Effect of branched-chain amino acids on muscle atrophy in cancer cachexia

Abstract

In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a significant suppression in the loss of body weight in mice bearing a cachexia-inducing tumour (MAC16), producing a significant increase in skeletal muscle wet weight, through an increase in protein synthesis and a decrease in degradation. Leucine attenuated the increased phosphorylation of PKR (double-stranded-RNA-dependent protein kinase) and eIF2alpha (eukaryotic initiation factor 2alpha) in skeletal muscle of mice bearing the MAC16 tumour, due to an increased expression of PP1 (protein phosphatase 1). Weight loss in mice bearing the MAC16 tumour was associated with an increased amount of eIF4E bound to its binding protein 4E-BP1 (eIF4E-binding protein 1), and a progressive decrease in the active eIF4G-eIF4E complex due to hypophosphorylation of 4E-BP1. This may be due to a reduction in the phosphorylation of mTOR (mammalian target of rapamycin), which may also be responsible for the decreased phosphorylation of p70(S6k) (70 kDa ribosomal S6 kinase). There was also a 5-fold increase in the phosphorylation of eEF2 (eukaryotic elongation factor 2), which would also decrease protein synthesis through a decrease in translation elongation. Treatment with leucine increased phosphorylation of mTOR and p70(S6k), caused hyperphosphorylation of 4E-BP1, reduced the amount of 4E-BP1 associated with eIF4E and caused an increase in the eIF4G-eIF4E complex, together with a reduction in phosphorylation of eEF2. These changes would be expected to increase protein synthesis, whereas a reduction in the activation of PKR would be expected to attenuate the increased protein degradation.

Figure 1. Effect of PIF alone or in combination with amino acids on protein synthesis in murine myotubes after 4 h of incubation

The amino acids (2 mmol/l) were added 2 h prior to PIF (4.2 nmol/l). The concentration of the amino acids in DMEM was: 0.8 mmol/l for Leu, Ile and Val; 0.4 mmol/l for Phe; 0.34 mmol/l for Arg; and 0.2 mmol/l for Met. The results are means±S.E.M. of three separate determinations. *P<0.05, **P<0.01 or ***P<0.001 compared with control. †††P<0.001 compared with PIF alone.

Figure 2. Effect of PIF alone or in combination with BCAAs on phosphorylated and total eIF2α

(A) Western blot analysis of phospho-eIF2α (p EIF2α) in relation to total eIF2α in cytosolic extracts of murine myotubes after incubation with PIF (4.2 nmol/l) for various periods of time, either alone or in the presence of leucine (2 mmol/l). (B) Effect of the BCAA (2 mmol/l) alone or together with PIF on the phosphorylation of eIF2α after 4 h incubation determined by Western blotting. The amino acids were added to the myotubes 2 h prior to PIF. The densitometric analysis represents the means±S.E.M. for three separate Western blots, and is presented as the ratio of the phosphorylated to total eIF2α (ph/tot). *P<0.05 and ***P<0.001 compared with control; †P<0.05 and †††P<0.001 compared with PIF alone.

Figure 3. Effect of PIF (A) or Ang II (B) alone or in combination with leucine, salubrinal and a PKR inhibitor on protein synthesis in murine myotubes after 4 h of incubation

Both the inhibitors and leucine (2 mmol/l) were added to the myotubes 2 h prior to PIF (4.2 nmol/l) or Ang II (0.5 μmol/l). Salubrinal (Sal; 15 μmol/l) is a phospho-eIF2α phosphatase inhibitor. PKR (I), PKR inhibitor {210 nmol/l; 8-[1-(1H-imidazol-4-yl)-meth-(Z)-yl idene]-6,8-dihydrothiazol[5,4-e]indol-7-one}. *P<0.05, **P<0.01 and ***P<0.001 compared with control; †††P<0.001 compared with PIF or Ang II alone.

Figure 4. Effect of PIF alone or in combination with BCAAs on protein degradation in myotubes after 24 h of incubation

Details of the procedure are given in the Experimental section. The amino acids (2 mmol/l) were added 2 h prior to PIF (4.2 nmol/l). Results are means±S.E.M. of three separate determinations. ***P<0.001 compared with control; ††P<0.01 compared with PIF alone.

Figure 5. Effect of administration of BCAAs on muscle loss in cachexia in mice bearing the MA16 tumour

Effect of daily per os administration of leucine (■), isoleucine (▲) and valine (△) (all at 1g/kg of body weight) in comparison with PBS controls (●) on body weight change (A) and tumour volume (B) of mice bearing the MAC16 tumour. At the end of the experiment, mean weight of the soleus muscles (C), the rate of protein synthesis in gastrocnemius muscles (D) and the rate of protein degradation in soleus muscles (E) were determined. The details of the experiment are given in the Experimental section. Values are means±S.E.M. of six animals per treatment. *P<0.05, **P<0.01 and ***P<0.001 compared with control.

Figure 6. Effect on phosphorylated and total PKR, eIF2α, mTOR and p70^S6k and total PP1 in gastrocnemius muscles from non-tumour-bearing mice, and mice bearing the MAC16 tumour treated with either PBS or leucine

Western blots of phospho-PKR (p PKR) and total PKR (A), phospho-eIF2α (p eIF2α) and total eIF2α (B), PP1 with actin loading control (C), phospho-mTOR (p mTOR) and total mTOR (D) and phospho-p70^S6k (P70S6K) and total p70^S6k (E) in gastrocnemius muscle of non-tumour-bearing mice (NTB), and mice bearing the MAC16 tumour treated with either PBS (MAC16) or leucine (1 g/kg of body weight) (MAC16+Leu). Samples were taken at the end of the experiment shown in Figure 5. Representative Western blots are shown, and the densitometric analysis shown underneath the blots represents means±S.E.M. for three separate Western blots. The values represent the ratio of the phosphorylated (ph) to total (tot) forms. *P<0.05 and ***P<0.001 compared with non-tumour-bearing mice; †P<0.05, ††P<0.01 and †††P<0.001 compared with mice bearing the MAC16 tumour treated with PBS.

Figure 7. Effect of weight loss on the amount of eIF4E available for active eIF4G–eIF4E complex formation

Total amounts of 4E-BP1 (A) and eIF4G (B) associated with eIF4E, and phosphorylation states of 4E-BP1 (p 4E-BP1) (C), eIF4E (p eIF4E) (D), eEF2 (p eEF2) (E), mTOR (p mTOR) (F) and p70^S6k (p P70S6K) (G) in gastrocnemius muscle of mice bearing the MAC16 tumour with increasing levels of weight loss, were determined by Western blotting. In (C), the amount of 4E-BP1 in the γ-phosphorylated form is shown as a percentage of the total 4E-BP1. The γ-form is the most highly phosphorylated and has the slowest electrophoretic mobility. Representative Western blots are shown, and the densitometric analysis, shown underneath, represents the means±S.E.M. for three separate Western blots. The ratio of phospho (ph) to total (tot) forms is shown in (F), (G) and (H). *P<0.05, **P<0.01 and ***P<0.001 compared with animals without weight loss.

Figure 8. Effect on phosphorylated and total 4E-BP1, eIF4E and eEF2 from non-tumour-bearing mice, and mice bearing the MAC16 tumour treated with either PBS or leucine

Western blots showing the amount of 4E-BP1 (A) and eIF4G (B) associated with eIF4E, and phosphorylation states of 4E-BP1 (p 4E-BP1) (C), eIF4E (p eIF4E) (D) and eEF2 (p eEF2) (E) in gastrocnemius muscle of non-tumour-bearing mice (NTB), and in mice bearing the MAC16 tumour treated with PBS (MAC16) or leucine (MAC16+Leu). The details of the experiment are given in the Experimental section and in the legend to Figure 5. Samples were taken at the end of the experiment depicted in Figure 5. Representative Western blots are shown, and the densitometric analysis, shown underneath, represents the means±S.E.M. for three separate Western blots. The ratio of phospho (ph) to total (tot) forms is shown in (F). *P<0.05 and **P<0.01 compared with non-tumour-bearing animals; †P<0.05 and †††P<0.001 compared with mice bearing the MAC16 tumour treated with PBS.