β3 adrenergic receptor β3 ar  (Santa Cruz Biotechnology)

Journal: Food Science and Human Wellness

Article Title: Ginsenoside F1 administration promotes UCP1-dependent fat browning and ameliorates obesity-associated insulin resistance

doi: 10.1016/j.fshw.2023.03.025

Figure Lengend Snippet: Fig. 1 Validation of ginsenoside F1 as a novel UCP1 activator. (a) Workflow for obtaining novel UCP1 activator. (b) Relative luciferase activity of HEK293T cells co-transfected with -2.5 kb UCP1-luciferase and renilla plasmid. Indicated ginsenosides (30 μmol/L) were added to HEK293T cells for 24 h, n = 3. (c) Structure of F1. (d) Optimization of ginsenoside F1 concentration, n = 5-6. (e) Ucp1 mRNA level was assessed in differentiated 3T3-L1 cells after ginsenoside F1 or PPT (100 μmol/L) treated by qRT-PCR, n = 4-5. (f) UCP1 protein expression was detected by western blotting in primary adipocytes and differentiated 3T3-L1 cells after ginsenoside F1 or PPT (100 μmol/L) treated. Data were presented as mean ± SD. The one-way ANOVA followed by the LSD post hoc test used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001 compared with ctrl group.

Article Snippet: The following primary antibodies were used: GAPDH (Servicebio, GB12002, 1:1 000), actin (BD biosciences, 612657, 1:5 000), UCP1 (Sigma, U6382, 1:1 000), PKA (Cell Signaling Technology, 4782S, 1:1000), p-PKA (Cell Signaling Technology, 5661T, 1:1000), p-CREB (Abcam, ab32096, 1:1 000), CREB (Cell Signaling Technology, 9197T, 1:1 000), PGC-1α (Abcam, ab54481, 1:1 000), PPARγ (Cell Signaling Technology, 2435T, 1:1 000), TGR5 (Novus Biologicals, NBP223669SS, 1:1 000), β3-AR (Biorbyt, orb221343, 1:1 000), p-IR (Cell signaling Technology, 3026, 1:1 000), IR (Cell signaling Technology , 3025, 1:1 000), Primary antibodies were incubated overnight at 4 °C.

Techniques: Biomarker Discovery, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Concentration Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: Food Science and Human Wellness

Article Title: Ginsenoside F1 administration promotes UCP1-dependent fat browning and ameliorates obesity-associated insulin resistance

doi: 10.1016/j.fshw.2023.03.025

Figure Lengend Snippet: Fig. 2 Ginsenoside F1 binds to β3-AR to enhance UCP1 expression via cAMP/PKA/CREB pathway. (a) Intracellular uptake of F1 in cells was analyzed using UPLC-MS. (b) 3T3-L1-derived adipocytes were treated with ginsenoside F1 (100 μmol/L) for 3 h, then analyzed PKA/CREB pathway using western blotting, n = 4. (c) PKA/CREB signaling was analyzed in 3T3-L1 adipocytes treated with F1 (100 μmol/L) using western blotting in the presence or absence of PKA inhibitor H 89. (d) The binding ability of F1 to β3-AR was evaluated in pull down assay. (e) Thermogenesis signaling was analyzed in 3T3-L1 adipocytes treated with F1 (100 μmol/L) using western blotting in the presence or absence of the β3-AR inhibitor SR 59230A. (f) Thermogenesis signaling was analyzed using western blotting. The 3T3-L1 adipocytes were transfected with negative control (NC) or β3-AR siRNAs 24 h prior to F1 treatment (100 μmol/L). (g) Mitochondria respiration was analyzed in 3T3-L1 adipocytes treated with F1 (100 μmol/L) in the presence or absence of the β3-AR inhibitor SR 59230A, or treated with norepinephrine (NE) using Agilent Seahorse XFp Analyzer. (h) ATP content was detected in 3T3-L1 adipocytes treated with F1 (100 μmol/L) using kits, n = 5. (i) UCP1 expression was analyzed in 3T3-L1 adipocytes treated with ginsenoside F1 (100 μmol/L) or different concentration of norepinephrine (NE) using western blotting. (j) Glucose uptake of the 3T3-L1 adipocytes (j1) and hepatocytes Hepa1-6 cells (j2) cultured in NE, ginsenoside F1 (100 μmol/L) or combination of NE and F1 (100 μmol/L), n = 4. (k) Glucose uptake of the 3T3-L1 adipocytes (k1) and hepatocytes Hepa1-6 cells (k2) cultured in NE, ginsenoside F1 (100 μmol/L) or combination of NE and F1 (100 μmol/L) with or without siRNA treatment. (l) Scheme for F1 increasing thermogenesis via UCP1 activation. Data were presented as mean ± SD. Two-way Student’s t-test (b-h) and one-way ANOVA followed by the LSD post hoc test (i-k) used for statistical analysis. #P < 0.05, ###P < 0.001 compared with ctrl group; *P < 0.05, **P < 0.01, ***P < 0.001 compared with F1 group.

Article Snippet: The following primary antibodies were used: GAPDH (Servicebio, GB12002, 1:1 000), actin (BD biosciences, 612657, 1:5 000), UCP1 (Sigma, U6382, 1:1 000), PKA (Cell Signaling Technology, 4782S, 1:1000), p-PKA (Cell Signaling Technology, 5661T, 1:1000), p-CREB (Abcam, ab32096, 1:1 000), CREB (Cell Signaling Technology, 9197T, 1:1 000), PGC-1α (Abcam, ab54481, 1:1 000), PPARγ (Cell Signaling Technology, 2435T, 1:1 000), TGR5 (Novus Biologicals, NBP223669SS, 1:1 000), β3-AR (Biorbyt, orb221343, 1:1 000), p-IR (Cell signaling Technology, 3026, 1:1 000), IR (Cell signaling Technology , 3025, 1:1 000), Primary antibodies were incubated overnight at 4 °C.

Techniques: Expressing, Derivative Assay, Western Blot, Binding Assay, Pull Down Assay, Transfection, Negative Control, Concentration Assay, Cell Culture, Activation Assay