Abstract

   Disclosure: E.A. Oclon: None. A. Gurgul: None. I. Jasielczuk: None. T.
   Szmatola: None. D.A. Zieba-Przybylska: None.

   Accumulated toxic lipids induce ER stress, oxidative stress, and
   mitochondrial dysfunction, leading to cell death. Despite a growing
   emphasis on the role of lipotoxicity in neurons, the mechanisms behind
   these disruptions, particularly those affecting protein turnover,
   remain unclear. This study aimed to investigate the effects of
   lipotoxicity on protein turnover-related pathways in hypothalamic
   neurons using RNA-seq to analyze gene expression changes. The
   mHypoA-2/12 hypothalamic cell line was cultured in high-glucose DMEM
   with 10% FBS and antibiotics. Cells were seeded in 24-well plates and
   incubated at 37°C with 5% CO₂. Palmitic acid (PA) was dissolved in
   serum-free DMEM with 5% fatty acid-free BSA for treatment, while
   controls received serum-free DMEM with 5% BSA. Cellular parameters were
   assessed, including viability, caspase-3/7 activity, and calcium
   levels. Total RNA was extracted, quantified, and analyzed using RNA-seq
   (QuantSeq 3’ mRNA-Seq). Sequencing was performed with a minimum depth
   of 6 million reads per sample. Differential expression, clustering, and
   pathway enrichment were analyzed with DESeq2 and iDEP.96. Statistical
   analyses were performed in JASP using one-way ANOVA with Tukey post hoc
   or Kruskal-Wallis with Dunn post hoc as appropriate. PA treatment (5
   mM, 24 h) reduced cell survival by 60.8% (p < 0.001), increased
   caspase-3/7 activity (2.1-fold, p < 0.001), and elevated calcium levels
   (p < 0.01), indicating cellular stress and apoptosis activation.
   Pathway enrichment analysis revealed upregulation of processes involved
   in protein turnover and stress responses, including protein folding
   (GO:0006457; p = 7e-14), ER stress response (GO:0034976; p = 1.08e-08),
   and ER-associated degradation (ERAD; GO:0036503; p = 2.32e-07). Key
   genes related to protein turnover, such as Hspa5 (logFC: 1.38, p <
   0.001), Hspa1b (logFC: 4.69, p = 0.005), and Hsp90aa1 (logFC: 1.21, p <
   0.001), were significantly upregulated, indicating a robust cellular
   response to manage protein misfolding under lipotoxic stress. Our
   findings show that lipotoxicity in hypothalamic neurons activates
   processes related to protein turnover, mainly through the upregulation
   of chaperone-mediated protein folding and quality control. This
   suggests a cellular attempt to manage protein misfolding under
   lipotoxic stress. Increased apoptosis, decreased cell viability, and
   elevated calcium levels further support the cytotoxic effects of
   certain lipids, indicating that the stress may overwhelm the protective
   mechanisms to manage protein misfolding, ultimately leading to neuronal
   dysfunction. The activation of ER stress-related pathways, including
   protein folding, ER-associated degradation (ERAD), and the unfolded
   protein response, highlights the significant cellular effort to manage
   misfolded proteins. This research was financially supported by the
   Grant no. UMO-2023/51/B/NZ9/00651.

   Presentation: Sunday, July 13, 2025
     __________________________________________________________________

   Articles from Journal of the Endocrine Society are provided here
   courtesy of The Endocrine Society
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