Dihydrotestosterone stimulates amino acid uptake and the expression of LAT2 in mouse skeletal muscle fibres through an ERK1/2-dependent mechanism

Abstract

Dihydrotestosterone (DHT) has acute/non-genomic actions in adult mammalian skeletal muscles whose physiological functions are still poorly understood. Therefore, the primary aim of this study was to investigate the acute/non-genomic effects of DHT on amino acid uptake as well as the cellular signal transduction events underlying these actions in mouse fast- and slow-twitch skeletal muscle fibre bundles. 14C-Labelled amino acids were used to investigate the effects of DHT and testosterone (T) on amino acid uptake and pharmacological interventions were used to determine the cellular signal transduction events mediating these actions. While T had no effect on the uptake of isoleucine (Ile) and α-methylaminoisobutyric acid (MeAIB) in both fibre types, DHT increased their uptake in the fast-twitch fibre bundles. This effect was reversed by inhibitors of protein translation, the epidermal growth factor receptor (EGFR), system A, system L, mTOR and MEK. However, it was relatively insensitive to inhibitors of transcription, androgen receptors and PI3K/Akt. Additionally, DHT treatment increased the expression of LAT2 and the phosphorylation of the EGFR in the fast-twitch fibre bundles and that of ERK1/2, RSK1/2 and ATF2 in both fibre types. Also, it decreased the phosphorylation of eEF2 and increased the incorporation of Ile into proteins in both fibre types. Most of these effects were reversed by EGFR and MEK inhibitors. From these findings we suggest that another physiological function of the acute/non-genomic actions of DHT in isolated mammalian skeletal muscle fibres is to stimulate amino acid uptake. This effect is mediated through the EGFR and involves the activation of the MAPK pathway and an increase in LAT2 expression.

Figure 1. DHT but not T increases amino acid uptake in fast-twitch skeletal muscle fibre bundles

Bar graphs showing the effects of treating fast- (filled bars) and slow- (open bars) twitch fibre bundles with physiological levels of DHT (A and B) and T (C and D) on the uptake of l-[U-¹⁴C]Ile (A and C) and α-[1-¹⁴C]MeAIB (B and D). Note that treating the fibre bundles with DHT, but not T, significantly (P = 0.001) increases the uptake of both l-[U-¹⁴C]Ile and α-[1-¹⁴C]MeAIB) in the fast-twitch fibre bundles. In contrast, DHT had little effect on the uptake of both amino acids in the slow-twitch bundles. *P < 0.05 when compared to control.

Figure 2. The effects of DHT on amino acid uptake in mouse skeletal muscle fibre bundles are partly mediated through a system A amino acid transporter

A, bar graphs showing the effects of treating fast-twitch (filled bars) and slow-twitch (open bars) muscle fibre bundles with DHT and MeAIB, as shown, on the uptake of l-[U-¹⁴C]Ile. B, a representative Western blot and summary data showing the effects of DHT on the expression of SNAT2 in cytosolic proteins extracted from fast- (F, filled bars) and slow- (S, open bars) twitch muscle fibre bundles. Note that treating the muscle fibre bundles with MeAIB significantly (P < 0.05) reduces the basal as well as the DHT-induced increase in Ile uptake in both fibre types. Furthermore the antibody labels a single band with a molecular mass of ∼60 kDa and DHT treatment has no effect on the expression of SNAT2. FT and ST are the fast- and slow-twitch preparations treated with DHT, respectively. *P < 0.05 compared to control; †P < 0.05 compared to DHT treatment.

Figure 3. The effects of DHT on amino acid uptake in mouse skeletal muscle fibre bundles are mediated through LAT2

A, bar graphs showing the effects of treating fast- (filled bars) and slow- (open bars) twitch muscle fibre bundles with DHT and BCH, as shown, on the uptake of l-[U-¹⁴C]Ile. B, a typical Western blot and summary data showing the effects of DHT on the expression of LAT2 in membrane proteins extracted from fast- (F, filled bars) and slow- (S, open bars) twitch fibre bundles. Note that treating the muscle fibre bundles with BCH significantly (P < 0.05) reduces the basal as well as the DHT-induced increase in Ile uptake in both fibre types. Additionally, the antibody labels a single band with a molecular mass of ∼60 kDa and that DHT treatment increases the expression of LAT2 in the fast-twitch fibres only. FT and ST are the fast- and slow-twitch preparations treated with DHT, respectively. *P < 0.05 compared to control; †P < 0.05 compared to DHT treatment.

Figure 4. The effects of DHT on amino acid uptake in mouse skeletal muscle fibre bundles are partly mediated through the androgen receptor

Bar graphs showing the effects of treating fast- (filled bars) and slow- (clear bars) twitch skeletal muscle fibre bundles with the standard Ringer solution or the Ringer solution plus physiological levels of DHT alone or DHT plus the androgen receptor inhibitors cyproterone (A) and flutamide (B). Note that treatment of the fibre bundles with cyproterone alone significantly (P = 0.001 and 0.005 in the fast- and slow-twitch fibres, respectively) increases the basal uptake of l-[U-¹⁴C]Ile in both fibre types. In contrast, flutamide on its own significantly (P = 0.028) increases amino acid uptake in the fast-twitch fibre bundles only. Additionally, pre-treating the fibre bundles with both compounds significantly (P < 0.05) reduces the DHT-induced increase in Ile uptake. However, these effects were greater in the fibre bundles pre-treated with flutamide than in those pre-treated with cyproterone. *P < 0.05 compared to control; †P < 0.05 compared to DHT treatment.

Figure 5. The effects of DHT on amino acid uptake in mouse skeletal muscle fibre bundles are mediated through the epidermal growth factor receptor (EGFR)

A, bar graphs showing the effects of treating fast- (filled bars) and slow- (clear bars) twitch muscle fibre bundles with either DHT or tyrphostin AG1478 as shown in the figure. Note that treating the fibre bundles with AG1478 significantly (P < 0.05) reduces the basal uptake of (l-[U-¹⁴C] Ile) in both fibre types. Also, it completely abolishes the DHT-induced increase in amino acid uptake in the fast-twitch muscle fibre bundles. B, a Western blot showing the effects of treating the muscle fibre bundles with DHT alone or DHT plus AG1478 on the phosporylation of the EGFR in membrane proteins. Note that the antibody labels a single band with a molecular mass of ∼170 kDa and that treating the bundles with DHT increases the phosphorylation of EGFR and this effect is completely reversed by AG1478. *P < 0.05 compared to control; †P < 0.05 compared to DHT treatment.

Figure 6. The effects of DHT on amino acid uptake in mouse skeletal muscle fibre bundles are mediated through the ERK pathway

A, bar graphs showing the effects of treating fast- (filled bars) and slow- (open bars) twitch muscle fibre bundles with either DHT or the MEK inhibitor PD98059, as shown in the figure, on the uptake of l-[U-¹⁴C]Ile. Note that treating the fibre bundles with PD98059 significantly reduces the basal uptake of Ile in both fibre types. Also, it completely abolishes the DHT-induced increase in Ile. B, Western blots showing the effects of treating muscle fibre bundles with DHT on the phosphorylation of ERK1/2, RSK1/2 and MSK1/2. Note that treating the muscle fibre bundles with DHT increases the phosphorylation of ERK1/2 and RSK1/2 but not that of MSK1/2. C, Western blots showing the effects of treating small muscle fibre bundles with DHT and PD98059 (as shown) on the cytosolic concentrations of phosphorylated ERK1/2 and RSK1/2. Note that the pERK antibody labels two bands with molecular masses of ∼42 and 44 kDa, whereas that of pRSK labels two bands with molecular masses of ∼78 and 80 kDa. Furthermore, treating the bundles with DHT increases the phosphorylation of ERK1/2 and RSK1/2 and that pre-treatment of the fibre bundles with PD98059 completely abolishes the effects of DHT. D, typical Western blots showing the effects of DHT on the cytosolic concentrations of phosphorylated JNK and p38. Note that the pJNK antibody labels a single band with a molecular mass of ∼60 kDa, whereas that of pp38 labels a band with a molecular mass of ∼42 kDa. Furthermore, DHT treatment has no effect on the phosphorylation of either kinase. *P < 0.05 compared to control; †P < 0.05 compared to DHT treatment.

Figure 7. The effects of DHT on amino acid uptake in mouse skeletal muscle fibre bundles are not mediated through the Akt

A and B, bar graphs showing the effects of treating fast- (filled bars) and slow- (open bars) twitch muscle fibre bundles, with the compounds shown below each panel, on the uptake of l-[U-¹⁴C]Ile. Note that treating the fibre bundles with either deguelin or rapamycin alone significantly (P = 0.033 for deguelin and 0.025 for rapamycin) reduces the basal uptake of l-[U-¹⁴C]Ile in both fibre types. Furthermore, rapamycin but not deguelin abolished the DHT-induced increase in Ile uptake. *P < 0.05 compared to control; †P < 0.05 compared to DHT treatment.

Figure 8. Actinomycin D blunts the effects of DHT on amino acid uptake in mouse skeletal muscle fibre bundles

A, bar graphs showing the effects of treating fast- (filled bars) and slow- (open bars) twitch muscle fibre bundles with Ringer solution containing either DHT or actinomycin D on the uptake of l-[U-¹⁴C]Ile. Note that treating the muscle fibre bundles with actinomycin D alone had no effect on the basal uptake of Ile in both fibre types. Moreover, it blunts without completely abolishing the DHT-induced increase in Ile uptake. B, representative Western blots showing the effects of DHT on the expression of pc-Jun, myoD, cmyc and pATF2. Note that DHT does not significantly (P > 0.05) affect the phosphorylation/expression of any of the transcriptional factors except ATF2. *P < 0.05 compared to control; †P < 0.05 compared to DHT treatment.

Figure 9. Actinomycin D blunts the effects of DHT on amino acid uptake in mouse skeletal muscle fibre bundles

A, bar graphs showing the effects of treating fast- (filled bars) and slow- (open bars) twitch muscle fibre bundles with Ringer solution containing either DHT or cycloheximide on the uptake of l-[U-¹⁴C]Ile. Note that treating the muscle fibre bundles with cycloheximide significantly (P < 0.05) reduces the basal uptake of Ile in both fibre types. Moreover, pre-treatment of the fibre bundles with it completely abolishes the DHT-induced increase in Ile uptake. B, representative Western blots showing the effects of DHT on the phosphorylation of eEF2. Note that the antibody labels a single band with a molecular mass of ∼100 kDa. Moreover, treatment of the muscle fibre bundles with DHT completely abolishes the phosphorylation of the protein in both fibre types. C, summary data showing the effects of treating muscle fibre bundles with DHT on Ile incorporation into the muscle fibre bundles. Note that treating the muscle fibre bundles with DHT significantly (P = 0.004) increases the incorporation of Ile in the fast-twitch fibre bundles. In contrast it leads to a slight decrease (9.8 ± 5.6%, n = 3 fibres) in the incorporation of Ile in the slow-twitch fibre bundles. *P < 0.05 compared to control; †P < 0.05 compared to DHT treatment.

Figure 10. Cell signalling pathway mediating the effects of DHT on amino acid transport in mammalian skeletal muscle fibres

A schematic diagram showing the cellular signalling pathway we suggest mediates the acute effects of DHT on amino acid in mammalian skeletal muscle fibre bundles. Our hypothesis is that DHT, through an unknown mechanism, activates the EGFR and this leads to the activation of RSK1/2 by ERK1/2. The activated RSK1/2 then increases the activity and expression of LAT2 (blue circle with a cross), which in turn increases the transport of Ile into the muscle fibre bundles. Additionally, RSK1/2 enhances the mRNA translation into proteins. Note that the transport of Ile is coupled to that of small neutral amino acids such as glutamine (Gln) that are transported into the cell by system A amino acid transporters such as SNAT2 (red circle with a cross). Thus, an increase in the activity of LAT2 indirectly increases the activity of SNAT2. The eventual effects of all these processes are to increase protein synthesis and hence skeletal muscle mass especially in fast-twitch muscle fibres.