a bioactive metabolite of leucine, is widely recognized for its anabolic and anti-catabolic effects in skeletal muscle. However, the molecular mechanisms underlying these effects, particularly in relation to circadian regulation, remain incompletely understood. Here, we investigated the impact of HMB on dexamethasone-induced muscle atrophy in C2C12 myotubes, with a focus on anabolic signaling and circadian clock regulation. C2C12 myotubes were treated with HMB or HMB after dexamethasone-induced atrophy. HMB treatment significantly improved cell viability, surface area and fiber diameter by reducing expression of CBL-B, MuRF1 and Atrogin1, key mediators of muscle proteolysis, and increasing myogenin expression compared with atrophic conditions. While HMB did not activate AKT or mTOR, it robustly increased phosphorylation of P70S6K and S6 through a phospholipase D (PLD)-dependent mechanism. HMB restored disrupted circadian clock gene expression induced by dexamethasone, including normalization of expression patterns. HMB also enhanced circadian rhythmic amplitude and advanced phase timing, indicating improved clock robustness. These findings identify circadian regulation as a novel target of HMB action and demonstrate that HMB preserves muscle homeostasis through coordinated modulation of anabolic signaling and intrinsic circadian machinery. This study provides mechanistic insight into how HMB protects against muscle atrophy and highlights circadian regulation as an important contributor to skeletal muscle health.<\/jats:p>","DOI":"10.3390\/ijms27146189","type":"journal-article","created":{"date-parts":[[2026,7,10]],"date-time":"2026-07-10T15:13:36Z","timestamp":1783696416000},"page":"6189","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["\u03b2-Hydroxy-\u03b2-methylbutyrate (HMB) Counteracts Atrophy and Restores Circadian Rhythms in Myotubes"],"prefix":"10.3390","volume":"27","author":[{"given":"Meytal","family":"Cohen-Or","sequence":"first","affiliation":[{"name":"Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nava","family":"Chapnik","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natalie","family":"Avital-Cohen","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9061-3780","authenticated-orcid":false,"given":"Oren","family":"Froy","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1186\/s12576-020-00768-9","article-title":"The ubiquitin-proteasome system in regulation of the skeletal muscle homeostasis and atrophy: From basic science to disorders","volume":"70","author":"Kitajima","year":"2020","journal-title":"J. Physiol. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4798","DOI":"10.1128\/MCB.01347-08","article-title":"Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor 1 signaling during muscle atrophy caused by unloading","volume":"29","author":"Nakao","year":"2009","journal-title":"Mol. Cell. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"4294","DOI":"10.1111\/febs.12253","article-title":"Mechanisms regulating skeletal muscle growth and atrophy","volume":"280","author":"Schiaffino","year":"2013","journal-title":"FEBS J."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2911","DOI":"10.1113\/jphysiol.2013.253203","article-title":"Effects of leucine and its metabolite beta-hydroxy-beta-methylbutyrate on human skeletal muscle protein metabolism","volume":"591","author":"Wilkinson","year":"2013","journal-title":"J. Physiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"973","DOI":"10.1016\/j.exger.2013.06.005","article-title":"beta-Hydroxy-beta-methylbutyrate (HMB) enhances the proliferation of satellite cells in fast muscles of aged rats during recovery from disuse atrophy","volume":"48","author":"Alway","year":"2013","journal-title":"Exp. Gerontol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"4741","DOI":"10.1073\/pnas.0600678103","article-title":"The role of phospholipase D and phosphatidic acid in the mechanical activation of mTOR signaling in skeletal muscle","volume":"103","author":"Hornberger","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1177\/0748730414561638","article-title":"Circadian rhythms, the molecular clock, and skeletal muscle","volume":"30","author":"Harfmann","year":"2015","journal-title":"J. Biol. Rhythm."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/j.molmet.2013.10.005","article-title":"Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock","volume":"3","author":"Dyar","year":"2014","journal-title":"Mol. Metab."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"19090","DOI":"10.1073\/pnas.1014523107","article-title":"CLOCK and BMAL1 regulate MyoD and are necessary for maintenance of skeletal muscle phenotype and function","volume":"107","author":"Andrews","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1186\/s13395-015-0039-5","article-title":"The endogenous molecular clock orchestrates the temporal separation of substrate metabolism in skeletal muscle","volume":"5","author":"Hodge","year":"2015","journal-title":"Skelet. Muscle"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"101155","DOI":"10.1016\/j.arr.2020.101155","article-title":"Do not neglect the role of circadian rhythm in muscle atrophy","volume":"63","author":"Zhang","year":"2020","journal-title":"Ageing Res. Rev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"101050","DOI":"10.1016\/j.molmet.2020.101050","article-title":"Day-night rhythm of skeletal muscle metabolism is disturbed in older, metabolically compromised individuals","volume":"41","author":"Wefers","year":"2020","journal-title":"Mol. Metab."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4389","DOI":"10.1039\/D3FO04174C","article-title":"beta-Hydroxy-beta-methylbutyrate (HMB) leads to phospholipase D2 (PLD2) activation and alters circadian rhythms in myotubes","volume":"15","author":"Chapnik","year":"2024","journal-title":"Food Funct."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"109926","DOI":"10.1016\/j.jnutbio.2025.109926","article-title":"beta-Hydroxy-beta-methylbutyrate (HMB) increases muscle mass and diminishes weight gain in high-fat-fed mice","volume":"142","author":"Chapnik","year":"2025","journal-title":"J. Nutr. Biochem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4836","DOI":"10.1039\/C8FO00898A","article-title":"beta-Hydroxy-beta-methylbutyrate modulates lipid metabolism in adipose tissues of growing pigs","volume":"9","author":"Duan","year":"2018","journal-title":"Food Funct."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"368","DOI":"10.1016\/j.nutres.2014.02.003","article-title":"beta-Hydroxy-beta-methylbutyrate facilitates PI3K\/Akt-dependent mammalian target of rapamycin and FoxO1\/3a phosphorylations and alleviates tumor necrosis factor alpha\/interferon gamma-induced MuRF-1 expression in C2C12 cells","volume":"34","author":"Kimura","year":"2014","journal-title":"Nutr. Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1152\/physrev.00019.2003","article-title":"Cellular and molecular regulation of muscle regeneration","volume":"84","author":"Charge","year":"2004","journal-title":"Physiol. Rev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"22583","DOI":"10.1074\/jbc.R114.566091","article-title":"Phospholipase D and the maintenance of phosphatidic acid levels for regulation of mammalian target of rapamycin (mTOR)","volume":"289","author":"Foster","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"755","DOI":"10.1016\/j.bbamcr.2008.12.017","article-title":"Beta-hydroxy-beta-methylbutyrate (HMB) stimulates myogenic cell proliferation, differentiation and survival via the MAPK\/ERK and PI3K\/Akt pathways","volume":"1793","author":"Kornasio","year":"2009","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/j.tcb.2013.07.002","article-title":"Molecular architecture of the mammalian circadian clock","volume":"24","author":"Partch","year":"2014","journal-title":"Trends Cell Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"220215","DOI":"10.1098\/rsob.220215","article-title":"Nutrient-sensitive protein O-GlcNAcylation shapes daily biological rhythms","volume":"12","author":"Liu","year":"2022","journal-title":"Open Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"16219","DOI":"10.1073\/pnas.1408886111","article-title":"A circadian gene expression atlas in mammals: Implications for biology and medicine","volume":"111","author":"Zhang","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Su, Y., Wang, M., Chen, J., Bao, Y., Wen, R., Ren, H.W., and Luan, Z.L. (2025). Progress in understanding how clock genes regulate aging and associated metabolic processes. Front. Physiol., 16.","DOI":"10.3389\/fphys.2025.1654369"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e2424046122","DOI":"10.1073\/pnas.2424046122","article-title":"Control of circadian muscle glucose metabolism through the BMAL1-HIF axis in obesity","volume":"122","author":"Chaikin","year":"2025","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"447","DOI":"10.1007\/s41999-024-01129-0","article-title":"The role of the circadian timing system in sarcopenia in old age: A scoping review","volume":"16","author":"Palmese","year":"2025","journal-title":"Eur. Geriatr. Med."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e170998","DOI":"10.1172\/JCI170998","article-title":"Circadian disruption, clock genes, and metabolic health","volume":"134","author":"Schrader","year":"2024","journal-title":"J. Clin. Investig."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"216","DOI":"10.1017\/S0029665117003962","article-title":"Circadian rhythms, nutrition and implications for longevity in urban environments","volume":"77","author":"Froy","year":"2018","journal-title":"Proc. Nutr. Soc."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/14\/6189\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T04:16:55Z","timestamp":1784175415000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/14\/6189"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,10]]},"references-count":27,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["ijms27146189"],"URL":"https:\/\/doi.org\/10.3390\/ijms27146189","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,10]]}}}