Fig 2: (A) Fluorescent images of transfected C2C12 myoblast cells. Immunocytochemistry shows SNAT2 (i, red indicated combined de novo and transfected SNAT2), nuclei staining (ii, blue, DAPI), plasmid transfected SNAT2 (iii, green, SNAT2-eGFP), and a merged image (iv) showing co-localisation of de novo SNAT2 and SNAT2-eGFP overexpression. Typical transfection rate of cells was approximately 16%. Scale 100 µm. (B) Western blot of myoblast cells probed using a polyclonal antibody for GFP (top) and actin (bottom) used as a loading control, showing control (untransfected) and SNAT2-eGFP transfected cell protein expression of GFP at approximately 83 kDa (i.e. SNAT2 + GFP fusion protein weight combined), n = 3 separate repeats per group separated by a dotted line for clarity. (C) Densitometry analysis of the western blot (B) showing a significant difference in the transfected group expression of GFP associated SNAT2 24 h after SNAT2-eGFP plasmid transfection (Student's t-test, n = 3, P < 0.05, data presented as mean ± SEM).

Fig 3: (A) Fluorescent images of SNAT2-eGFP transfected myoblast cells showing, (i) SNAT2-eGFP transfected (white arrows indicate perinuclear puncta of SNAT2-eGFP, whilst SNAT2-eGFP also extended to other cellular regions without puncta), (ii) immunocytochemistry from de novo SNAT2 (includes SNAT2-eGFP protein), (iii) nuclei stained using DAPI, (iv) merged image (i–iv, white arrows indicate perinuclear puncta of SNAT2-eGFP and co-localisation of de novo SNAT2 and SNAT2-eGFP). Image (v) shows an increased exposure fluorescent image using image (ii) to show transfected cells overexpressing SNAT2 and de novo SNAT2 expression from nearby untransfected cells (an untransfected example is indicated by the white arrow) both being targeted by a primary antibody to SNAT2. Scale 20 µm. (B) Fluorescent image, of two examples, of myoblast cells previously transfected with the plasmid for SNAT2-eGFP and thereafter differentiated into myotubes (pictured) for 5 days showing retention of SNAT2-eGFP. White arrows indicate SNAT2 puncta in close proximity to the nuclei with additional SNAT2 puncta being more cytoplasmic and distant (red arrows). Further cytoplasmic fluorescence of SNAT2-eGFP extended along the length of the myotubes. Scale 25 µm.

Fig 4: The glutamate/glutamine cycle. High-affinity excitatory amino acid transporters (EAATs) of astrocytes primarily clear Glu in the synaptic cleft that is released from presynaptic neurons. GLT1 (glutamate transporter 1/EAAT2/SLC1A2) is the major regulator that transports Glu to astrocytes. In astrocytes, Glu is converted into Gln by glutamine synthetase (GS). Synthesized Gln is released from the astrocytes through sodium-coupled neutral amino acid transporters (SNAT3/SLC38A3 and SNAT5/SLC38A5). Released Gln is transported into neurons via SNAT1 and SNAT2 (SLC38A1 and SLC38A2) and converted back to Glu by phosphate-activated glutaminase (PAG or GLS1/2, mainly GLS2 in brain) for synaptic transmission of glutamatergic neurons. Glutamate is further transformed to ?-aminobutyric acid (GABA) by glutamic acid decarboxylases (GAD1 and GAD2) in the GABAergic neurons. Glu can be synthesized in the astrocytes from glucose via the tricarboxylic acid (TCA) cycle, followed by transamination of a-ketoglutarate (a-KG). Pyruvate carboxylase (PC) converts pyruvate to oxaloacetate that is converted to a-KG via the TCA cycle. Glutamate dehydrogenase (GDH) connects the TCA and Glu-Gln cycles by reversibly converting Glu into a-KG and vice versa. Expressional changes by chronic immobilization stress (CIS) and Gln supplementation are marked by red and green arrows, respectively.

Fig 5: (A) Myoblast cells were exposed to C14‐MeAIB for 1 h, and thereafter, uptake of the C14‐MeAIB was measured using scintillation counting; cells were either not transfected control (light grey) or transfected with SNAT2‐eGFP (dark grey). A small but not significant increase in C14‐MeAIB transport was recorded in the transfected myoblasts (Student's t‐test, n = 3 both groups, data presented as mean ± SEM). (B) The effect of the hormone DHT was investigated upon untransfected myoblast cells that were treated as control, DHT (4.5 nM) or pre‐treated with the androgen receptor antagonist flutamide (3 μM) alone or for 1 h prior to an identical DHT exposure. DHT treatment alone resulted in a significant (*) 46 ± 10% increase in C14‐MeAIB transport. Flutamide treatment irrespective of DHT exposure was not different from control (n = 4, one‐way ANOVA, P < 0.05, data presented as mean ± SEM). (C) Myoblast cells were either untreated or exposed to 4.5 nM DHT for 1 h and thereafter exposed to C14‐MeAIB for one additional hour, and the uptake of C14‐MeAIB was measured. This was carried out to control (not transfected) and SNAT2‐eGFP transfected groups. DHT significantly increased C14‐MeAIB transport in untransfected and transfected myoblasts (n = 3, one‐way ANOVA where a > b, P < 0.05). Inset histogram shows the percentage change in the DHT stimulated transport of C14‐MeAIB calculated from the main figure. Transfected myoblasts showed a significant enhancement of C14‐MeAIB transport (207.7 ± 26.6%, dark grey) versus control untransfected cells (119.8 ± 5%, light grey, n = 3, P < 0.03, Student's t‐test, data presented as mean ± SEM). (D) Myoblast cells were either fed or starved for 150 min and then exposed to C14‐MeAIB for 1 h and uptake of C14‐MeAIB measured in control (not transfected) and SNAT2‐eGFP transfected groups. Starvation significantly increased C14‐MeAIB transport in untransfected and transfected myoblasts (n = 3, one‐way ANOVA, P < 0.05) versus their not starved controls. Inset histogram reports the percentage change in starvation induced transport of C14‐MeAIB shown in the main figure, and no significant increase in C14‐MeAIB transport after transfection (111.6 ± 61.3%, dark grey) was found versus control untransfected cells (52.2 ± 27%, light grey, n = 3, Student's t‐test, P > 0.05, data presented as mean ± SEM).