In:
Journal of the Endocrine Society, The Endocrine Society, Vol. 6, No. Supplement_1 ( 2022-11-01), p. A258-A258
Abstract:
Sedentary lifestyles and changes in diet are fueling the worldwide epidemic of obesity and the prevalence of NAFLD and insulin resistance (IR). It has been predicted that NAFLD and its more advanced form, nonalcoholic steatohepatitis (NASH) will be the next epidemic in chronic liver diseases, and NAFLD/NASH will soon replace viral hepatitis as the primary cause of end-stage liver failure and transplantation. NAFLD is a significant risk factor for IR and cardiovascular diseases, and it highlights the importance of finding a way to control the epidemic. CARF is a multifunctional stress-responsive gene and was found to be reduced in response to metabolic stress in the fatty liver of diet-induced obesity mice (DIO). We also showed that CARF expression was down-regulated in palmitate (PA)-treated HepG2 cells. In this study, we aim to understand the consequences of the reduced expression of CARF on the development of NAFLD. Methods We performed RNA seq, RT-PCR and western blotting (WB) to evaluate the effect of CARF knockdown on lipid metabolism in HepG2 cells. Differentially expressed genes were analyzed by gene sort enrichment analysis (GSEA) and Ingenuity Pathway Analysis (IPA) to identify the metabolic pathways affected by CARF depletion. Apoptosis was assessed by TUNEL assay. To induce ER stress, HepG2 cells were treated with thapsigargin and evaluated its effect on CARF. Results By performing Sh-RNA mediated CARF knockdown, RNA-seq analysis, RT-PCR, western blotting (WB), we were able to show that ER-stress and de-novo lipogenesis pathways were significantly affected in HepG2 cells. We showed that GRP78, CHOP, PERK, ERN1a, genes associated with ER-stress were upregulated in CARF-depleted HepG2 cells. Additionally, thapsigargin-induced ER stress was found to reduce the expression of CARF along with the increased expression of ER stress marker genes. We also showed that overexpression of CARF mitigated the thapsigargin-induced ER stress suggesting that CARF protects against ER stress in HepG2 cells. In addition, the genes associated with triglyceride biosynthesis GPAT3, GPAM were significantly upregulated, indicating that lipogenesis was triggered by silencing of CARF in HepG2 cells. BODIPY staining and TG assay confirmed that silencing of CARF enhanced triglyceride biosynthesis in HepG2 cells. Furthermore, we showed that the expression of antioxidant genes GPX2, GPX3, and TXRND3 decreased, resulting in enhanced oxidative stress and higher apoptosis in CARF depleted cells. Conclusion We identified novel roles of CARF regulating cellular ER-Stress, lipid metabolism, and oxidative stress, and its impairment in response to metabolic stress could lead to the development of NAFLD in obese patients with metabolic abnormalities. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 12:42 p.m. - 12:47 p.m.
Type of Medium:
Online Resource
ISSN:
2472-1972
DOI:
10.1210/jendso/bvac150.530
Language:
English
Publisher:
The Endocrine Society
Publication Date:
2022
detail.hit.zdb_id:
2881023-5
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