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The catalytic subunit of the system L1 amino acid transporter (slc7a5) facilitates nutrient signalling in mouse skeletal muscle.

Poncet N, Mitchell FE, Ibrahim AF, McGuire VA, English G, Arthur JS, Shi YB, Taylor PM - PLoS ONE (2014)

Bottom Line: The SLC7A5 (LAT1) catalytic subunit of high-affinity System L1 functions as a glycoprotein-associated heterodimer with the multifunctional protein SLC3A2 (CD98).These changes were associated with >50% decrease in S6K Thr389 phosphorylation in muscles from MS-Slc7a5-KO mice, indicating reduced mTOR-S6K pathway activation, despite no significant differences in lean tissue mass between groups on the same diet.MS-Slc7a5-KO mice on 30% protein diet exhibited mild insulin resistance (e.g. reduced glucose clearance, larger gonadal adipose depots) relative to control animals.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee, United Kingdom.

ABSTRACT
The System L1-type amino acid transporter mediates transport of large neutral amino acids (LNAA) in many mammalian cell-types. LNAA such as leucine are required for full activation of the mTOR-S6K signalling pathway promoting protein synthesis and cell growth. The SLC7A5 (LAT1) catalytic subunit of high-affinity System L1 functions as a glycoprotein-associated heterodimer with the multifunctional protein SLC3A2 (CD98). We generated a floxed Slc7a5 mouse strain which, when crossed with mice expressing Cre driven by a global promoter, produced Slc7a5 heterozygous knockout (Slc7a5+/-) animals with no overt phenotype, although homozygous global knockout of Slc7a5 was embryonically lethal. Muscle-specific (MCK Cre-mediated) Slc7a5 knockout (MS-Slc7a5-KO) mice were used to study the role of intracellular LNAA delivery by the SLC7A5 transporter for mTOR-S6K pathway activation in skeletal muscle. Activation of muscle mTOR-S6K (Thr389 phosphorylation) in vivo by intraperitoneal leucine injection was blunted in homozygous MS-Slc7a5-KO mice relative to wild-type animals. Dietary intake and growth rate were similar for MS-Slc7a5-KO mice and wild-type littermates fed for 10 weeks (to age 120 days) with diets containing 10%, 20% or 30% of protein. In MS-Slc7a5-KO mice, Leu and Ile concentrations in gastrocnemius muscle were reduced by ∼40% as dietary protein content was reduced from 30 to 10%. These changes were associated with >50% decrease in S6K Thr389 phosphorylation in muscles from MS-Slc7a5-KO mice, indicating reduced mTOR-S6K pathway activation, despite no significant differences in lean tissue mass between groups on the same diet. MS-Slc7a5-KO mice on 30% protein diet exhibited mild insulin resistance (e.g. reduced glucose clearance, larger gonadal adipose depots) relative to control animals. Thus, SLC7A5 modulates LNAA-dependent muscle mTOR-S6K signalling in mice, although it appears non-essential (or is sufficiently compensated by e.g. SLC7A8 (LAT2)) for maintenance of normal muscle mass.

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Both Slc7a5 gene expression and SLC7A5 transport activity are reduced in Slc7a5+/− mouse tissues.(A) Slc7a5 mRNA expression in liver, heart and intestine (as indicated) from Elalpha-Cre Slc7a5+/+ (n = 5–9) and Slc7a5+/− (n = 4–5) mice as determined by qPCR and normalized to β-Actin. Intestine was determined not significant (N.S.) although p<0.1. (B) Slc7a5 mRNA expression in diaphragm from Slc7a5+/+ and Slc7a5+/− mice as determined by qPCR and normalized to β-Actin (n = 6). Uptake of 3H-phenylalanine into diaphragm from Slc7a5+/+ and Slc7a5+/− mice (n = 5). *and ***indicate p<0.05 and p<0.001 respectively by unpaired t-test. (C) Representative Western blot of SLC7A5 protein in heart and soleus muscle lysates from from Slc7a5+/+ and Slc7a5+/− mice. Blot quantitation shown in lower panels.
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pone-0089547-g002: Both Slc7a5 gene expression and SLC7A5 transport activity are reduced in Slc7a5+/− mouse tissues.(A) Slc7a5 mRNA expression in liver, heart and intestine (as indicated) from Elalpha-Cre Slc7a5+/+ (n = 5–9) and Slc7a5+/− (n = 4–5) mice as determined by qPCR and normalized to β-Actin. Intestine was determined not significant (N.S.) although p<0.1. (B) Slc7a5 mRNA expression in diaphragm from Slc7a5+/+ and Slc7a5+/− mice as determined by qPCR and normalized to β-Actin (n = 6). Uptake of 3H-phenylalanine into diaphragm from Slc7a5+/+ and Slc7a5+/− mice (n = 5). *and ***indicate p<0.05 and p<0.001 respectively by unpaired t-test. (C) Representative Western blot of SLC7A5 protein in heart and soleus muscle lysates from from Slc7a5+/+ and Slc7a5+/− mice. Blot quantitation shown in lower panels.

Mentions: Slc7a5 mRNA expression was significantly reduced (by around half) in heart, skeletal muscle (e.g. diaphragm) and liver of Slc7a5+/− mice in comparison with Slc7a5+/+ littermates (Figure 2). Reduced SLC7A5 protein expression was also detected in heart and soleus muscle of Slc7a5+/− mice relative to Slc7a5+/+ littermates (Figure 2C). We confirmed a reduction in functional SLC7A5 expression in diaphragm muscle by demonstrating that 5 µM phenylalanine uptake was significantly lower in Slc7a5+/− diaphragm compared to wild-type controls (Figure 2B). Nevertheless, there were no significant differences between mass of tissues including skeletal muscles (gastrocnemius, soleus), heart or liver for Slc7a5+/− mice and wild-type littermates (data not shown), nor in AA content of tissues or plasma (Table S1).


The catalytic subunit of the system L1 amino acid transporter (slc7a5) facilitates nutrient signalling in mouse skeletal muscle.

Poncet N, Mitchell FE, Ibrahim AF, McGuire VA, English G, Arthur JS, Shi YB, Taylor PM - PLoS ONE (2014)

Both Slc7a5 gene expression and SLC7A5 transport activity are reduced in Slc7a5+/− mouse tissues.(A) Slc7a5 mRNA expression in liver, heart and intestine (as indicated) from Elalpha-Cre Slc7a5+/+ (n = 5–9) and Slc7a5+/− (n = 4–5) mice as determined by qPCR and normalized to β-Actin. Intestine was determined not significant (N.S.) although p<0.1. (B) Slc7a5 mRNA expression in diaphragm from Slc7a5+/+ and Slc7a5+/− mice as determined by qPCR and normalized to β-Actin (n = 6). Uptake of 3H-phenylalanine into diaphragm from Slc7a5+/+ and Slc7a5+/− mice (n = 5). *and ***indicate p<0.05 and p<0.001 respectively by unpaired t-test. (C) Representative Western blot of SLC7A5 protein in heart and soleus muscle lysates from from Slc7a5+/+ and Slc7a5+/− mice. Blot quantitation shown in lower panels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3935884&req=5

pone-0089547-g002: Both Slc7a5 gene expression and SLC7A5 transport activity are reduced in Slc7a5+/− mouse tissues.(A) Slc7a5 mRNA expression in liver, heart and intestine (as indicated) from Elalpha-Cre Slc7a5+/+ (n = 5–9) and Slc7a5+/− (n = 4–5) mice as determined by qPCR and normalized to β-Actin. Intestine was determined not significant (N.S.) although p<0.1. (B) Slc7a5 mRNA expression in diaphragm from Slc7a5+/+ and Slc7a5+/− mice as determined by qPCR and normalized to β-Actin (n = 6). Uptake of 3H-phenylalanine into diaphragm from Slc7a5+/+ and Slc7a5+/− mice (n = 5). *and ***indicate p<0.05 and p<0.001 respectively by unpaired t-test. (C) Representative Western blot of SLC7A5 protein in heart and soleus muscle lysates from from Slc7a5+/+ and Slc7a5+/− mice. Blot quantitation shown in lower panels.
Mentions: Slc7a5 mRNA expression was significantly reduced (by around half) in heart, skeletal muscle (e.g. diaphragm) and liver of Slc7a5+/− mice in comparison with Slc7a5+/+ littermates (Figure 2). Reduced SLC7A5 protein expression was also detected in heart and soleus muscle of Slc7a5+/− mice relative to Slc7a5+/+ littermates (Figure 2C). We confirmed a reduction in functional SLC7A5 expression in diaphragm muscle by demonstrating that 5 µM phenylalanine uptake was significantly lower in Slc7a5+/− diaphragm compared to wild-type controls (Figure 2B). Nevertheless, there were no significant differences between mass of tissues including skeletal muscles (gastrocnemius, soleus), heart or liver for Slc7a5+/− mice and wild-type littermates (data not shown), nor in AA content of tissues or plasma (Table S1).

Bottom Line: The SLC7A5 (LAT1) catalytic subunit of high-affinity System L1 functions as a glycoprotein-associated heterodimer with the multifunctional protein SLC3A2 (CD98).These changes were associated with >50% decrease in S6K Thr389 phosphorylation in muscles from MS-Slc7a5-KO mice, indicating reduced mTOR-S6K pathway activation, despite no significant differences in lean tissue mass between groups on the same diet.MS-Slc7a5-KO mice on 30% protein diet exhibited mild insulin resistance (e.g. reduced glucose clearance, larger gonadal adipose depots) relative to control animals.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee, United Kingdom.

ABSTRACT
The System L1-type amino acid transporter mediates transport of large neutral amino acids (LNAA) in many mammalian cell-types. LNAA such as leucine are required for full activation of the mTOR-S6K signalling pathway promoting protein synthesis and cell growth. The SLC7A5 (LAT1) catalytic subunit of high-affinity System L1 functions as a glycoprotein-associated heterodimer with the multifunctional protein SLC3A2 (CD98). We generated a floxed Slc7a5 mouse strain which, when crossed with mice expressing Cre driven by a global promoter, produced Slc7a5 heterozygous knockout (Slc7a5+/-) animals with no overt phenotype, although homozygous global knockout of Slc7a5 was embryonically lethal. Muscle-specific (MCK Cre-mediated) Slc7a5 knockout (MS-Slc7a5-KO) mice were used to study the role of intracellular LNAA delivery by the SLC7A5 transporter for mTOR-S6K pathway activation in skeletal muscle. Activation of muscle mTOR-S6K (Thr389 phosphorylation) in vivo by intraperitoneal leucine injection was blunted in homozygous MS-Slc7a5-KO mice relative to wild-type animals. Dietary intake and growth rate were similar for MS-Slc7a5-KO mice and wild-type littermates fed for 10 weeks (to age 120 days) with diets containing 10%, 20% or 30% of protein. In MS-Slc7a5-KO mice, Leu and Ile concentrations in gastrocnemius muscle were reduced by ∼40% as dietary protein content was reduced from 30 to 10%. These changes were associated with >50% decrease in S6K Thr389 phosphorylation in muscles from MS-Slc7a5-KO mice, indicating reduced mTOR-S6K pathway activation, despite no significant differences in lean tissue mass between groups on the same diet. MS-Slc7a5-KO mice on 30% protein diet exhibited mild insulin resistance (e.g. reduced glucose clearance, larger gonadal adipose depots) relative to control animals. Thus, SLC7A5 modulates LNAA-dependent muscle mTOR-S6K signalling in mice, although it appears non-essential (or is sufficiently compensated by e.g. SLC7A8 (LAT2)) for maintenance of normal muscle mass.

Show MeSH
Related in: MedlinePlus