Plasma membrane ion channels are important for cell homeostasis with their combined properties often defining the function of that cell; however, for white fat adipocytes (WFA) their expression and associated roles remain unclear. Given the importance of chloride channels in WFA membrane potential (Pulbutr et al., 2007; Bentley et al., 2014), we aimed to identify these at the molecular level. We explored this issue in adipocytes isolated from different adipose depots of adult rats, mice and 3T3-L1 cells: a common adipocyte cell line model. RNAseq revealed the expression of various chloride channel isoforms in the epididymal WFA of adult male rats. Among these, putative plasma membrane chloride channels were the volume-regulated chloride channels Lrrc8a/b/c/d and Ttyh2, the calcium-activated chloride channels, Ano1 and Ttyh3 and the glutamate aspartate transporter, Eaat1. Relative expression was confirmed by RT-qPCR. Data are given as mean ratios (95% CI, number of determinations) relative to rSWFA. Statistical significance, with p<0.01 to account for repeated measures, was determined by ANOVA with Dunnet’s multiple comparison test relative to rSWFA. Protein expression was determined by western blot in undifferentiated and differentiated 3T3-L1 cells with statistical significance determined by unpaired T-test. Confocal immunofluorescence was used to identify the cellular location of chloride channels on WFA. To investigate the role of chloride channels in adipogenesis, the effect of their inhibitors on 3T3-L1 cells was determined by measuring Nile Red accumulation analysed using One-way ANOVA.
RT-qPCR showed no difference in depot expression for Lrrc8a, Lrrc8c, Lrrc8d, Ttyh2 and ANO1, whereas, in rMWFA and rPWFA, Lrrc8b was expressed by 4.9 (2.5 to 13, n=5) and 3.3 (1.6 to 9, n=5) fold greater, respectively. Western blot demonstrated that Lrrc8a and Ttyh3 were expressed in differentiated 3T3-L1 cells by 4.1 (3.0 to 5.1, n=16) and 5.4 (-6.4 to 17.2, n=3) compared to undifferentiated 3T3-L1 cells, respectively. Data also showed that Eaat1 was exclusively expressed in differentiated 3T3-L1 cells while Ano1 was exclusively expressed in undifferentiated 3T3-L1 cells. Immunofluorescence showed that Eaat1, Ano1, Ttyh2 and Ttyh3 are primarily located to the plasma membrane, while Lrrc8a was all around the cells. During 3T3-L1 differentiation, 25µM DCPIB, a selective blocker of the volume-regulated anion channels Lrrc8a and Ttyh2, and 5μM quercetin, which inhibits Ca2+-activated Cl– channels Ttyh3 and Ano1, significantly reduced adipogenesis by 34% (16% to 53%, n=5) and 19% (6% to 31%, n=5) respectively. However, 50µM DIDS, which also inhibits Ttyh3, Ano1, and 10µM UCPH101, which inhibits Eaat1, were without effect.
This study provides evidence for the existence of chloride channels with various expression patterns among different WFA depots and in 3T3-L1 cells. Lrrc8a, Ttyh2, Ttyh3 and Eaat1 were all expressed in the plasma membrane of murine WFA, Lrrc8a was also found intracellularly. Ano1 was expressed only in rat WFA. The observation that DCPIB and Quercetin significantly inhibited adipogenesis suggests that chloride channels play a role in adipocyte differentiation.