Chaperone-mediated autophagy protects against hyperglycemic stress

Area of Science:

• Cellular Biology
• Molecular Biology
• Autophagy Research

Background:

• Chaperone-mediated autophagy (CMA) is crucial for cellular homeostasis and metabolism, with defects linked to human diseases.
• While well-studied in mammals, functional evidence for CMA in fish, like rainbow trout (RT), is recent.
• Rainbow trout is a model for glucose intolerance and possesses two Lamp2A paralogs, key regulators of CMA.

Purpose of the Study:

• To investigate the function and regulation of chaperone-mediated autophagy (CMA) in rainbow trout (RT) under high-glucose conditions.
• To validate a CMA fluorescent reporter in an RT cell line and assess CMA flux.
• To elucidate the role of CMA in protecting against glucose-induced metabolic stress in RT.

Main Methods:

• Validated a KFERQ-PA-mCherry1 fluorescent reporter in RT hepatoma cells (RTH-149) to track CMA.
• Exposed cells to high-glucose (HG) or control glucose levels and analyzed reporter translocation and degradation.
• Investigated the involvement of mitochondrial reactive oxygen species (ROS) and the Nfe2l2/Nrf2 pathway using specific inhibitors and activators.

Main Results:

• High-glucose (25 mM) significantly increased CMA flux in RT cells, evidenced by reporter translocation to lysosomes and reduced half-life.
• CMA activation by HG was dependent on KFERQ, Lamp2A, mitochondrial ROS generation, and the Nfe2l2/Nrf2 transcription factor.
• CMA demonstrated a protective role against HG-induced stress, mediated predominantly by one of the two RT Lamp2A paralogs.

Conclusions:

• This study provides definitive evidence for functional chaperone-mediated autophagy (CMA) in rainbow trout.
• High-glucose exposure activates CMA in RT cells via ROS and Nrf2, highlighting its role in metabolic regulation.
• CMA acts protectively against glucose-induced stress in RT, with Lamp2A playing a critical mediating role.