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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 āˆ¼40% 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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Related in: MedlinePlus

Altered dietary protein intake affects plasma and intramuscular AA concentrations and mTORC1 pathway signalling in MS-Slc7a5-KO mice.Mean Ā± SEM for nā€Š=ā€Š6ā€“10 (WT) and 3ā€“5 (MS-Slc7a5-KO) male mice. (A) There were significant effects of dietary protein content (F (2, 27)ā€Š=ā€Š4.97, pā€Š=ā€Š.014) on gastrocnemius leucine concentration. Statistically-significant differences between groups were only detected for MS-Slc7a5-KO animals on different protein diets (**D, p<0.01) as indicated. (B) There were significant effects of both genotype (F (1, 23)ā€Š=ā€Š5.55, pā€Š=ā€Š.027) and dietary protein content (F (2, 23)ā€Š=ā€Š4.49, pā€Š=ā€Š.023) on plasma leucine concentration. Statistically-significant differences between genotype (***G, p<0.001) and dietary protein (***D, p<0.001; wild-type only) groups are indicated. (C) Upper panel shows quantitation of S6K phosphorylation normalised to effect of insulin injection (wild-type mouse on standard chow diet), lower panel shows representative western blot for phospho-S6K and total S6K. Animals injected with NaCl and insulin were used as a negative and positive control, respectively. 2-way ANOVA shows significant effects of both genotype (F (1, 27)ā€Š=ā€Š9.61, pā€Š=ā€Š.004) and dietary protein content (F (2, 27)ā€Š=ā€Š4.97, pā€Š=ā€Š.015) on S6K phosphorylation. Statistically-significant differences between genotype (**G, p<0.01) and diet (**D, p<0.001; wild-type only) groups are indicated.
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pone-0089547-g006: Altered dietary protein intake affects plasma and intramuscular AA concentrations and mTORC1 pathway signalling in MS-Slc7a5-KO mice.Mean Ā± SEM for nā€Š=ā€Š6ā€“10 (WT) and 3ā€“5 (MS-Slc7a5-KO) male mice. (A) There were significant effects of dietary protein content (F (2, 27)ā€Š=ā€Š4.97, pā€Š=ā€Š.014) on gastrocnemius leucine concentration. Statistically-significant differences between groups were only detected for MS-Slc7a5-KO animals on different protein diets (**D, p<0.01) as indicated. (B) There were significant effects of both genotype (F (1, 23)ā€Š=ā€Š5.55, pā€Š=ā€Š.027) and dietary protein content (F (2, 23)ā€Š=ā€Š4.49, pā€Š=ā€Š.023) on plasma leucine concentration. Statistically-significant differences between genotype (***G, p<0.001) and dietary protein (***D, p<0.001; wild-type only) groups are indicated. (C) Upper panel shows quantitation of S6K phosphorylation normalised to effect of insulin injection (wild-type mouse on standard chow diet), lower panel shows representative western blot for phospho-S6K and total S6K. Animals injected with NaCl and insulin were used as a negative and positive control, respectively. 2-way ANOVA shows significant effects of both genotype (F (1, 27)ā€Š=ā€Š9.61, pā€Š=ā€Š.004) and dietary protein content (F (2, 27)ā€Š=ā€Š4.97, pā€Š=ā€Š.015) on S6K phosphorylation. Statistically-significant differences between genotype (**G, p<0.01) and diet (**D, p<0.001; wild-type only) groups are indicated.

Mentions: In order to provide further insight into possible impairment of nutrient-signalling by muscle-specific Slc7a5 knockout, MS-Slc7a5-KO and control mice were challenged with diets of 10%, 20% or 30% protein (low, control and high protein diets respectively) from age 40 days for a 60-day period. Dietary protein compositions were chosen to maintain equivalent energy intake by minimizing possible effects on food intake. No difference was observed in growth rates or food intake between genotypes or diets even though a trend for small (<5%) reductions in heart and skeletal muscle size was noted for MS-Slc7a5-KO mice on the different diets (Figure S3). In wild-type mice, the leucine concentration in gastrocnemius muscle was maintained at around 0.2 nmol/mg across the 10ā€“30% dietary protein range, despite a progressive increase in plasma leucine concentration. In contrast, intramuscular leucine concentration in MS-Slc7a5-KO animals increased in relation to dietary protein content whereas plasma leucine concentration remained unaltered (Figures 6A and B). A similar pattern was observed for isoleucine (Figure S4). On the 30% diet, leucine, isoleucine and glutamine accumulated in muscle of MS-Slc7a5-KO mice relative wild-type mice (Figures 6 and S4). In non-fasted wild-type mice, intramuscular (gastrocnemius) mTOR-S6K activity increased with dietary protein intake (Figures 6 and S5) and was consistently higher than mTOR-S6K activity in muscle from MS-Slc7a5-KO mice on both high- and low-protein diets (Figures 6C and S5). This was observed in gastrocnemius muscle of both male and female mice and also in soleus muscle (Figures S5A and S5B respectively).


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)

Altered dietary protein intake affects plasma and intramuscular AA concentrations and mTORC1 pathway signalling in MS-Slc7a5-KO mice.Mean Ā± SEM for nā€Š=ā€Š6ā€“10 (WT) and 3ā€“5 (MS-Slc7a5-KO) male mice. (A) There were significant effects of dietary protein content (F (2, 27)ā€Š=ā€Š4.97, pā€Š=ā€Š.014) on gastrocnemius leucine concentration. Statistically-significant differences between groups were only detected for MS-Slc7a5-KO animals on different protein diets (**D, p<0.01) as indicated. (B) There were significant effects of both genotype (F (1, 23)ā€Š=ā€Š5.55, pā€Š=ā€Š.027) and dietary protein content (F (2, 23)ā€Š=ā€Š4.49, pā€Š=ā€Š.023) on plasma leucine concentration. Statistically-significant differences between genotype (***G, p<0.001) and dietary protein (***D, p<0.001; wild-type only) groups are indicated. (C) Upper panel shows quantitation of S6K phosphorylation normalised to effect of insulin injection (wild-type mouse on standard chow diet), lower panel shows representative western blot for phospho-S6K and total S6K. Animals injected with NaCl and insulin were used as a negative and positive control, respectively. 2-way ANOVA shows significant effects of both genotype (F (1, 27)ā€Š=ā€Š9.61, pā€Š=ā€Š.004) and dietary protein content (F (2, 27)ā€Š=ā€Š4.97, pā€Š=ā€Š.015) on S6K phosphorylation. Statistically-significant differences between genotype (**G, p<0.01) and diet (**D, p<0.001; wild-type only) groups are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0089547-g006: Altered dietary protein intake affects plasma and intramuscular AA concentrations and mTORC1 pathway signalling in MS-Slc7a5-KO mice.Mean Ā± SEM for nā€Š=ā€Š6ā€“10 (WT) and 3ā€“5 (MS-Slc7a5-KO) male mice. (A) There were significant effects of dietary protein content (F (2, 27)ā€Š=ā€Š4.97, pā€Š=ā€Š.014) on gastrocnemius leucine concentration. Statistically-significant differences between groups were only detected for MS-Slc7a5-KO animals on different protein diets (**D, p<0.01) as indicated. (B) There were significant effects of both genotype (F (1, 23)ā€Š=ā€Š5.55, pā€Š=ā€Š.027) and dietary protein content (F (2, 23)ā€Š=ā€Š4.49, pā€Š=ā€Š.023) on plasma leucine concentration. Statistically-significant differences between genotype (***G, p<0.001) and dietary protein (***D, p<0.001; wild-type only) groups are indicated. (C) Upper panel shows quantitation of S6K phosphorylation normalised to effect of insulin injection (wild-type mouse on standard chow diet), lower panel shows representative western blot for phospho-S6K and total S6K. Animals injected with NaCl and insulin were used as a negative and positive control, respectively. 2-way ANOVA shows significant effects of both genotype (F (1, 27)ā€Š=ā€Š9.61, pā€Š=ā€Š.004) and dietary protein content (F (2, 27)ā€Š=ā€Š4.97, pā€Š=ā€Š.015) on S6K phosphorylation. Statistically-significant differences between genotype (**G, p<0.01) and diet (**D, p<0.001; wild-type only) groups are indicated.
Mentions: In order to provide further insight into possible impairment of nutrient-signalling by muscle-specific Slc7a5 knockout, MS-Slc7a5-KO and control mice were challenged with diets of 10%, 20% or 30% protein (low, control and high protein diets respectively) from age 40 days for a 60-day period. Dietary protein compositions were chosen to maintain equivalent energy intake by minimizing possible effects on food intake. No difference was observed in growth rates or food intake between genotypes or diets even though a trend for small (<5%) reductions in heart and skeletal muscle size was noted for MS-Slc7a5-KO mice on the different diets (Figure S3). In wild-type mice, the leucine concentration in gastrocnemius muscle was maintained at around 0.2 nmol/mg across the 10ā€“30% dietary protein range, despite a progressive increase in plasma leucine concentration. In contrast, intramuscular leucine concentration in MS-Slc7a5-KO animals increased in relation to dietary protein content whereas plasma leucine concentration remained unaltered (Figures 6A and B). A similar pattern was observed for isoleucine (Figure S4). On the 30% diet, leucine, isoleucine and glutamine accumulated in muscle of MS-Slc7a5-KO mice relative wild-type mice (Figures 6 and S4). In non-fasted wild-type mice, intramuscular (gastrocnemius) mTOR-S6K activity increased with dietary protein intake (Figures 6 and S5) and was consistently higher than mTOR-S6K activity in muscle from MS-Slc7a5-KO mice on both high- and low-protein diets (Figures 6C and S5). This was observed in gastrocnemius muscle of both male and female mice and also in soleus muscle (Figures S5A and S5B respectively).

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 āˆ¼40% 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