Figure 7. Intracellular lactate concentration mediates piperine induction of UCP1 in an AMPK-dependent manner.

(A) ¹H-NMR-based metabolic profiling analysis shows the levels of lactate, fumarate, and malate in piperine-treated or untreated cell metabolite extracts. (B) C2C12 cells were treated with 30 μM piperine. Lactate was detected using a lactate colorimetric absorbance assay. (C) C2C12 cells were treated with piperine (30 μM). Quantitative RT-PCR analysis was performed with specific primers. β-actin was used as an endogenous control. (D) C2C12 cells were transiently transfected with AMPKα2 siRNA (100 nM) and then treated with piperine (30 μM). Quantitative RT-PCR analysis was performed. (E) C2C12 cells were transiently transfected with AMPKα2 siRNA (100 nM) and then treated with piperine (30 μM). Expression of UCP1 and β-actin was quantified by Western blot analysis. (F) C2C12 cells were treated with 30 μM piperine. BAT of C57BL/6 mouse were obtained from Korea University. The cells and BAT were lysed, and the expression of UCP and β-actin was quantified by Western blot analysis. The level of β-actin was measured as a control for protein loading. (G) C2C12 myoblasts were stimulated with sodium-L-lactate (30 mM). Cell lysates were analyzed by Western blot analysis with antibodies. (H) C2C12 myoblasts were stimulated with sodium-L-lactate. Cell lysates were analyzed by Western blot analysis. (I) Total mRNA was extracted from C2C12 and 3T3-L1 cells, and quantitative RT-PCR was conducted. (J) 3T3-L1 pre-adipocytes were stimulated with piperine. Cell lysates were analyzed by Western blot analysis. (K) 3T3-L1 pre-adipocytes were stimulated with piperine. Cell lysates were analyzed by Western blot analysis. (L) C2C12 cells were pre-treated with 1 mM GDP for 30 min and treated with 30 μM piperine for 24 h. Mitochondrial oxygen consumption rate (OCR) in cells was measured using the XF Seahorse flux analyzer in response to 1 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone/antimycin A. The proton leak calculated from left figure result. (M) C2C12 cells were pre-treated with 1 mM GDP for 30 min and treated with 30 μM piperine for 3 h. Mitochondria lysates were analyzed by Western blot analysis. *P < 0.05, **P < 0.01 compared with the untreated controls. Results are from three independently replicated experiments. Cropped images of full-length blots are shown.