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Vitamin D deficiency down-regulates Notch pathway contributing to skeletal muscle atrophy in old wistar rats.

Domingues-Faria C, Chanet A, Salles J, Berry A, Giraudet C, Patrac V, Denis P, Bouton K, Goncalves-Mendes N, Vasson MP, Boirie Y, Walrand S - Nutr Metab (Lond) (2014)

Bottom Line: A reduction in Notch pathway activity may explain the age-related decrease in satellite cell proliferation, as this pathway has been implicated in satellite cell self-renewal.An unpaired student's t-test was performed to test the effect of the experimental conditions.The D-depleted group showed -39%, -31% drops in expression of two markers known to modulate proliferation (Bmp4, Fgf-2 mRNA levels) and -56% drop in one marker of cell proliferation (PCNA protein expression) compared to controls (p < 0.05).

View Article: PubMed Central - PubMed

Affiliation: Université d'Auvergne, Unité de Nutrition Humaine, Equipe ECREIN, CLARA, CRNH Auvergne; INRA, UMR 1019, UNH, CRNH Auvergne, Clermont Université, 63000 Clermont-Ferrand, France ; Université d'Auvergne, Unité de Nutrition Humaine, Equipe NuTriM, CRNH Auvergne; INRA, UMR 1019, UNH, CRNH Auvergne, Clermont Université, 63000 Clermont-Ferrand, France ; INRA, UMR1019, UNH, CRNH Auvergne, 63000 Clermont-Ferrand, France.

ABSTRACT

Background: The diminished ability of aged muscle to self-repair is a factor behind sarcopenia and contributes to muscle atrophy. Muscle repair depends on satellite cells whose pool size is diminished with aging. A reduction in Notch pathway activity may explain the age-related decrease in satellite cell proliferation, as this pathway has been implicated in satellite cell self-renewal. Skeletal muscle is a target of vitamin D which modulates muscle cell proliferation and differentiation in vitro and stimulates muscle regeneration in vivo. Vitamin D status is positively correlated to muscle strength/function, and elderly populations develop a vitamin D deficiency. The aim of this study was to evaluate how vitamin D deficiency induces skeletal muscle atrophy in old rats through a reduction in Notch pathway activity and proliferation potential in muscle.

Methods: 15-month-old male rats were vitamin D-depleted or not (control) for 9 months (n = 10 per group). Rats were 24-month-old at the end of the experiment. Gene and/or protein expression of markers of proliferation, or modulating proliferation, and of Notch signalling pathway were studied in the tibialis anterior muscle by qPCR and western blot. An unpaired student's t-test was performed to test the effect of the experimental conditions.

Results: Vitamin D depletion led to a drop in concentrations of plasma 25-hydroxyvitamin D in depleted rats compared to controls (-74%, p < 0.01). Tibialis anterior weight was decreased in D-depleted rats (-25%, p < 0.05). The D-depleted group showed -39%, -31% drops in expression of two markers known to modulate proliferation (Bmp4, Fgf-2 mRNA levels) and -56% drop in one marker of cell proliferation (PCNA protein expression) compared to controls (p < 0.05). Notch pathway activity was blunted in tibialis anterior of D-depleted rats compared to controls, seen as a down-regulation of cleaved Notch (-53%, p < 0.05) and its target Hes1 (-35%, p < 0.05).

Conclusions: A 9-month vitamin D depletion induced vitamin D deficiency in old rats. Vitamin D depletion induces skeletal muscle atrophy in old rats through a reduction in Notch pathway activity and proliferation potential. Vitamin D deficiency could aggravate the age-related decrease in muscle regeneration capacity.

No MeSH data available.


Related in: MedlinePlus

Effect of vitamin D depletion on Notch-1 transcript levels and protein expression in old rats. (A) mRNA expression of full length Notch-1 in tibialis anterior of control and depleted rats was analyzed using a RT2 Profiler Custom PCR Array following the manufacturer’s protocol. Full length Notch-1 mRNA in control and vitamin D depleted samples was normalized using expression of Tbp as a housekeeping gene and was relative to control group according to the 2-ΔΔCT method (n = 7 for control group and n = 9 for depleted group). mRNA expression of full length Notch was unaffected by vitamin D depletion. Data presented are means ± SEM; *p < 0.05. (B) Protein expression of transmembrane fragment Notch-1 (TMNotch-1) was analyzed in tibialis anterior by western blotting and quantified using Multi Gauge V3.2 software. Expression of the total amount of p38 was used to normalize protein loading between samples (n = 7 for control group and n = 9 for depleted group). Protein expression of cleaved Notch (TMNotch) was decreased in old D-depleted rats compared to controls. A.U = arbitrary units. Data presented are means ± SEM; *p < 0.05.
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Fig6: Effect of vitamin D depletion on Notch-1 transcript levels and protein expression in old rats. (A) mRNA expression of full length Notch-1 in tibialis anterior of control and depleted rats was analyzed using a RT2 Profiler Custom PCR Array following the manufacturer’s protocol. Full length Notch-1 mRNA in control and vitamin D depleted samples was normalized using expression of Tbp as a housekeeping gene and was relative to control group according to the 2-ΔΔCT method (n = 7 for control group and n = 9 for depleted group). mRNA expression of full length Notch was unaffected by vitamin D depletion. Data presented are means ± SEM; *p < 0.05. (B) Protein expression of transmembrane fragment Notch-1 (TMNotch-1) was analyzed in tibialis anterior by western blotting and quantified using Multi Gauge V3.2 software. Expression of the total amount of p38 was used to normalize protein loading between samples (n = 7 for control group and n = 9 for depleted group). Protein expression of cleaved Notch (TMNotch) was decreased in old D-depleted rats compared to controls. A.U = arbitrary units. Data presented are means ± SEM; *p < 0.05.

Mentions: The Notch pathway regulates skeletal muscle proliferation. Data from the PCR array showed that the expression of Notch intermediates was altered in the vitamin D-depleted group. Delta-1 mRNA level was significantly reduced by 63% in the TA of vitamin D-depleted rats compared to controls (Figure 5A). Unexpectedly, despite a significant reduction in its transcript level, Delta-1 protein expression remained unchanged between the two groups (control vs. depleted: 91.28 ± 19.23 vs. 85.27 ± 16.03, A.U., p = NS, Figure 5B). Although mRNA expression of full length Notch was unaffected by vitamin D depletion (control vs. depleted: 1 ± 0.17 vs. 0.86 ± 0.15, p = NS Figure 6A), protein expression of cleaved Notch (TMNotch) was decreased in old D-depleted rats compared to controls (-53%, p < 0.05, Figure 6B). Following the activation of the Notch pathway, the intracellular domain of the Notch receptor translocates to the nucleus where it acts as a transcription factor to promote the transcription of its target genes. Therefore, we analysed the mRNA levels of its target Hes1. In addition, as already shown, the variation of the mRNA expression of Notch targets reflects the Notch pathway activity[21, 24]. We observed that the expression of Hes1 was significantly reduced after vitamin D depletion in old rats (-35%, p < 0.05, Figure 7).Figure 5


Vitamin D deficiency down-regulates Notch pathway contributing to skeletal muscle atrophy in old wistar rats.

Domingues-Faria C, Chanet A, Salles J, Berry A, Giraudet C, Patrac V, Denis P, Bouton K, Goncalves-Mendes N, Vasson MP, Boirie Y, Walrand S - Nutr Metab (Lond) (2014)

Effect of vitamin D depletion on Notch-1 transcript levels and protein expression in old rats. (A) mRNA expression of full length Notch-1 in tibialis anterior of control and depleted rats was analyzed using a RT2 Profiler Custom PCR Array following the manufacturer’s protocol. Full length Notch-1 mRNA in control and vitamin D depleted samples was normalized using expression of Tbp as a housekeeping gene and was relative to control group according to the 2-ΔΔCT method (n = 7 for control group and n = 9 for depleted group). mRNA expression of full length Notch was unaffected by vitamin D depletion. Data presented are means ± SEM; *p < 0.05. (B) Protein expression of transmembrane fragment Notch-1 (TMNotch-1) was analyzed in tibialis anterior by western blotting and quantified using Multi Gauge V3.2 software. Expression of the total amount of p38 was used to normalize protein loading between samples (n = 7 for control group and n = 9 for depleted group). Protein expression of cleaved Notch (TMNotch) was decreased in old D-depleted rats compared to controls. A.U = arbitrary units. Data presented are means ± SEM; *p < 0.05.
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Related In: Results  -  Collection

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Fig6: Effect of vitamin D depletion on Notch-1 transcript levels and protein expression in old rats. (A) mRNA expression of full length Notch-1 in tibialis anterior of control and depleted rats was analyzed using a RT2 Profiler Custom PCR Array following the manufacturer’s protocol. Full length Notch-1 mRNA in control and vitamin D depleted samples was normalized using expression of Tbp as a housekeeping gene and was relative to control group according to the 2-ΔΔCT method (n = 7 for control group and n = 9 for depleted group). mRNA expression of full length Notch was unaffected by vitamin D depletion. Data presented are means ± SEM; *p < 0.05. (B) Protein expression of transmembrane fragment Notch-1 (TMNotch-1) was analyzed in tibialis anterior by western blotting and quantified using Multi Gauge V3.2 software. Expression of the total amount of p38 was used to normalize protein loading between samples (n = 7 for control group and n = 9 for depleted group). Protein expression of cleaved Notch (TMNotch) was decreased in old D-depleted rats compared to controls. A.U = arbitrary units. Data presented are means ± SEM; *p < 0.05.
Mentions: The Notch pathway regulates skeletal muscle proliferation. Data from the PCR array showed that the expression of Notch intermediates was altered in the vitamin D-depleted group. Delta-1 mRNA level was significantly reduced by 63% in the TA of vitamin D-depleted rats compared to controls (Figure 5A). Unexpectedly, despite a significant reduction in its transcript level, Delta-1 protein expression remained unchanged between the two groups (control vs. depleted: 91.28 ± 19.23 vs. 85.27 ± 16.03, A.U., p = NS, Figure 5B). Although mRNA expression of full length Notch was unaffected by vitamin D depletion (control vs. depleted: 1 ± 0.17 vs. 0.86 ± 0.15, p = NS Figure 6A), protein expression of cleaved Notch (TMNotch) was decreased in old D-depleted rats compared to controls (-53%, p < 0.05, Figure 6B). Following the activation of the Notch pathway, the intracellular domain of the Notch receptor translocates to the nucleus where it acts as a transcription factor to promote the transcription of its target genes. Therefore, we analysed the mRNA levels of its target Hes1. In addition, as already shown, the variation of the mRNA expression of Notch targets reflects the Notch pathway activity[21, 24]. We observed that the expression of Hes1 was significantly reduced after vitamin D depletion in old rats (-35%, p < 0.05, Figure 7).Figure 5

Bottom Line: A reduction in Notch pathway activity may explain the age-related decrease in satellite cell proliferation, as this pathway has been implicated in satellite cell self-renewal.An unpaired student's t-test was performed to test the effect of the experimental conditions.The D-depleted group showed -39%, -31% drops in expression of two markers known to modulate proliferation (Bmp4, Fgf-2 mRNA levels) and -56% drop in one marker of cell proliferation (PCNA protein expression) compared to controls (p < 0.05).

View Article: PubMed Central - PubMed

Affiliation: Université d'Auvergne, Unité de Nutrition Humaine, Equipe ECREIN, CLARA, CRNH Auvergne; INRA, UMR 1019, UNH, CRNH Auvergne, Clermont Université, 63000 Clermont-Ferrand, France ; Université d'Auvergne, Unité de Nutrition Humaine, Equipe NuTriM, CRNH Auvergne; INRA, UMR 1019, UNH, CRNH Auvergne, Clermont Université, 63000 Clermont-Ferrand, France ; INRA, UMR1019, UNH, CRNH Auvergne, 63000 Clermont-Ferrand, France.

ABSTRACT

Background: The diminished ability of aged muscle to self-repair is a factor behind sarcopenia and contributes to muscle atrophy. Muscle repair depends on satellite cells whose pool size is diminished with aging. A reduction in Notch pathway activity may explain the age-related decrease in satellite cell proliferation, as this pathway has been implicated in satellite cell self-renewal. Skeletal muscle is a target of vitamin D which modulates muscle cell proliferation and differentiation in vitro and stimulates muscle regeneration in vivo. Vitamin D status is positively correlated to muscle strength/function, and elderly populations develop a vitamin D deficiency. The aim of this study was to evaluate how vitamin D deficiency induces skeletal muscle atrophy in old rats through a reduction in Notch pathway activity and proliferation potential in muscle.

Methods: 15-month-old male rats were vitamin D-depleted or not (control) for 9 months (n = 10 per group). Rats were 24-month-old at the end of the experiment. Gene and/or protein expression of markers of proliferation, or modulating proliferation, and of Notch signalling pathway were studied in the tibialis anterior muscle by qPCR and western blot. An unpaired student's t-test was performed to test the effect of the experimental conditions.

Results: Vitamin D depletion led to a drop in concentrations of plasma 25-hydroxyvitamin D in depleted rats compared to controls (-74%, p < 0.01). Tibialis anterior weight was decreased in D-depleted rats (-25%, p < 0.05). The D-depleted group showed -39%, -31% drops in expression of two markers known to modulate proliferation (Bmp4, Fgf-2 mRNA levels) and -56% drop in one marker of cell proliferation (PCNA protein expression) compared to controls (p < 0.05). Notch pathway activity was blunted in tibialis anterior of D-depleted rats compared to controls, seen as a down-regulation of cleaved Notch (-53%, p < 0.05) and its target Hes1 (-35%, p < 0.05).

Conclusions: A 9-month vitamin D depletion induced vitamin D deficiency in old rats. Vitamin D depletion induces skeletal muscle atrophy in old rats through a reduction in Notch pathway activity and proliferation potential. Vitamin D deficiency could aggravate the age-related decrease in muscle regeneration capacity.

No MeSH data available.


Related in: MedlinePlus