Ursodeoxycholic acid induces sarcopenia associated with decreased protein synthesis and autophagic flux - PubMed (original) (raw)

Ursodeoxycholic acid induces sarcopenia associated with decreased protein synthesis and autophagic flux

Josué Orozco-Aguilar et al. Biol Res. 2023.

Abstract

Background: Skeletal muscle generates force and movements and maintains posture. Under pathological conditions, muscle fibers suffer an imbalance in protein synthesis/degradation. This event causes muscle mass loss and decreased strength and muscle function, a syndrome known as sarcopenia. Recently, our laboratory described secondary sarcopenia in a chronic cholestatic liver disease (CCLD) mouse model. Interestingly, the administration of ursodeoxycholic acid (UDCA), a hydrophilic bile acid, is an effective therapy for cholestatic hepatic alterations. However, the effect of UDCA on skeletal muscle mass and functionality has never been evaluated, nor the possible involved mechanisms.

Methods: We assessed the ability of UDCA to generate sarcopenia in C57BL6 mice and develop a sarcopenic-like phenotype in C2C12 myotubes and isolated muscle fibers. In mice, we measured muscle strength by a grip strength test, muscle mass by bioimpedance and mass for specific muscles, and physical function by a treadmill test. We also detected the fiber's diameter and content of sarcomeric proteins. In C2C12 myotubes and/or isolated muscle fibers, we determined the diameter and troponin I level to validate the cellular effect. Moreover, to evaluate possible mechanisms, we detected puromycin incorporation, p70S6K, and 4EBP1 to evaluate protein synthesis and ULK1, LC3 I, and II protein levels to determine autophagic flux. The mitophagosome-like structures were detected by transmission electron microscopy.

Results: UDCA induced sarcopenia in healthy mice, evidenced by decreased strength, muscle mass, and physical function, with a decline in the fiber's diameter and the troponin I protein levels. In the C2C12 myotubes, we observed that UDCA caused a reduction in the diameter and content of MHC, troponin I, puromycin incorporation, and phosphorylated forms of p70S6K and 4EBP1. Further, we detected increased levels of phosphorylated ULK1, the LC3II/LC3I ratio, and the number of mitophagosome-like structures. These data suggest that UDCA induces a sarcopenic-like phenotype with decreased protein synthesis and autophagic flux.

Conclusions: Our results indicate that UDCA induces sarcopenia in mice and sarcopenic-like features in C2C12 myotubes and/or isolated muscle fibers concomitantly with decreased protein synthesis and alterations in autophagic flux.

Keywords: Autophagic flux; Bile acids; Protein synthesis; Sarcopenia; Ursodeoxycholic acid.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Muscle function declines in mice treated with UDCA after 6 weeks. C57BL/6 male mice were treated orally with UDCA 200 mg/kg corporal body weight for 6 weeks (1.04% NaCl pH 8.4 to the control group). Mice were monitored daily, and muscular evaluations were made at the beginning and during the sixth week. a The hindlimb strength was measured by a dynamometer. Values represent the difference between the initial measure before UDCA treatment and the end of treatment. b The forelimb strength was measured by a dynamometer. Values represent the difference between the initial measure before UDCA treatment and the end of treatment. c The forelimb strength was measured by a weightlifting test. Values represent the difference between the initial score normalized before UDCA treatment and the end of treatment. d After euthanizing, the TA muscle was dissected to evaluate the electromyography performance. The area under the curve was calculated for each animal using GraphPad Prism 8.0 software and represented individually. e The maximum distance reached by each animal on a treadmill was recorded. Values represent the difference between the initial distance before UDCA treatment and the end of treatment. The result shows the individual value of the subject, with the mean ± SEM for each group. (n = 5 mice per group, no paired t-test, *p < 0.05 with respect to the control group). SEM standard error of the mean, UDCA ursodeoxycholic acid

Fig. 2

After 6 weeks, UDCA treatment diminishes the fiber diameter and troponin I level in adult mice. C57BL/6 male mice were treated orally with UDCA 200 mg/kg corporal body weight for 6 weeks (1.04% NaCl pH 8.4 to the control group). Mice were monitored daily and were euthanized during the sixth week. a TA muscle cross-sections were stained with laminin to delimit the sarcolemma. The scale bar indicates 100 μm. b The minimal Feret’s diameters were calculated using the MyoVision software. Fiber diameters were grouped from 5 to 80 μm to quantify the total fiber percentage in each group. c Accumulative frequency analysis to UDCA and control group were plotted. d The area under the curve was calculated in accumulative frequency to fiber diameters using GraphPad Prism 8.0 software. e TA muscle was homogenized to evaluate troponin I protein levels by western blot analysis, using β-actin as the loading control, and the molecular weights are depicted in kilodaltons (kDa). f Quantitative analysis for troponin I protein levels is shown. The result shows the mean ± SEM for each group (n = 5 mice per group, no paired t-test, *p < 0.05 with respect to the control group). SEM standard error of the mean, UDCA ursodeoxycholic acid

Fig. 3

UDCA reduces the diameter of C2C12 myotubes in a dose-dependent manner. Myotubes were incubated with 0–50–100–200 μM UDCA for 72 h. a MHC was detected by indirect immunofluorescence and used to delimit the myotube diameter. Images were captured by fluorescence microscopy. The scale bar indicates 100 μm. b The diameter of the myotubes was measured using ImageJ software. The quantification was performed, and the individual values for myotube were plotted. c The myotubes were classified using the control group median (32.13 μm). Myotubes with large or small diameters were determined if their diameter was larger or smaller than the threshold value. The values indicate the percentage of myotubes expressed as the mean ± SEM of three independent experiments (one‐way ANOVA, post‐hoc Dunnet, *p < 0.05 with respect to the control). ANOVA analysis of variance, MHC myosin heavy chain, SEM standard error of the mean, UDCA ursodeoxycholic acid

Fig. 4

UDCA reduces sarcomeric proteins in C2C12 myotubes. C2C12 myoblasts were differentiated for 4–5 days and incubated with 0, 50, 100, 200, and 400 μM UDCA for 72 h. a MHC and troponin I protein levels were detected by western blot analysis, using β‐actin as the loading control. Molecular weights are shown in kDa. Densitometry analysis for (b) MHC and (c) troponin I is represented as a fold of change expressed as the mean ± SD of three independent experiments (no paired t-test, *p < 0.05 with respect to the control group). MHC myosin heavy chain, SD standard deviation, UDCA ursodeoxycholic acid

Fig. 5

Isolated muscle fiber diameter is reduced by UDCA treatment. Muscle fibers were isolated from C57BL/6 male mice and incubated with 200 μM UDCA for 72 h. a Indirect immunofluorescence detected EDL muscle fiber MHC to delimit the fiber diameter. Fluorescent microscopy images were captured. The scale bar indicates 50 μm. b The diameter of the isolated muscle fiber was measured using ImageJ software. The quantification was performed, and the individual values for fiber were plotted. c FDB muscle fiber MHC was detected by indirect immunofluorescence to delimit the fiber diameter. Fluorescent microscopy images were captured. The scale bar indicates 50 μm. d The diameter of the isolated muscle fiber was measured using ImageJ software. The quantification was performed, and the individual values for fiber were plotted. The result shows the value of each fiber, with the mean ± SD for each group (n = 27 EDL muscle fibers, 60 FDB muscle fibers, no paired t-test with Welch’s correction, *p < 0.05 with respect to the control group). EDL extensor digitorum longus, FDB flexor digitorum brevis, MHC myosin heavy chain, SD standard deviation, UDCA ursodeoxycholic acid

Fig. 6

UDCA decreases puromycin incorporation into C2C12 myotubes. C2C12 myoblasts were differentiated for 4–5 days and incubated with 200 μM UDCA for 24 h. a Puromycin incorporation was detected by western blot analysis, using β‐actin as a loading control. Molecular weight is indicated in kDa. b A densitometric analysis of the puromycin incorporation bands was performed. The values are shown as a fold of change and expressed as the mean ± SD of three independent experiments (no paired t-test, *p < 0.05 with respect to the control group). SD standard deviation, UDCA ursodeoxycholic acid

Fig. 7

UDCA decreases p70S6K and 4EBPP1 phosphorylation and p70S6K total protein levels into C2C12 myotubes. C2C12 myoblasts differentiated for 4–5 days were incubated with 200 μM UDCA for 72 h. a p70S6K phosphorylation and total levels were detected by western blot analysis, using p70S6K total levels or GAPDH as a loading control, respectively. Molecular weight is indicated in kDa. b Densitometric analysis of p-p70S6K (Thr389) and (c) p70S6K total protein levels were performed. d 4EBP1 phosphorylation and total levels were detected by western blot analysis, using 4EBP1 total levels or β‐actin as a loading control, respectively. Molecular weight is indicated in kDa. e Densitometric analysis of p-4EBP1 (Ser65) and f 4EBP1 total protein levels was performed. The values are shown as a fold of change and expressed as the mean ± SD of three independent experiments (no paired t-test, *p < 0.05 with respect to the control group). SD standard deviation, UDCA ursodeoxycholic acid

Fig. 8

UDCA increases ULK1 levels and LC3II/LC3I ratio, and decreases autophagy flux in C2C12 myotubes. C2C12 myoblasts were differentiated for 4–5 days and incubated with 200 μM UDCA. a After 12 h, ULK1 phosphorylation and total levels were detected by western blot analysis, using ULK1 total levels or tubulin as a loading control, respectively. Molecular weight is indicated in kDa. b Densitometric analysis of p-ULK1 (Ser317) and c ULK1 total protein levels were performed. d After 72 h, LC3I and LC3II protein levels were detected by western blot analysis in the absence or presence of chloroquine. β‐actin was used as a loading control. Molecular weight is indicated in kDa. e A densitometric analysis of the LC3II/LC3I ratio was performed. f Autophagic flux was calculated using the subtraction of LC3II levels in the presence and absence of chloroquine [(LC3II + CQ) − (LC3II-CQ)]). The values are shown as a fold of change and expressed as the mean ± SD of three independent experiments (no paired t-test with Welch’s correction, *p < 0.05 with respect to the control group). SD standard deviation, UDCA ursodeoxycholic acid

Fig. 9

UDCA increases the presence of mitophagosome-like structures in C2C12 myotubes. C2C12 myoblasts differentiated for 4–5 days were incubated with 200 μM UDCA for 72 h. a Representative transmission electron microscopy images of mitophagosome-like structures in myotubes. b Quantification of mitophagosome-like structures number by myotube area was performed. The values are shown as a percentage of presence and expressed as the mean ± SD of four independent experiments (no paired t-test, *p < 0.05 with respect to the control group). SD standard deviation, UDCA ursodeoxycholic acid

References

1. Hilscher MB, Kamath PS, Eaton JE. Cholestatic liver diseases: a primer for generalists and subspecialists. Mayo Clin Proc. 2020;95(10):2263–2279. doi: 10.1016/j.mayocp.2020.01.015. - DOI - PubMed
1. Kobayashi Y, Hara N, Sugimoto R, Mifuji-Moroka R, Tanaka H, Eguchi A, et al. The associations between circulating bile acids and the muscle volume in patients with non-alcoholic fatty liver disease (NAFLD) Intern Med. 2017;56(7):755–762. doi: 10.2169/internalmedicine.56.7796. - DOI - PMC - PubMed
1. Sauerbruch T, Hennenberg M, Trebicka J, Beuers U. Bile acids, liver cirrhosis, and extrahepatic vascular dysfunction. Front Physiol. 2021;12:718783. doi: 10.3389/fphys.2021.718783. - DOI - PMC - PubMed
1. Allen SL, Quinlan JI, Dhaliwal A, Armstrong MJ, Elsharkawy AM, Greig CA, et al. Sarcopenia in chronic liver disease: mechanisms and countermeasures. Am J Physiol Gastrointest Liver Physiol. 2021;320(3):G241–G257. doi: 10.1152/ajpgi.00373.2020. - DOI - PMC - PubMed
1. Hanai T, Shiraki M, Ohnishi S, Miyazaki T, Ideta T, Kochi T, et al. Rapid skeletal muscle wasting predicts worse survival in patients with liver cirrhosis. Hepatol Res. 2016;46(8):743–751. doi: 10.1111/hepr.12616. - DOI - PubMed

Ursodeoxycholic acid induces sarcopenia associated with decreased protein synthesis and autophagic flux - PubMed (original) (raw)