Journal: Molecular Metabolism

Article Title: Myosin 1c: A novel regulator of glucose uptake in brown adipocytes

doi: 10.1016/j.molmet.2021.101247

Figure Lengend Snippet: Myosin 1c inhibition induces glucose uptake exclusively in BAT . (A) The effect of 5 μM of PClP, a Myo1c inhibitor, on insulin (1 μM)-stimulated glucose uptake in white adipocytes normalized to basal. Cells were pretreated with PClP for 30 min prior to stimulation with insulin for 2 h (n = 3). (B) The effect of 5 μM of PClP on glucose uptake in brown adipocytes in response to insulin (1 μM) and isoprenaline (1 μM) normalized to basal. Cells were pretreated with PClP for 30 min prior to stimulation with insulin and isoprenaline for 5 h (n = 4). (C) Glucose uptake in brown adipocytes with isoprenaline (1 μM) and in response to antibodies neutralizing Myo1c and Myo1b for 8-10 h normalized to IgG (n = 4). (D) In vivo glucose uptake in BAT, WAT, and gastrocnemius in C57Bl/6N mice treated with PClP (5 mg/kg body weight i.p.) or vehicle (DMSO) for 2.5 h (n = 4 mice). Each value represents mean ± SEM. Statistics for (C) were calculated using one-way ANOVA with Dunn's multiple comparisons post-test. Statistics for the remaining data presented were calculated using two-way ANOVA followed by Tukey's multiple comparisons post-test. ∗P < 0.05 and ∗∗P < 0.01 compared to controls. # represents statistical differences within groups for P < 0.05.

Article Snippet: Briefly, scrambled (sc-37007) or Myo1c siRNA (sc-44614) purchased from SCBT was delivered into brown adipocytes on days 5–6 using Lipofectamine RNAiMAX (Thermo Fisher Scientific) dissolved in Opti-MEM (Gibco) at a final concentration of 90 nM.

Techniques: Inhibition, In Vivo

Journal: Molecular Metabolism

Article Title: Myosin 1c: A novel regulator of glucose uptake in brown adipocytes

doi: 10.1016/j.molmet.2021.101247

Figure Lengend Snippet: Inhibition of myosin 1c leads to GLUT1 transcription, synthesis, and translocation in brown adipocytes . (A) Gene expression of Slc2a1 and Slc2a4 in brown (n = 5–7) and white adipocytes (n = 4) treated with PClP (5 μM) or isoprenaline for 2 h. (B) Representative immunoblot with the relative quantification of GLUT1 protein in brown adipocytes treated with isoprenaline for 5 h (n = 5). (C) Representative immunoblot with the relative quantification of GLUT1 and myosin 1c protein in brown adipocytes treated with Myo1c siRNA (90 nM) and isoprenaline for 48 h (n = 5). (D) Representative TIRF images and their relative quantification showing GLUT1 proteins at the plasma membrane of brown adipocytes following stimulation with PClP (5 μM) or isoprenaline (1 μM) for 5 h, scale bar 10 μm (n = 4). (E) Representative TIRF images and their relative quantification showing GLUT4 proteins at the plasma membrane of brown adipocytes following stimulation with PClP (5 μM) or insulin (1 μM) for 5 h, scale bar 10 μm (n = 4). Each value represents mean ± SEM. Statistics were calculated using a paired t-test. ∗P < 0.05 and ∗∗ compared to controls.

Article Snippet: Briefly, scrambled (sc-37007) or Myo1c siRNA (sc-44614) purchased from SCBT was delivered into brown adipocytes on days 5–6 using Lipofectamine RNAiMAX (Thermo Fisher Scientific) dissolved in Opti-MEM (Gibco) at a final concentration of 90 nM.

Techniques: Inhibition, Translocation Assay, Gene Expression, Western Blot, Quantitative Proteomics, Clinical Proteomics, Membrane

Journal: Molecular Metabolism

Article Title: Myosin 1c: A novel regulator of glucose uptake in brown adipocytes

doi: 10.1016/j.molmet.2021.101247

Figure Lengend Snippet: Myosin 1c inhibition induces PKA-mediated activation of CREB, p38, and ATF2 independent of an increase in cAMP . (A) cAMP production measured as pmol/well in brown adipocytes stimulated with PClP (5 μM) or isoprenaline (1 μM) for 15 min (n = 3). (B) Immunoblots and their relative quantifications showing the effect of Myo1c knockdown on p-CREB (S133) and p-p38 (T180/Y182) (n = 4). (C) Immunoblots and their relative quantifications showing the effect of PClP (5 μM) and isoprenaline (1 μM) stimulation on p-CREB (S133), p-p38 (T180/Y182), and p-ATF2 (T71) in brown adipocytes (n = 5–6). (D) Immunoblots and their relative quantifications showing p-CREB (S133) in brown adipocytes stimulated with either PClP (5 μM) or isoprenaline (1 μM) for 15 min in the presence or absence of H89 (40 μM), a PKA inhibitor (n = 3). Statistics for (D) were calculated using 2-way ANOVA followed by Fischer's LSD post-test. Statistics for the remaining data presented were calculated using a paired t-test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 for differences as compared to controls. # and ## represent statistical differences within groups.

Article Snippet: Briefly, scrambled (sc-37007) or Myo1c siRNA (sc-44614) purchased from SCBT was delivered into brown adipocytes on days 5–6 using Lipofectamine RNAiMAX (Thermo Fisher Scientific) dissolved in Opti-MEM (Gibco) at a final concentration of 90 nM.

Techniques: Inhibition, Activation Assay, Western Blot, Knockdown