Pharmaceutical inhibition of mTOR in the common marmoset: effect of rapamycin on regulators of proteostasis in a non-human primate

Abstract

Background: Inhibition of mechanistic target of rapamycin (mTOR) has emerged as a viable means to lengthen lifespan and healthspan in mice, although it is still unclear whether these benefits will extend to other mammalian species. We previously reported results from a pilot experiment wherein common marmosets (Callithrix jacchus) were treated orally with rapamycin to reduce mTOR signaling in vivo in line with previous reports in mice and humans. Further, long-term treatment did not significantly alter body weight, daily activity, blood lipid concentrations, or glucose metabolism in this cohort.

Methods: In this study, we report on the molecular consequences of rapamycin treatment in marmosets on mechanisms that regulate protein homeostasis (proteostasis) in vivo. There is growing appreciation for the role of proteostasis in longevity and for the role that mTOR plays in regulating this process. Tissue samples of liver and skeletal muscle from marmosets in our pilot cohort were assessed for expression and activity of components of the ubiquitin-proteasome system, macroautophagy, and protein chaperones.

Results: Rapamycin treatment was associated with increased expression of PSMB5, a core subunit of the 20S proteasome, but not PSMB8 which is involved in the formation of the immunoproteasome, in the skeletal muscle and liver. Surprisingly, proteasome activity measured in these tissues was not affected by rapamycin. Rapamycin treatment was associated with an increased expression of mitochondria-targeted protein chaperones in skeletal muscle, but not liver. Finally, autophagy was increased in skeletal muscle and adipose, but not liver, from rapamycin-treated marmosets.

Conclusions: Overall, these data show tissue-specific upregulation of some, but not all, components of the proteostasis network in common marmosets treated with a pharmaceutical inhibitor of mTOR.

Keywords: autophagy; healthspan; immunoproteasome; proteasome; protein chaperone.

Figures

Fig. 1

(a) Immunoblot showing phosphorylated and total ribosomal protein S6 from skeletal muscle of control (lanes indicated with ‘C’) and rapamycin-treated (lanes indicated with ‘R’) marmosets. (b) Quantification of relative levels of phosphorylation of ribosomal protein S6 (S6) in skeletal muscle generated from immunoblot in A. Data are presented as mean values (± SEM) for indicated groups (c–d). Plot comparing relative levels of phosphorylated/total S6 ratio in muscle with that found in adipose and liver collected from the same rapamycin-treated marmosets (n = 7 total rapamycin-treated animals). Circles represent values generated from samples collected from an individual animal and line is regression line. Values in each panel give Pearson's correlation coefficient for indicated relationship and p value. Data from liver and adipose were presented previously in (23,24).

Fig. 2

Quantification of relative abundance of proteasome subunits PSMB5 and PSMB8 in skeletal muscle (a) or liver (b) from control and rapamycin-treated marmosets. Data are presented as values for each protein normalized using Ponceau S staining of immunoblot as a loading control. (c) Representative immunoblot for skeletal muscle data presented in (a). (d) Rate of 20S or 26S-mediated cleavage of fluorescent peptide (Suc-LLVY-AMC) with or without proteasome inhibitor MG132 in samples from skeletal muscle of control (n = 5) and rapamycin-treated (n = 7) marmosets. Data are presented as mean values (± SEM) for indicated groups. Asterisks represent p < 0.05 for Student's t-test comparing control to rapamycin values.

Fig. 3

Quantification of relative levels of indicated molecular chaperones in skeletal muscle (a) or liver (b) from control (n = 5) and rapamycin-treated (n = 7) marmosets. Data are presented as mean values (± SEM) for indicated groups. Data are presented as values for each protein normalized using Ponceau S staining of immunoblot as a loading control. Asterisks represent p < 0.05 for Student's t-test comparing control to rapamycin values. (c) Representative immunoblot for skeletal muscle data presented in (a).

Fig. 4

(a) Quantification of LC3B-II levels (left) and LC3B-II/LC3B-I ratio (right) in skeletal muscle, liver, or adipose tissue from control and rapamycin-treated marmosets. Data are presented as mean values (± SEM) for indicated groups and normalized to control-treated samples in each case for clarity of presentation. Asterisks represent p < 0.05 for Student's t-test comparing control to rapamycin values. (b) Representative immunoblots for data presented in (a).