Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Alpha‐Ketoisocaproate Attenuates Muscle Atrophy in Cancer Cachexia Models

doi: 10.1002/jcsm.70044

Figure Lengend Snippet: KIC regulates protein turnover and myostatin expression. (A) C2C12 myotubes were treated with CHX (10 μM) or myostatin (10 ng/mL), with or without l ‐leucine (1 mM) or KIC (1 mM), for 24 h, followed by puromycin (10 μg/mL) treatment for 1 h. Protein synthesis was assessed by detecting puromycin incorporation via IB, normalized to β‐actin expression ( n = 3). (B) C2C12 myotubes were treated with MG132 (10 μM) or myostatin (10 ng/mL), with or without l ‐leucine (1 mM) or KIC (1 mM), for 24 h. Protein degradation was assessed by detecting ubiquitin‐conjugated proteins via IB, normalized to β‐actin expression ( n = 3). (C) Relative myostatin mRNA levels in C2C12 myotubes treated with 1 mM l ‐leucine metabolites ( l ‐leucine, KIC, and HMB) for 24 h were evaluated using RT‐PCR and normalized to those of GAPDH ( n = 3). Myostatin protein expression (D) in C2C12 myotubes treated with KIC at the indicated doses (0.1–1 mM) for 48 h or (E) in C2C12 myotubes treated with KIC (1 mM) for the indicated times (3–48 h), analysed via IB and normalized to β‐actin expression ( n = 3). (F) MuRF1 and MAFbx protein expression in myostatin (20 ng/mL) treated‐C2C12 myotubes, with or without KIC (1 mM) for 24 h, analysed via IB and normalized to β‐actin expression ( n = 3). Results are expressed as mean ± SEM Statistical analysis was performed using one‐way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control. ANOVA, analysis of variance; CHX, cycloheximide; HMB, β‐hydroxy‐β‐methylbutyrate; IB, immunoblot; KIC, alpha‐ketoisocaproate; RT‐PCR, real‐time polymerase chain reaction; SEM, standard error of the mean.

Article Snippet: Anti‐myosin heavy chain (MyHC) (#MAB4470SP, 1:200, ICC) and recombinant myostatin (#788‐G8) were purchased from R&D Systems (USA).

Techniques: Expressing, Ubiquitin Proteomics, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Alpha‐Ketoisocaproate Attenuates Muscle Atrophy in Cancer Cachexia Models

doi: 10.1002/jcsm.70044

Figure Lengend Snippet: KIC inhibits myotube atrophy in C26‐CM‐treated C2C12 myotubes. (A) Experimental scheme for the CAC mimetic in vitro study. MuRF1, MAFbx, and myostatin protein expression in C2C12 myotubes cultured with DM containing 30% C26‐CM for the indicated times (1–48 h) was analysed using IB and normalized to β‐actin expression ( n = 3). (B–C) C2C12 myotubes were incubated in DM containing 30% C26‐CM with or without KIC (0.1–1 mM) for 48 h. (B) Relative mRNA levels of MuRF1 , MAFbx , and myostatin were assessed using RT‐PCR and normalized to those of GAPDH ( n = 3). (C) Protein expression of MuRF1, MAFbx, and myostatin was analysed using IB and normalized to that of β‐actin ( n = 3). (D–F) C2C12 myotubes were pretreated with myostatin (20 ng/mL) for 1 h, then incubated in DM containing 30% C26‐CM with or without KIC (0.3 mM) for 48 h. (D) MuRF1 and MAFbx protein expression was analysed using IB and normalized to that of β‐actin ( n = 3). (E) Protein degradation was evaluated by detecting ubiquitin‐conjugated proteins using IB, normalized to β‐actin expression (n = 3). (F) MyHC ICC and morphological analysis of C2C12 myotubes. MyHC (green) and DAPI (blue) were used for visualisation. The myotube diameter was quantified using ImageJ ( n = 50). The fusion index was calculated as the percentage of nuclei within MyHC‐positive myotubes ( n = 5). The results are expressed as the mean ± SEM Statistical analysis was performed using one‐way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control. Scale bar, 100 μm. ANOVA, analysis of variance; CM, conditioned medium; DM, differentiation medium; IB, immunoblot; ICC, immunocytochemistry; KIC, alpha‐ketoisocaproate; RT‐PCR, real‐time polymerase chain reaction; SEM, standard error of the mean.

Article Snippet: Anti‐myosin heavy chain (MyHC) (#MAB4470SP, 1:200, ICC) and recombinant myostatin (#788‐G8) were purchased from R&D Systems (USA).

Techniques: In Vitro, Expressing, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction, Ubiquitin Proteomics, Control, Western Blot, Immunocytochemistry, Real-time Polymerase Chain Reaction

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Alpha‐Ketoisocaproate Attenuates Muscle Atrophy in Cancer Cachexia Models

doi: 10.1002/jcsm.70044

Figure Lengend Snippet: KIC inhibits myotube atrophy by mediating MCT1‐2 in C26‐CM‐treated C2C12 myotubes. (A) Comparison of the relative mRNA expression of MCT1 and MCT2 in C2C12 myotubes treated with 1 mM ketone bodies ( l ‐lactate, pyruvate, and KIC) for the indicated times (0.5–24 h). mRNA expression was evaluated by RT‐PCR and normalized to that of GAPDH ( n = 3). (B–C) C2C12 myotubes were pretreated with ARC (100 nM) for 1 h, then incubated with DM containing 30% C26‐CM with or without KIC (0.3 mM) for 48 h. (B) Protein expression of MuRF1, MAFbx, and myostatin was evaluated using immunoblotting and normalized using β‐actin expression ( n = 3). (C) MyHC ICC and morphological analysis of C2C12 myotubes. MyHC (green) and DAPI (blue) were used for visualisation. The myotube diameter was quantified using ImageJ ( n = 50). The fusion index was calculated as the percentage of nuclei within MyHC‐positive myotubes ( n = 5). Results are expressed as the mean ± SEM Statistical analysis was performed using one‐way ANOVA. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control. Scale bar, 100 μm. ANOVA, analysis of variance; ARC, AR‐C155858; CM, conditioned media; DM, differentiation media; IB, immunoblot; ICC, immunocytochemistry; KIC, alpha‐ketoisocaproate; RT‐PCR, real‐time polymerase chain reaction; SEM, standard error of the mean.

Article Snippet: Anti‐myosin heavy chain (MyHC) (#MAB4470SP, 1:200, ICC) and recombinant myostatin (#788‐G8) were purchased from R&D Systems (USA).

Techniques: Comparison, Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Western Blot, Control, Immunocytochemistry, Real-time Polymerase Chain Reaction

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Alpha‐Ketoisocaproate Attenuates Muscle Atrophy in Cancer Cachexia Models

doi: 10.1002/jcsm.70044

Figure Lengend Snippet: KIC phosphorylates Akt–FoxO3a in C2C12 myotubes. (A) Protein expression of p‐Akt (ser473) , Akt, p‐FoxO3a (ser253) , and FoxO3a in C2C12 myotubes cultured in DM containing KIC (0.3 mM) for the indicated times (1–24 h). (B) Protein expression of p‐Akt (ser473) , Akt, p‐FoxO3a (ser253) , and FoxO3a in C2C12 myotubes cultured in DM containing 30% C26‐CM for the indicated times (3–48 h). (C) Protein expression of p‐Akt (ser473) , Akt, p‐FoxO3a (ser253) , and FoxO3a in C2C12 myotubes were incubated in DM containing 30% C26‐CM with or without KIC (0.1–1 mM) for 48 h. (D) C2C12 myotubes were pretreated with ARC (100 nM) for 1 h, then incubated with DM containing 30% C26‐CM with or without KIC (0.3 mM) for 48 h. Protein expression of p‐Akt (ser473) , Akt, p‐FoxO3a (ser253) , and FoxO3a was evaluated using IB and normalized to that of Akt and FoxO3a ( n = 3). (E–F) C2C12 myotubes were pretreated with LY (20 μM) for 1 h, then incubated with DM containing 30% C26‐CM with or without KIC (0.3 mM) for 48 h. (E) Protein expression of p‐Akt (ser473) , Akt, p‐FoxO3a (ser253) , FoxO3a, MuRF1, MAFbx, and myostatin was evaluated using IB and normalized using Akt, FoxO3a, and β‐actin ( n = 3). (F) MyHC ICC and morphological analysis of C2C12 myotubes. MyHC (green) and DAPI (blue) were used for visualisation. Myotube diameter was quantified using ImageJ ( n = 50). The fusion index was calculated as the percentage of nuclei within MyHC‐positive myotubes ( n = 5). Results are expressed as the mean ± SEM Statistical analysis was performed using one‐way ANOVA. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control. Scale bar, 100 μm. ANOVA, analysis of variance; ARC, AR‐C155858; CM, conditioned media; DM, differentiation media; IB, immunoblot; ICC, immunocytochemistry; KIC, alpha‐ketoisocaproate; LY, LY294002; SEM, standard error of the mean.

Article Snippet: Anti‐myosin heavy chain (MyHC) (#MAB4470SP, 1:200, ICC) and recombinant myostatin (#788‐G8) were purchased from R&D Systems (USA).

Techniques: Expressing, Cell Culture, Incubation, Control, Western Blot, Immunocytochemistry

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Alpha‐Ketoisocaproate Attenuates Muscle Atrophy in Cancer Cachexia Models

doi: 10.1002/jcsm.70044

Figure Lengend Snippet: KIC inhibits myotube atrophy by enhancing p‐Akt and FoxO3a interaction and inhibiting FoxO3a translocation in C26‐CM‐treated C2C12 myotubes. (A) Localisation of FoxO3a was assessed using ICC in C2C12 myotubes incubated in DM containing 30% C26‐CM, with or without KIC (0.3 mM) for 48 h. Cells were stained for FoxO3a (green) and DAPI (blue) and visualized using confocal microscopy. (B) Protein–protein interaction between 14‐3‐3 proteins, p‐Akt (ser473) , and FoxO3a in C2C12 myotubes cultured in DM containing 30% C26‐CM for the indicated times (1–24 h) or in DM containing 30% C26‐CM with or without KIC (0.3 mM) for 24 h. Protein–protein interactions were evaluated by Co‐IP performed using an anti‐FoxO3a antibody ( n = 3). (C–F) C2C12 myotubes were transfected with Akt or FoxO3a siRNA (100 nM) or control siRNA (100 nM) and incubated for 24 h, followed by treatment with DM containing 30% C26‐CM, with or without KIC (0.3 mM) for 48 h. (C, E) Myostatin protein expression was evaluated by IB and normalized to that of β‐actin ( n = 3). (D, F) MyHC ICC and morphological analysis were performed using MyHC (green) and DAPI (blue) for visualisation. Myotube diameter was quantified using ImageJ ( n = 50), and the fusion index was calculated as the percentage of nuclei within MyHC‐positive myotubes ( n = 5). Results are expressed as the mean ± SEM Statistical analysis was performed using one‐way ANOVA. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control. Scale bar in (B) = 50 μm, and in (D, F) = 100 μm. ANOVA, analysis of variance; CM, conditioned media; Co‐IP, co‐immunoprecipitation; DM, differentiation media; IB, immunoblot; ICC, immunocytochemistry; KIC, alpha‐ketoisocaproate; RT‐PCR, real‐time polymerase chain reaction; SEM, standard error of the mean.

Article Snippet: Anti‐myosin heavy chain (MyHC) (#MAB4470SP, 1:200, ICC) and recombinant myostatin (#788‐G8) were purchased from R&D Systems (USA).

Techniques: Translocation Assay, Incubation, Staining, Confocal Microscopy, Cell Culture, Protein-Protein interactions, Co-Immunoprecipitation Assay, Transfection, Control, Expressing, Immunoprecipitation, Western Blot, Immunocytochemistry, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Alpha‐Ketoisocaproate Attenuates Muscle Atrophy in Cancer Cachexia Models

doi: 10.1002/jcsm.70044

Figure Lengend Snippet: KIC administration prevents CAC via the Akt–FoxO3 pathway in C26 cell‐injected mice. (A) Myostatin, TNF‐α, IFN‐γ, and IL‐6 levels in the serum were analysed using ELISA ( n = 12 per group). (B) Comparison of the relative mRNA expression of MuRF1 , MAFbx , and Myostatin in TA muscle evaluated using RT‐PCR and normalized using GAPDH expression ( n = 12 per group). (C) Protein expression of p‐Akt (ser473) , Akt, p‐FoxO3a (ser253) , FoxO3a, and myostatin in TA muscle evaluated using IB was normalized using Akt, FoxO3a, and β‐actin expression ( n = 3). (D) The lysate of TA muscle was fractionated into nuclear and cytoplasmic fractions. FoxO3a protein expression evaluated using IB was normalized using the cytoplasmic protein GAPDH and the nuclear protein Lamin B ( n = 3). (E) Protein–protein interaction between p‐Akt (ser473) and FoxO3a in TA muscle. Protein–protein interactions were evaluated by Co‐IP performed using an anti‐FoxO3a antibody ( n = 3). (F) Representative HE‐stained images of CSA in TA muscle show the fibre size distribution and average fibre CSA ( n = 12 per group). Results are expressed as the mean ± SEM One‐way ANOVA was used to determine statistical significance. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus sham; # p < 0.05, ## p < 0.01, and ### p < 0.001 versus C26. Sham: sham mice; C26: C26‐injected mice; C26 + KIC: C26‐injected mice treated with KIC (10 mg/kg) intraperitoneally. Scale bar, 100 μm. ANOVA, analysis of variance; CAC, cancer‐associated cachexia; Co‐IP, co‐immunoprecipitation; CSA, cross‐sectional area; ELISA, enzyme‐linked solvent assay; HE, haematoxylin–eosin; IB, immunoblot; KIC, alpha‐ketoisocaproate; SEM, standard error of the mean; TA, tibialis anterior.

Article Snippet: Anti‐myosin heavy chain (MyHC) (#MAB4470SP, 1:200, ICC) and recombinant myostatin (#788‐G8) were purchased from R&D Systems (USA).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Comparison, Expressing, Reverse Transcription Polymerase Chain Reaction, Protein-Protein interactions, Co-Immunoprecipitation Assay, Staining, Immunoprecipitation, Solvent, Western Blot

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Alpha‐Ketoisocaproate Attenuates Muscle Atrophy in Cancer Cachexia Models

doi: 10.1002/jcsm.70044

Figure Lengend Snippet: KIC administration prevents CAC in 4T1 cell‐injected mice. (A) Scheme of the experimental schedule for the CAC animal study and tumour‐free body weight determination. (B) Grip strength testing during days 0–28. Each result was expressed as the mean ± SEM of n = 10 mice in each group. Two‐way ANOVA was used to determine statistical significance. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus sham; # p < 0.05, ## p < 0.01, and ### p < 0.001 versus 4T1. (C) Skeletal muscle (TA, GCM, and QA) morphology and skeletal muscle weight of mice were evaluated at the time of sacrifice ( n = 10 per group). (D) The heart, kidney, white adipose tissue, and tumour weights were measured at the time of sacrifice ( n = 10 per group). (E) Myostatin levels in serum were analysed using ELISA ( n = 10 per group). One‐way ANOVA was used to determine statistical significance. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus sham; # p < 0.05, ## p < 0.01, and ### p < 0.001 versus 4T1. (F) Schematic diagram of the mechanism of action of KIC: KIC is transported into skeletal muscle cells via MCT1–2 and activates Akt. KIC inhibits FoxO3a activation in an Akt‐dependent manner, leading to the export of FoxO3a from the nucleus. Consequently, sequestration of FoxO3a from the nucleus alleviates cancer cachexia by inhibiting myostatin. Sham: sham mice; KIC: mice treated with KIC (10 mg/kg) intraperitoneally. 4 T1: 4T1‐injected mice; 4T1 + KIC: 4T1‐injected mice treated with KIC (10 mg/kg) intraperitoneally. ANOVA, analysis of variance; CAC, cancer‐associated cachexia; ELISA, enzyme‐linked solvent assay; GCM, gastrocnemius; KIC, alpha‐ketoisocaproate; QA, quadriceps; SEM, standard error of the mean; TA, tibialis anterior.

Article Snippet: Anti‐myosin heavy chain (MyHC) (#MAB4470SP, 1:200, ICC) and recombinant myostatin (#788‐G8) were purchased from R&D Systems (USA).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Activation Assay, Solvent

Journal: The Journal of Physiology

Article Title: Long non‐coding RNAs Kcnq1ot1 and Lncpint are involved in skeletal muscle atrophy induced by the space exposome

doi: 10.1113/jp288987

Figure Lengend Snippet: Figure 3. LINC-PINT and KCNQ1OT1 expression in a 3-D human cellular model of skeletal muscle atrophy A, Immunohistochemical images of the 3-D human cellular model after vehicle (upper image) or myostatin (lower image) treatment. Nuclei are shown in blue (DAPI (4’,6-diamidino-2-phenylindole) staining) and myosin heavy chain 1, a proxy of myotube maturity, in red (MyH1 staining). The images show a greater amount of MyH1 in vehicle- compared to myostatin-treated models (details described in Pérez-Díaz et al., 2025). Scale bar: 100 μm. B, LINC-PINT expression (qPCR) in 3-D human cellular models exposed to vehicle (yellow-coloured squares, n = 7) or myostatin (plum-coloured circles, n = 6) treatment. Full- and dashed lines in the truncated violin plots indicate median and first and third quartiles, respectively. C, KCNQ1OT1 expression (qPCR) in 3-D human cellular models exposed to vehicle (yellow-coloured squares, n = 11) or myostatin (plum-coloured circles, n = 11) treatment (∗P = 0.0132). Full- and dashed lines in the truncated violin plots indicate median and first and third quartiles, respectively.

Article Snippet: To simulate muscle atrophy in the 3-D cellular models, we treated mature 3-D models (10 days in differentiation media) with 100 ng/ml of myostatin (788-G8, R&D Systems) or a vehicle (Dulbecco’s Phosphate Buffered Saline, DPBS) for 5 days.

Techniques: Expressing, Immunohistochemical staining, Staining