# Sestrin2 is Induced Upon Cellular Stress but Has No Effect on Myotube Size or Amino Acid Sensing in C2C12 Myotubes

## TL;DR

The study finds that Sestrin2 is increased by cellular stress in muscle cells but does not affect muscle growth or amino acid sensing.

## Contribution

The study reveals that Sestrin2 is stress-inducible in muscle cells but does not regulate mTORC1 or muscle growth under basal conditions.

## Key findings

- Sestrin2 is induced by ER stress in C2C12 myotubes but not sestrins 1 or 3.
- Silencing Sestrin2 does not affect myotube size or mTORC1 nutrient sensing.
- Sestrin2 may protect muscle cells from ER stress but is not critical for basal anabolism.

## Abstract

Sestrins are a stress‐inducible family of proteins that function in cell survival and nutrient sensing through their regulation of mTORC1. Muscle wasting is associated with cellular stress, but to date, there is limited in vitro research investigating sestrins in skeletal muscle cells. Here we use C2C12 myotubes to understand how sestrin proteins (sestrin 1–3) are regulated by different forms of cellular stress linked to muscle wasting conditions. Furthermore, since sestrin2 is a well‐characterised protein but is lowly expressed in muscle tissue in the absence of stress, we also aimed to determine if silencing this protein impacted parameters of muscle growth or nutrient sensing by mTORC1 under basal conditions. Incubating C2C12 myotubes with the endoplasmic reticulum (ER) stress‐inducing agent tunicamycin, or a high concentration (1000 µM) of hydrogen peroxide (H2O2), increased sestrin2 protein levels with no change in sestrins 1 or 3. This increase was temporally associated with increased ER stress markers Ddit3 mRNA and ATF4 protein levels, and could be blocked by approximately half when myotubes were co‐incubated with H2O2 and the ER‐stress inhibitor 4‐Phenylbutyrate. siRNA silencing of sestrin2 blunted the phosphorylation of the mTORC1 effector S6K1, but did not acutely influence protein synthesis or myotube size. Similarly, silencing sestrin2 did not affect mTORC1 signalling in response to nutrient deprivation. These data indicate that sestrin2 is stress‐inducible and may play a role in protecting skeletal muscle from ER stress, but is less important in regulating mTORC1 and nutrient sensing in unstressed/basal conditions.

Endoplasmic reticulum stress is associated with muscle wasting, and specifically induces sestrin2 levels within C2C12 myotubes, but not sestrins 1 or 3. Silencing sestrin2 in myotube cultures does not acutely alter myotube size, or the ability of mTORC1 to sense the presence of amino acids. Sestrin2 is likely an important regulator of cellular health under stresses conditions but does not influence anabolism in unstressed muscle.