Vibration and β-hydroxy-β-methylbutyrate treatment suppresses intramuscular fat infiltration and adipogenic differentiation in sarcopenic mice

Abstract

Background: Sarcopenia is an aging-induced deterioration of skeletal muscle mass and function. Low-magnitude high-frequency vibration (LMHFV) was shown to improve muscle functions and β-hydroxy-β-methylbutyrate (HMB) to increase muscle mass and strength. Muscle-derived stem cells (MDSCs) are progenitor cells important for muscle regeneration. We hypothesized that LMHFV and HMB could retard sarcopenia by reducing fat infiltration through inhibiting adipogenesis in MDSCs.

Methods: Senescence-accelerated mouse P8 male mice were randomized into control (CTL), HMB, LMHFV (VIB), and combined (COM) groups. Interventions started at age of month 7 and assessed at 1, 2, and 3 months post-intervention by densitometry, histology, and functional tests. In vitro, MDSCs isolated from gastrocnemius of senescence-accelerated mouse P8 mice were characterized, randomized into CTL, VIB, HMB, and COM groups, and assessed by oil red O staining, mRNA, and protein expression.

Results: At 2 months post-intervention, percentage lean mass of HMB, VIB, and COM groups were significantly higher than CTL group. Twitch, tetanic, and specific tetanic forces of COM group were higher, while specific twitch force of both VIB and COM groups were higher. Grip strength of HMB, VIB, and COM groups were higher. Histologically, both VIB and COM groups presented lower oil red O area than CTL group. Type I muscle fibre in CTL group was higher than HMB, VIB, and COM groups. MDSC were detected in situ by immunofluorescence stain with stem cell antigen-1 signals confirmed with higher β-catenin expression in the COM group. The observations were also confirmed in vitro, MDSCs in the HMB, VIB, and COM groups presented lower adipogenesis vs. the CTL group. β-Catenin mRNA and protein expressions were lower in the CTL group while their relationship was further validated through β-catenin knock-down approach.

Conclusions: Our results showed that combined LMHFV and HMB interventions enhanced muscle strength and decreased percentage fat mass and intramuscular fat infiltration as compared with either treatment alone. Additive effect of LMHFV and HMB was demonstrated in β-catenin expression than either treatment in MDSCs and altered cell fate from adipogenesis to myogenesis, leading to inhibition of intramuscular lipid accumulation. Wnt/β-catenin signalling pathway was found to be the predominant regulatory mechanism through which LMHFV and HMB combined treatment suppressed MDSCs adipogenesis.

Keywords: Fat infiltration; HMB; LMHFV; MDSC; Sarcopenia; Wnt/β-catenin.

Conflict of interest statement

The authors declare no conflict of interest and certify that they comply with the ethical guidelines for authorship and publishing of the Journal of Cachexia, Sarcopenia, and Muscle.51

© 2020 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.

Figures

Figure 1

Body composition evaluation of SAMP8 mice by dual energy X‐ray absorptiometry (DXA). (A) Percentage lean mass and (B) percentage fat mass of SAMP8 mice in different groups at 1, 2, and 3 months. Percentage lean mass in CTL group was lower than HMB, VIB, and COM groups at month 2 post‐intervention, while the percentage fat mass in CTL group was higher than HMB, VIB, and COM groups at month 2. (C) Representative image of whole‐body DXA scanning (lean mass mode) (D) and (fat mass mode) demonstrating that the HMB, VIB, and COM groups showed higher percentage lean mass and lower fat mass at month 2, respectively. Lean and fat masses indicated by white arrows. *P < 0.05; **P < 0.01. (E) Serum myostatin level of COM group showed significantly higher than CTL group at month 3. (F) Serum adiponectin level of VIB group showed significantly higher than CTL group at month 3. *P < 0.05. HMB, β‐hydroxy‐β‐methylbutyrate; SAM, senescence‐accelerated mouse.

Figure 2

Morphological differences and fat infiltration of skeletal muscles in senescence‐accelerated mouse P8 mice upon different treatments. (A) H&E, oil red O (ORO), and myosin heavy chain (MHC) staining of muscle histology in different groups at month 3. CTL group presented more intramuscular fat tissue than HMB, VIB, and COM groups at month 3 by H&E where fat tissues are indicated by yellow arrows. HMB, VIB, and COM groups presented lower ORO signal (red area) than the CTL group at month 3. At the gastrocnemius, percentage area of type I fibre (blue) in the CTL group was significantly higher than all treatment groups with no significant difference detected in type IIa and IIb fibres at month 3. (B) CTL group showed higher percentage type I fibres than HMB, VIB, and COM groups by MHC immunofluorescence staining at month 3 (***P < 0.001). (C) Quantitative analysis revealed that ORO area of both VIB and COM groups was significantly higher than CTL group at month 3, *P < 0.05, **P < 0.01. HMB, β‐hydroxy‐β‐methylbutyrate.

Figure 3

In situ detection of muscle‐derived stem cells (MDSC) and β‐catenin expression. (A) MDSC was detected and identified by positive stem cell antigen‐1 (Sca‐1, green) signal, highlighted against basal lamina surrounded muscle fibres (laminin, red) with nuclei stained with DAPI (blue). All MDSC were observed to reside between muscle fibres scattered across the muscle section (white arrows). (B) Western blot analysis showed that total β‐catenin content were increased in the treatment groups compared with the CTL group after 3 months of treatment with only the combine treatment (COM) group reaching statistical significant difference (* n = 3, P < 0.05, analysis of variance with Bonferroni post hoc test).

Figure 4

Muscle strength of senescence‐accelerated mouse P8 mice upon different treatments at various time points. (A) Twitch force (F0) of COM group was significantly higher than CTL group at month 3 post‐intervention. No significant difference was found in other groups. (B) Tetanic force (Ft) of COM group was significantly higher than CTL group at month 3 with no significant difference detected in other groups. (C) Specific twitch force (SF0) of VIB and COM groups were significantly higher than CTL group at month 3 with no significant difference detected in other groups. (D) Specific tetanic force (SFt) of COM group was significantly higher than CTL group at month 3 with no significant difference detected in other groups. (E) The grip strength of HMB, VIB, and COM groups were significantly higher than CTL group at month 3. *P < 0.05; **P < 0.01; ***P < 0.001. HMB, β‐hydroxy‐β‐methylbutyrate.

Figure 5

Morphology and characterization of muscle‐derived stem cells (MDSC) at different stages of their isolation utilizing the pre‐plate technique. (A) PP1 cells isolated from muscle of SAMP8 mice. (B) PP3 cells from muscle of SAMP8 mice. (C) PP6 MDSCs isolated from muscle of SAMP8 mice. MDSCs are indicated by yellow arrows. (D) Alizarin red S staining at day 10 after osteogenic induced culture in MDSCs. (E) Oil red O staining at day 10 after adipogenic induced culture in MDSCs. (F) Immunofluorescence staining of MDSCs at day 5 after myogenic induction. Myotubes are indicated by white arrows, green area is myosin heavy chain IIa positive, and the blue area is nucleus stained by DAPI. SAM, senescence‐accelerated mouse.

Figure 6

ORO staining of MDSCs after adipogenic induction upon different treatments. (A) CTL group showed more adipogenic cells than HMB, VIB, and COM groups. (B) Quantitative data showed that all treatment groups presented significantly less ORO positive area than the CTL group. The COM group showed significantly less adipogenic cells than HMB group. (C) PPARγ mRNA expression level in CTL group was significantly higher than HMB, VIB, and COM groups. (D) PGC1α mRNA expression in CTL group was significantly higher than VIB and COM groups. PGC1α mRNA expression in COM group was significantly lower than HMB group. (E) C/EBPα mRNA expression in CTL group was significantly higher than COM group and marginally higher than HMB group. (F) Relative expression of β‐catenin mRNA in CTL group was significantly lower than HMB and COM groups. (G) Western blot results revealed that HMB, VIB, and COM groups had significantly higher β‐catenin protein level than CTL group. *P < 0.05; **P < 0.01; ***P < 0.001. HMB, β‐hydroxy‐β‐methylbutyrate; ORO, oil red O.

Figure 7

Treatment effects on adipogenesis differentiation in MDSC abolished by si‐β‐catenin knockdown. (A) CTL group showed more adipogenic cells than HMB, VIB, and COM groups after si‐NC knockdown, while no obvious difference was seen among groups after si‐β‐catenin knockdown. (B) Quantitative data showed that VIB and COM groups presented significantly less ORO area than CTL group after si‐NC knockdown, while no significant difference was seen among groups after si‐β‐catenin knockdown. (C) PPARγ mRNA expression in CTL group was significantly higher than COM group after si‐NC knockdown, while no significant difference was seen among groups after si‐β‐catenin knockdown. PGC1α mRNA expression in CTL group was significantly higher than COM group after si‐NC knockdown. No significant difference was seen among groups after si‐β‐catenin knockdown. C/EBPα mRNA expression in CTL group was significantly higher than COM group after si‐NC knockdown. No significant difference of C/EBPα mRNA was seen among groups after si‐β‐catenin knockdown. (F) Relative expression of β‐catenin mRNA in CTL group was significantly lower than HMB, VIB, and COM groups after si‐NC knockdown, but no significant difference was seen among groups after si‐β‐catenin knockdown. (G) Western blot result revealed that no significant difference of β‐catenin protein level was seen among groups after si‐β‐catenin knockdown, while HMB, VIB, and COM groups had significantly higher β‐catenin protein level than CTL group after si‐NC knockdown. *P < 0.05; **P < 0.01. HMB, β‐hydroxy‐β‐methylbutyrate; MDSC, muscle‐derived stem cell; ORO, oil red O.