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. 2013 Mar 15;10(1):28.
doi: 10.1186/1743-7075-10-28.

Supplementation of Carnitine Leads to an Activation of the IGF-1/PI3K/Akt Signalling Pathway and Down Regulates the E3 Ligase MuRF1 in Skeletal Muscle of Rats

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Free PMC article

Supplementation of Carnitine Leads to an Activation of the IGF-1/PI3K/Akt Signalling Pathway and Down Regulates the E3 Ligase MuRF1 in Skeletal Muscle of Rats

Janine Keller et al. Nutr Metab (Lond). .
Free PMC article

Abstract

Background: Recently, it has been shown that carnitine down-regulates genes involved in the ubiquitin-proteasome system (UPS) in muscle of pigs and rats. The mechanisms underlying this observation are yet unknown. Based on the previous finding that carnitine increases plasma IGF-1 concentration, we investigated the hypothesis that carnitine down-regulates genes of the UPS by modulation of the of the IGF-1/PI3K/Akt signalling pathway which is an important regulator of UPS activity in muscle.

Methods: Male Sprague-Dawley rats, aged four weeks, were fed either a control diet with a low native carnitine concentration or the same diet supplemented with carnitine (1250 mg/kg diet) for four weeks. Components of the UPS and IGF-1/PI3K/Akt signalling pathway in skeletal muscle were examined.

Results: Rats fed the diet supplemented with carnitine had lower mRNA and protein levels of MuRF1, the most important E3 ubiquitin ligase in muscle, decreased concentrations of ubiquitin-protein conjugates in skeletal muscle and higher IGF-1 concentration in plasma than control rats (P < 0.05). Moreover, in skeletal muscle of rats fed the diet supplemented with carnitine there was an activation of the PI3K/Akt signalling pathway, as indicated by increased protein levels of phosphorylated (activated) Akt1 (P < 0.05).

Conclusion: The present study shows that supplementation of carnitine markedly decreases the expression of MuRF1 and concentrations of ubiquitinated proteins in skeletal muscle of rats, indicating a diminished degradation of myofibrillar proteins by the UPS. The study moreover shows that supplementation of carnitine leads to an activation of the IGF-1/PI3K/Akt signalling pathway which in turn might contribute to the observed down-regulation of MuRF1 and muscle protein ubiquitination.

Figures

Figure 1
Figure 1
Relative mRNA abundance of FBXO32 and TRIM63 (A) and relative protein concentrations of atrogin-1 and MuRF1 (B,C) in M. quadriceps femoris of rats fed either a control diet (0 mg carnitine/kg diet; Control) or a diet supplemented with 1250 mg carnitine/kg diet (Carnitine). (A) Bars are means ± SD (n = 12/group). The normalized expression ratio in the control group is set to 1.0. * indicates a significant difference to the control group (P < 0.05). (B) Representative immunoblots specific to atrogin-1, MuRF1 and GAPDH as internal control are shown for one animal per group; immunoblots for the other animals revealed similar results. (C) Bars represent data from densitometric analysis and represent means ± SD (n = 6/group); bars are expressed relative to the protein level of the control group (= 1.00). * indicates a significant difference to the control group (P < 0.05).
Figure 2
Figure 2
Relative protein levels of ubiquitin-protein conjugates in M. quadriceps femoris of rats fed either a control diet (0 mg carnitine/kg diet; Control) or a diet supplemented with 1250 mg carnitine/kg diet (Carnitine). (A) A representative immunoblot specific to ubiquitin is shown for three animals per group; immunoblots for the other animals revealed similar results. Reversible staining of nitrocellulose membranes with Ponceau S revealed equal loading of protein. (B) Bars represent data from densitometric analysis and represent means ± SD (n = 6/group); bars are expressed relative to the protein level of the control group (= 1.00). * indicates a significant difference to the control group (P < 0.05).
Figure 3
Figure 3
(A) Relative mRNA abundance of IGF-1 in liver of rats fed either a control diet (0 mg carnitine/kg diet; Control) or a diet supplemented with 1250 mg carnitine/kg diet (Carnitine). Bars are means ± SD (n = 12/group). The normalized expression ratio in the control group is set to 1.0. * indicates a significant difference to the control group (P < 0.05). (B) Concentration of IGF-1 (ng/ml) in plasma of rats fed either a control diet (0 mg carnitine/kg diet; Control) or a diet supplemented with 1250 mg carnitine/kg diet (Carnitine). Bars represent mean ± SD (n = 12/group). * indicates a significant difference to the control group (P < 0.05).
Figure 4
Figure 4
Relative protein concentrations of total and phosphorylated Akt1 and FoxO1 and calculated phospho-Akt1/total Akt1 and phospho-FoxO1/total FoxO1 ratios in M. quadriceps femoris of rats fed either a control diet (0 mg carnitine/kg diet; Control) or a diet supplemented with 1250 mg carnitine/kg diet (Carnitine). (A) Representative immunoblots specific to total Akt1, phospho-Akt1, total FoxO1, phospho-FoxO1 and GAPDH as internal control are shown for one animal per group; immunoblots for the other animals revealed similar results. (B) Bars represent data from densitometric analysis and represent means ± SD (n = 6/group); bars are expressed relative to the protein level of the control group (= 1.00). * indicates a significant difference to the control group (P < 0.05).
Figure 5
Figure 5
Relative protein concentrations of total and phosphorylated mTOR at Ser2448 and Ser2481 and calculated phospho-mTOR (Ser2448)/total mTOR and phosphor-mTOR (Ser2481)/total mTOR ratios in M. quadriceps femoris of rats fed either a control diet (0 mg carnitine/kg diet; Control) or a diet supplemented with 1250 mg carnitine/kg diet (Carnitine). (A) Representative immunoblots specific to total mTOR, phosphor-mTOR (Ser2448), phosphor-mTOR (Ser2481) and α-Tubulin as internal control are shown for one animal per group; immunoblots for the other animals revealed similar results. (B) Bars represent data from densitometric analysis and represent means ± SD (n = 6/group); bars are expressed relative to the protein level of the control group (= 1.00). * indicates a significant difference to the control group (P < 0.05).

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