Oligonol, a Low-Molecular Weight Polyphenol Derived from Lychee, Alleviates Muscle Loss in Diabetes by Suppressing Atrogin-1 and MuRF1

Abstract

Stimulation of the ubiquitin-proteasome pathway-especially E3 ubiquitin ligases Atrogin-1 and MuRF1-is associated with muscle loss in diabetes. Elevated lipid metabolites impair myogenesis. Oligonol, a low molecular weight polyphenol derived from lychee, exhibited anti-diabetic and anti-obesity properties, suggesting it could be a proper supplement for attenuating muscle loss. Dietary (10 weeks) oligonol supplementation (20 or 200 mg/kg diet) on the skeletal muscle loss was investigated in diabetic db/db mice. Transcription factors NF-κB and FoxO3a involved in regulation of Atrogin-1 and MuRF1 were also investigated. Attenuation of muscle loss by oligonol (both doses) was associated with down-regulation of Atrogin-1 and MuRF1 gene expression. Oligonol supplementation decreased NF-κB expression in the nuclear fraction compared with db/db mice without oligonol supplement. Upregulation of sirtuin1 (SIRT1) expression prevented FoxO3a nuclear localization in db/db mice supplemented with oligonol. Marked increases in AMPKα activity and Ppara mRNA expression leading to lower lipid accumulation by oligonol provided additional benefits for attenuating muscle loss. Oligonol limited palmitate-induced senescent phenotype and cell cycle arrest and suppressed Atrogin-1 and MuRF1 mRNA expression in palmitate-treated C2C12 muscle cells, thus contributing to improving the impaired myotube formation. In conclusion, oligonol-mediated downregulation of Atrogin-1 and MuRF1 gene expression alleviates muscle loss and improves the impaired myotube formation, indicating that oligonol supplementation may be useful for the attenuation of myotube loss.

Keywords: Atrogin-1 and MuRF1; NF-κB; diabetes; flavanol-rich lychee fruit extract; muscle loss.

Figure 1

The cross-sectional area of tibialis anterior muscle in m/m, db/db mice, and db/db mice with oligonol (OLG) supplementation. (Aa–d) Myofibers were stained with hematoxylin-eosin (400×); (B) Mean cross-sectional area of tibialis anterior muscle; (C) The distribution of myofiber sizes in tibialis anterior muscles from m/m and db/db mice; and (D) db/db mice with or without oligonol (OLG) supplement.

Figure 2

Gene expression was analyzed using real-time PCR analysis in gastrocnemius muscle from m/m and db/db mice with or without oligonol (OLG) supplementation. Atrogin-1 (A) and MuRF1 (B) mRNA expression is expressed as mean ratio to control after normalization with 18S mRNA levels. Fold differences were calculated using the ΔΔCt method. Values presented are mean ± SEM (n = 5–6/group). Protein expression was analyzed using Western blotting with anti-NF-κB and -Histone H3 antibodies in gastrocnemius muscle (C, n = 4–5/group). Significance (p < 0.05) among groups is denoted by different letters.

Figure 3

Representative blots of nuclear FoxO3a (A), SIRT1 (B), phospho-Akt (Thr473), Akt (C), phospho-FoxO3a (Ser253), and total FoxO3a (D) in m/m and db/db mice with or without oligonol (OLG) supplement are shown (n = 5–6/group). Significance (p < 0.05) among groups is denoted by different letters.

Figure 4

Oil red O (ORO)-stained tibialis anterior muscle sections (Aa–d) in m/m and db/db mice with or without oligonol (OLG) supplementation (200×); (B) Quantification of neutral lipids stained with ORO solution in tibialis anterior muscle (n = 3/group); (C) Representative blots of AMPKα and phospho-AMPKα (Thr172) are shown (n = 5–6/group); (D) Ppara mRNA expression level is expressed as mean ratio to control after normalization with 18S mRNA levels (n = 5–6/group). Fold differences were calculated using the ΔΔCt method. Values presented are mean ± SEM. Significance (p < 0.05) among groups is denoted by different letters.

Figure 5

C2C12 myoblasts were pretreated with or without oligonol (10, 25, 50 μg/mL) for 16 h, followed by incubation with or without palmitate (PA, 250 μM) for 8 h. (Aa–e) Senescence-associated β-galactosidase positive cells exhibited blue-green color (400×); (Ba–e) The relative percentage of myoblasts at G0/G1, S, and G2/M phases of the cell cycle; (Ca–e) Myoblasts received different treatments during the proliferative phase were differentiated into myotubes; After 5 days of differentiation, crystal violet staining was used to examine myotube formation (100×); (D–F) Quantification of myotube number, diameter, and length, respectively; (G,H) Atrogin-1 and MuRF1 mRNA levels in differentiated C2C12 myotubes treated with PA and/or oligonol. Fold differences were calculated using the ΔΔCt method. Values presented are mean ± SEM (n = 3/group).