Hypotonic induced volume activated chloride current in 3T3-L1 preadipocytes and its effect on cell differentiation

Volume-activated chloride current (VACC), the most common and abundant anion current in organisms, is involved in various physiological and pathological processes. However, its expression and characteristics in preadipocytes remain unclear. Whole-cell patch clamp technique was used to investigate the presence and properties of VACC in 3T3-L1 preadipocytes, as well as the effects of chloride channel blockers (NPPB, DIDS, and tamoxifen) on this current under different osmotic conditions. Additionally, 3T3-L1 preadipocytes were treated with the chloride channel blocker TMEM16A to observe its effect on cell differentiation. Hypotonic stimulation significantly induced whole-cell currents in 3T3-L1 preadipocytes, which exhibited obviously outward-rectifying and volume-activated characteristics. Under hypotonic stimulation, NPPB, DIDS, and tamoxifen all inhibited the current, with NPPB showing a significantly stronger inhibitory effect than DIDS and tamoxifen. Moreover, growth-arrested 3T3-L1 preadipocytes at 2 days post-confluence were successfully induced, with mRNA levels of C/EBPα, C/EBPβ, PPARγ and FABP4 peaking on day 3 and decreasing by day 14. Protein levels of PPARγ, perilipin 1 and LRRC8A were significantly upregulated during differentiation. Treatment with TMEM16A significantly reduced adipocyte lipid droplet formation and FABP4 mRNA/protein levels, but increased C/EBPα and PPARγ mRNA levels, as well as LRRC8A protein levels after 3 days. Collectively, our results clarify the characteristics of VACC in 3T3-L1 preadipocytes, confirm the inhibitory effect of chloride channel blockers on 3T3-L1 preadipocyte differentiation, fill the gap in the understanding of VACC in preadipocytes, and lay a preliminary experimental foundation for further exploring the role of VACC in adipocyte differentiation.