Lysozyme Gene Expression in 3T3-L1 Cells Sustains Expression of Adipogenic Genes and Adipocyte Differentiation

Abstract

Substantial levels of lysozyme in adipose tissue in association to obesity have been recently demonstrated in mice and humans. In addition, experiments in mice suggest that lysozyme might impact on adipose tissue adipogenesis. To further investigate the relationship between lysozyme and adipogenesis, in the present study, we aimed to study lysozyme (Lyz2) during 3T3-L1 adipocyte differentiation and its possible role in adipogenesis. Time course experiment during 3T3-L1 adipocyte differentiation indicated that Lyz2 gene expression decreased at day 4, which was caused by isobutylmethylxanthine administration, and recovered at the end of the process (day 8). Importantly, the impact of isobutylmethylxanthine-induced downregulation of Lyz2 gene expression on adipogenesis was not comparable to that observed in the full cocktail, questioning whether the reduction in lysozyme at early stage of adipocyte differentiation is relevant to this process. In fact, the depletion in Lyz2 expression had a negative impact on adipogenesis, and rosiglitazone administration failed to compensate for the anti-adipogenic effect observed in Lyz2 gene knockdown cells. Otherwise, when Lyz2 gene knockdown cells were co-cultured with control cells, these cells had higher expression of adipogenic genes than those co-cultured with themselves at the end of adipocyte differentiation. In conclusion, this study suggests that lysozyme expression in 3T3-L1 cells sustains expression of adipogenic genes and adipocyte differentiation.

Keywords: 3T3-L1; adipogenesis; gene knockdown; lentiviral particles; lysozyme.

FIGURE 1

Lyz2 gene expression during 3T3-L1 adipocyte differentiation. (A) Lyz2, Cebpb, Pparg, Cebpa, Adipoq, Plin1, Slc2a4 and Fabp4 gene expression during 3T3-L1 adipocyte differentiation. **p < 0.01 and ***p < 0.001 compared to Day 0; ^† p < 0.05, ^†† p < 0.01 and ^††† p < 0.001 compared to Day 4. (B) The effect of individual compound administration (INS, DEX or IBMX) of differentiation cocktail on Lyz2, Cebpb, Pparg, Cebpa, Adipoq, Plin1, Slc2a4 and Fabp4 gene expression during 4 days. **p < 0.01 and ***p < 0.001 compared to complete differentiation cocktail (INS/DEX/IBMX); ^† p < 0.05, ^†† p < 0.01 and ^††† p < 0.001 compared to Day 0.

FIGURE 2

The impact of Lyz2 gene knockdown on expression of adipogenic genes during 3T3-L1 adipocyte differentiation. (A–H) Lyz2, Cebpb, Pparg, Cebpa, Adipoq, Plin1, Slc2a4 and Fabp4 gene expression during 3T3-L1 adipocyte differentiation at day 0, 4 and 8. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to shRNA_SCR.

FIGURE 3

(A) The impact of Lyz2 gene knockdown on intracellular lipid accumulation, which was measured as Oil Red O staining area (%), at day 8 of adipocyte differentiation. The lens magnification used was ×10. **p < 0.01 compared to shRNA_SCR. (B) The impact of rosiglitazone (0.5 μmol/L) administration during adipocyte differentiation on Lyz2, Cebpb, Cebpa, Adipoq, Plin1 and Slc2a4 gene expression at day 8 in shRNA_SCR and shRNA_Lyz2 cells. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to vehicle; ^† p < 0.05 and ^††† p < 0.001 compared to shRNA_SCR.

FIGURE 4

The effect of coculture Lyz2 gene knockdown cells (shRNA_Lyz2) with lysozyme-producing cells (shRNA_SCR) on Lyz2, Cebpb, Pparg, Cebpa, Adipoq, Plin1, Slc2a4 and Fabp4 gene expression at day 8 of adipocyte differentiation. *p < 0.05 compared to shRNA_Lyz2 cells co-cultured with themselves.