Abstract

Testosterone is a primary hormone for male sexual maturation, but it is also responsible for bone and skeletal muscle mass growth both for men and women. Recent data from our group suggest that testosterone supplementation (TS) in mice can lead to increased heart size and that this is sex‐specific, being observed only in females. Usually, muscle hypertrophy is related to the unbalance in between protein synthesis and degradation (PD). One of the major pathways to PD is the constitutive ubiquitin‐proteasome complex, composed by the catalytic subunits beta 1, 2 and 5 (β1, β2 and β5, respectively). Under stressful environments, like inflammation, oxidation, infections and injury repairing, the catalytic subunits can be replaced by its inducible forms, beta 1, 2 and 5 (β1i, β2i and β5i, respectively) known as the immunoproteasome (IP). As such, we investigated the involvement of protein degradation mechanisms in TS mice. Young (20 week‐old) male and female C57BL6 adult mice were exposed to TS diet or non‐supplemented (control) for 21 days. The animals were then euthanized and hearts were collected and pulverized in liquid nitrogen. Constitutive proteasome activity in the hearts was measured using the fluorogenic substrates for β1 activity Z‐LLE‐AMC, β2 activity Boc‐LSTRAMC and β5 activity Suc‐LLVY‐AMC and using the pan‐proteasome inhibitor bortezomib negative control. Immunoproteasome activity (β1i and β5i) was determined by using the fluorogenic substrates and immunoproteasome inhibitors. For western blot analysis immunoproteasome subunits content was determined using anti‐β1i, β2i and β5i antibodies. Oxidized protein content was determined by western blot using an oxyblot. The Western blots were normalized by total protein staining using Ponceau S. We investigated if TS females show changes in constitutive proteasome catalytic activity. We found no significant differences in β1, β2 and β5 proteasome activities among TS and control both for female and male mice. However, testosterone‐supplemented female mice exhibited a significant increase in β1i (21.16% increase) and β5i (56.25% increase) activities when compared to control mice, whereas male mice showed no significant changes in IP catalytic activities after testosterone supplementation. These changes don’t seem to be related to changes in the total IP catalytic amounts, since western blot for IP subunits showed no significant differences after TS both for female mice. When compared to control female mice, testosterone supplementation had a trend to increase (26.84% increase; p = 0.084) the levels of oxidized proteins. Together, these data suggest that testosterone supplementation could lead to an increased overall activity of β1i and β2i that appears to be sex‐specific. This may be related to an increased oxidative state in muscle cells. Further experiments are needed to determine the underlying mechanisms of how the immunoproteasome could be involved in testosterone‐supplementation sex‐related heart size increase.