(P31-042-23) An Improved In Vitro 3T3-L1 Adipocyte Model of Inflammation and Insulin Resistance

Objectives: Obesity is a significant public health concern characterized by chronic inflammation in adipose tissue and increased risk of insulin resistance (IR). Tumor necrosis factor alpha (TNF-α)/Hypoxia-treated 3T3-L1 adipocytes have been used to model inflamed and IR adipose tissue in a diet-induced animal model of obesity. However, TNF-α/Hypoxia treatment induces significant cell death in the adipocytes. New in vitro models of adipose tissue inflammation are needed in which adipocytes are viable yet maintain the inflammatory state. Here, we examined the treatment time with TNF-α in the presence of hypoxia to reduce cell death while maintaining inflammation and IR in 3T3-L1 adipocytes.

Methods: To develop the IR model, adipocytes were treated with 20 ng/ml TNF-α (12h or 24h) and incubated in a hypoxic chamber with 1% O2/5% CO2. To develop a long-term IR phenotype, 2.5mM glucose/10% FBS were added 24 h post-treatment. Viability and markers of IR and inflammation were assessed using Live/Dead staining and RT-qPCR, respectively.

Results: Treatment for 12 h with TNF-α in the presence of hypoxia resulted in a significant increase (P < 0.05) in the percentage of live cells by 1.85-fold compared to 24 h treated cells. However, IR and inflammation were maintained in the 12 h treated adipocytes; the expression of the insulin sensitive (IS) genes adiponectin (0.08 ± 0.1-fold) and solute carrier family 2 member 4 (SCLA2A4, aka GLUT4) (0.07 ± 0.1-fold) were significantly decreased (P < 0.05), while the expression of the inflammatory genes C-C Motif Chemokine Ligand 2 (CCL2) (228 ± 11-fold) and Interleukin 6 (21 ± 4-fold) were significantly increased (P < 0.05). In the long-term culture model, no significant differences were found in the viability of the 12 h TNF-α/24 h hypoxia treated cells across the different time points. At 24 h, the IS genes adiponectin and GLUT4 decreased by 0.03 ± 0.0 and 0.02 ± 0.0-fold, respectively, and remained downregulated at 48 and 72 h. CCL2 expression increased by 109.2 ± 9.6-fold at 24 h (p < 0.001). The fold increase decreased to 13.05 ± 3.82 after 72 h, however CCL2 expression was not statistically different after 48 h.

Conclusions: This study provides a new approach to modeling inflammation and IR in adipocytes in vitro and can be used to further examine obesity-linked IR and inflammation.

Funding Sources: NIFA award (ALA044-1-18037); NIH award (UL1TR003096-01)