# Zinc Depletion Increases Susceptibility to AMPK-Induced Atrophic Responses in C2C12 Myotubes

## TL;DR

Zinc deficiency makes muscle cells more vulnerable to atrophy caused by AMPK activation, despite not affecting AMPK itself.

## Contribution

The study reveals that zinc availability modulates downstream atrophic responses to AMPK signaling in muscle cells.

## Key findings

- Zinc-depleted cells showed greater atrophy and increased expression of atrophy-related genes after AMPK activation.
- Zinc supplementation increased intracellular zinc levels and altered zinc transporter expression in response to AMPK activation.
- AMPK activation levels were similar across zinc conditions, but downstream effects varied with zinc status.

## Abstract

Background: AMP-activated protein kinase (AMPK) acts as a key energy sensor that negatively regulates skeletal muscle mass. Zinc is an essential trace element that is required for myogenic differentiation and protein synthesis, while zinc deficiency has been associated with muscle atrophy in vivo. However, how zinc status modulates AMPK activation itself or alters downstream responses to AMPK signaling in muscle cells remains unclear. Methods: C2C12 myotubes were cultured under zinc-depleted (ZnD), zinc-sufficient (20 μM; Zn20), or zinc-supplemented (40 μM; Zn40) conditions. AMPK was activated by AICAR, and zinc status–dependent responses were evaluated using molecular and morphological analyses. Results: AICAR increased intracellular zinc levels in Zn20 and Zn40 but not in ZnD. Zinc transporter expression exhibited gene-specific regulation: Zip3 was upregulated across all zinc conditions, Zip14 was significantly induced in ZnD and Zn40, and Zip10 was selectively upregulated in Zn40. AICAR induced myotube atrophy in all groups; however, the reduction in myotube diameter was significantly greater under zinc-depleted conditions. Zinc depletion was associated with transcriptional upregulation of FoxO1, FoxO3, Atrogin-1, and MuRF1 in response to AICAR, while AMPK activation and suppression of S6K1 phosphorylation occurred to a similar extent regardless of zinc status. Conclusions: These findings indicate that zinc availability does not alter AMPK activation itself but modulates downstream atrophic responses to AMPK signaling. Under conditions of AMPK activation, adequate zinc availability is accompanied by increased intracellular zinc levels and stress-responsive ZIP regulation, which may limit excessive atrophic gene induction, whereas zinc depletion increases susceptibility to AMPK-induced atrophic responses in skeletal muscle cells.