Black raspberry water extract (BRWE) suppresses adipocyte development in human adipose tissue-derived mesenchymal stem cells (hMSCs) and zebrafish. (A,B) Accumulation of intracellular lipid (lipid droplets) was measured with Oil-Red O assay in hMSCs differentiated with the presence or absence of BRWE (5 and 10 μg·mL⁻¹) for 15 days. (C) mRNA levels of Pparg and Cebpa were measured using a Real-time RT-PCR assay. (D,E) Cell lysates were subjected to SDS-PAGE, and immunoblot analysis was performed using each antibody to PPARγ and C/EBPα. β-actin was used as a loading control. (F) Bright field images in the top row show both DMSO- or BRWE-treated zebrafish larvae at 17 dpf. Fluorescent adipocytes stained with Nile red are shown in the bottom row. Larvae are shown in lateral views with rostral to the right, dorsal to the top. Adipocytes are marked by white arrows. (G) Florescent intensities in Figure 1F were quantified using the ImageJ software and presented as a graph in arbitrary units. All data are expressed as the mean ± SEM. (Figure 1B,C) ^#p < 0.05, significantly different from DM (Wh)-untreated pre-adipocytes; * p < 0.05, ** p < 0.01, and *** p < 0.001, significantly different from DM (Wh)-stimulated adipocytes. (G) Unpaired t-test was used to analyze different versus the phosphate buffered-saline (PBS) group, and statistical significance was set as * p < 0.05. BRWE, black raspberry water extract.

BRWE increases browning of inguinal white adipose tissue in cold-stressed C57BL/6J mice. C57BL/6J mice were treated with BRWE (100 mg·kg⁻¹, oral administration) or phosphate buffered-saline (PBS) for 2 weeks and then placed at 4 °C for 5 h; (A,B) rectal temperature was measured after cold exposure: (C) representative infrared thermal image. (D) Body weight change and (E) adipose tissue weights were measured: (F) representative photograph of adipose tissues after cold exposure for 5 h. (G) H&E staining of iWAT (200× magnification, scale bar = 100 µm) was performed and (H) lipid droplet size were measured. (I,J) Immuno-fluorescent staining of iWAT (1000× magnification, scale bar = 25 µm) with anti-UCP1 antibody (green) was performed. (K,L) Western blot analysis of thermogenesis- and beige-related factors including PRDM16, PGC1α, UCP1, and TBX1 were performed. β-actin was used as a loading control. All data are expressed as the mean ± SEM. ^#p < 0.05, significantly different from blank group; * p < 0.05, ** p < 0.01, and *** p < 0.001, significantly different from control group. Each experiment was repeated at least 3 times, and similar results were obtained. BRWE, black raspberry water extract; iWAT, inguinal white adipose tissue.

BRWE promotes browning of adipocytes in 3T3-L1 adipocytes. Two days after achieving confluence, 3T3-L1 pre-adipocyte cells were stimulated by DM (Be) containing 0.5 mM IBMX, 167 μM insulin, T3 20 μM, and 0.5 μM troglitazone with/without various concentrations of BRWE. After 6 days, intracellular lipid droplets were measured with (A,B) Oil-Red O assay and the absorbance was measured at 500 nm. (C) The mRNA and (D,E) protein levels of thermogenesis-related factors in 3T3-L1 with BRWE were measured. (F) Expression of intracellular UCP1 and PGC1α in 3T3-L1 were evaluated by immunofluorescence staining. The changes were observed at 200× (scale bar = 100 μM). Florescent intensities were quantified using the ImageJ software and presented as a graph in arbitrary units. (G) Mitotracker staining was performed and observed at 200× (scale bar = 150 μM). (H) Protein levels of NRF1, CIDEA, and CPT1B were measured. All data are expressed as the mean ± SEM. ^#p < 0.05, significantly different from DM (Be)-untreated pre-adipocytes; * p < 0.05 and ** p < 0.01, significantly different from DM (Be)-stimulated adipocytes cells. Each experiment was repeated at least 3 times, and similar results were obtained. BRWE, black raspberry water extract.