Endocrine Abstracts

JOINT157

Background: Chronic low-grade inflammation in adipose tissue is closely linked to metabolic diseases and organ complications in overweight and obese individuals. Dysfunctional adipocytes that release inflammatory adipokines are responsible for initiating and maintaining this inflammation. Although it is a low-grade inflammation, it adversely affects distant organs and contributes to obesity-related complications. Sirtuin 1 (SIRT1), a crucial nutrient-sensing histone deacetylase, is enhanced by caloric restriction and attenuated by overfeeding. When SIRT1 levels drop, inflammatory factors are produced in white adipose tissue, suggesting that reduced SIRT1 connects overnutrition with adipose tissue inflammation.

Aim: This study aimed to identify microRNAs that negatively regulate SIRT1 expression and are upregulated in differentiated and hypertrophied adipocytes and explore their relationship with inflammatory parameters.

Methods: A bioinformatics study was conducted to discover microRNAs conserved among mammals, targeting the 5’-UTR of SIRT1. 3T3-L1 cells were cultured in DMEM medium containing 10% FBS and differentiated into mature and hypertrophied adipocytes using MDI induction medium (comprising 1-methyl-3-isobutylxanthine, dexamethasone, and insulin). The differentiation cycle continued for 14 days in a culture medium containing 10% FBS. The expression of selected miRNAs was evaluated by real-time PCR. The microRNA with the highest expression was selected for subsequent experiments. Transfection of 3T3-L1 cells was performed using polyethyleneimine (PEI) and confirmed by fluorescence microscopy. Real-time PCR and Western blotting analyzed SIRT1 gene and protein expression levels, respectively. Luciferase reporter gene assay assessed the direct interaction between microRNA and SIRT1. The gene expression of inflammatory cytokines was measured by real-time PCR.

Results: Bioinformatics analysis identified mmu-miR-448, mmu-miR-181-5p, mmu-miR-186-5p, mmu-miR-653-5p, and mmu-miR-199-5p aligning with the 5’-UTR of SIRT1. Among these, miR-186 had the highest expression in differentiated and hypertrophied adipocytes. Upregulation of miR-186 by its mimic oligonucleotide led to decreased SIRT1 levels, while inhibition by miR-186 anti-sense sequence increased SIRT1 expression. miR-186 caused a significant elevation in the expression of inflammatory genes, including IL-6, IL-1β, TNF-α, and MCP-1, indicating a strong relationship between miR-186-induced SIRT1 inhibition and inflammation.

Conclusion: Differentiation and hypertrophy of adipocytes are accompanied by changes in microRNA expression, affecting various biological outcomes. Notably, inhibition of SIRT1 by microRNAs such as miR-186 may contribute to the increased inflammation observed in obesity. These findings suggest that overcoming the negative regulation of SIRT1 by miR-186 could be a promising strategy to alleviate obesity-associated inflammation.