P2X7 Activation Enhances Lipid Accumulation During Adipocytes Differentiation Through Suppressing the Expression of Sirtuin-3, Sirtuin-5, and Browning Genes

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  FIGURE 1. P2X7 expression and lipid accumulation during adipocyte differentiation. (A) The protein levels of P2X7 during adipocyte differentiation (D0-D6) were measured by western blot analysis with quantification to β-actin. (B) The mRNA levels of P2X7 during adipocyte differentiation (D0, D2, and D4) were measured by qPCR. *p < 0.05 vs. preadipocytes (D0). (C) Cells were treated with A438079 (10 μM) or BzATP (50 μM) at D0 and again at the end of D2 upon the renewal of the differentiation media. After two-stage adipocyte differentiation, the intracellular lipid was stained by Oil Red O. Scale bar: 100 μm. The Oil Red O stain was extracted in isopropanol and was used to measure the value at 510 nm *p < 0.05 vs. vehicle-treated adipocytes (D4) and # p < 0.05 vs. adipocytes treated with BzATP (D4). (D) Cell viability of adipocytes at D4 was measured by Trypan Blue cell counting. (E) 3T3-L1 preadipocytes were treated with A438079 or BzATP for 24-48 h. Cell viability was determined by flow cytometry. The (F) droplet numbers and (G) droplet sizes in vehicle-, A438079-, and BzATP-treated cells were measured at D4 using SGcapture software (Optical Gaging Products). All quantitative data are…

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  FIGURE 2. P2X7 does not affect adipocyte differentiation but enhances lipid metabolism. 3T3-L1 preadipocytes were differentiated in the two-stage model for four days. Vehicle, A438079 (10 μM) or BzATP (50 μM) were administered at D0 and again at the end of D2 upon the renewal of the differentiation media. (A) The protein levels of PPARγ and C/EBPα at D0, the end of D2, and the end of D4 during adipocyte differentiation were determined by western blot analysis and quantified by normalizing to β-actin from three independent experiments. *p < 0.05 vs. vehicle-treated preadipocytes at D0. ns: non-significant. (B) The gene expression of PPARγ2, C/EBPβ, and C/EBPα during adipocyte differentiation was measured by qPCR. *p < 0.05 vs. vehicle-treated adipocytes. (C) Levels of intracellular triglyceride and released glycerol in culture medium at the end of D4 were determined by triglyceride assay kit (Abcam) and adipolysis assay kit (Millipore), respectively. *p < 0.05 vs. vehicle-treated adipocytes at D4 and # p < 0.05 vs. BzATP-treated adipocytes at D4. All quantitative data are presented as mean ± SEM from at least three independent experiments.

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  FIGURE 3. P2X7 activation at the second stage of adipocyte differentiation is sufficient to increase lipid accumulation without affecting adipogenic genes. (A) In the two-stage differentiation model, cells were treated with vehicle or BzATP (50 μM) at the end of D2. After 48 h (at the end of D4), cells were stained with Oil Red O to determine intracellular lipid accumulation. *p < 0.05 vs. vehicle-treated adipocytes (D4). Scale bar: 100 μm. (B) Cells after two-day first-stage differentiation were treated with BzATP (10, 25, 50 μM) for 24 h (determined at the end of D3) and 48 h (determined at the end of D4). The protein levels of PPARγ and C/EBPα were determined by western blot analysis and quantified by normalizing to β-actin from three independent experiments. *p < 0.05 vs. vehicle-treated preadipocytes at D0. ns: non-significant. (C) Cells after two-day first-stage differentiation were treated with BzATP (50 μM) for 24 h in the presence or absence of insulin (1 μg/ml). The mRNA levels of PPARγ2 and C/EBPα were determined by qPCR. *p < 0.05 vs. vehicle-treated adipocytes. ns: non-significant. (D) The cell lysate and cultured medium described in (A) were collected to determine…

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  FIGURE 4. P2X7 does not affect lipid metabolism of adipocytes after four-day differentiation. After two-stage differentiation, 3T3-L1 adipocytes were cultured in a complete DMEM containing 10% FBS without insulin, but with A438079 (10 μM) or BzATP (50 μM) for additional three days. (A) Cells at indicated differentiation day were stained with Oil Red O and quantified to determine lipid accumulation. (B) Intracellular triglyceride level and glycerol release at D7 were determined. (C) The cell viability of differentiated adipocytes at D7 was measured by Trypan Blue cell counting. Data are presented as mean ± SEM from at least three independent experiments.

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  FIGURE 5. P2X7 activation downregulates the expression of PRDM16, PGC-1α, and UCP1 genes during adipocyte differentiation. (A) Western blot analysis of browning marker PRDM16, PGC-1α, and UCP1 during adipocyte differentiation with quantification by normalizing to β-actin. *p < 0.05 vs. preadipocytes at D0. (B) Cells were treated with BzATP (50 μM) at D0 and again at the end of D2 upon the renewal of the differentiation media. The mRNA levels of PRDM16, PGC-1α, and UCP1 at D0, D2, and D4 were measured by qPCR. *p < 0.05 vs. vehicle-treated adipocytes at D2 or D4. (C) Cells after two-day differentiation (at the end of D2) were treated with insulin (1 μg/ml) with or without BzATP (10, 25, 50 μM) for 24 h (determined at the end of D3) and 48 h (determined at the end of D3). The protein levels were determined by western blot analysis with quantification by normalizing to β-actin. *p < 0.05 vs. vehicle-treated adipocytes at D4. (D) Differentiated adipocytes at the end of D2 were treated with insulin (1 μg/ml) and/or BzATP (50 μM) for 24 h. The mRNA levels of PRDM16, PGC-1α, and UCP1 were determined by qPCR. *p < 0.05 vs. vehicle-treated adipocytes and # p < 0.05 vs. insulin-treated adipocytes.…

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  FIGURE 6. P2X7 activation downregulates the expression of SIRT3/5 genes during adipocyte differentiation. (A,B) Cells were treated with BzATP (50 μM) at D0 and again at the end of D2 upon the renewal of the differentiation media. (A) The protein levels of SIRT3 and SIRT5 at D2 and D4 were determined by western blot analysis with quantification by normalizing to β-actin. *p < 0.05 vs. differentiated adipocytes at D2. # p < 0.05 vs. vehicle-treated adipocytes at D4. (B) The mRNA levels of SIRT3 and SIRT5 at D4 were determined by qPCR. *p < 0.05 vs. preadipocytes (D0) and # p < 0.05 vs. vehicle-treated adipocytes at D4. (C) Cells after two-day differentiation (at the end of D2) were treated with BzATP (50 μM) for 24 h (determined at the end of D3) in the presence or absence of insulin (1 μg/ml). The mRNA levels of SIRT3 and SIRT5 at D3 were determined by qPCR. *p < 0.05 vs. vehicle-treated adipocytes (D3) and # p < 0.05 vs. adipocytes treated with insulin (D3). All quantitative data are presented as mean ± SEM from at least three independent experiments.