Journal: Journal of Lipid Research

Article Title: Sympathetic tone dictates the impact of lipolysis on FABP4 secretion

doi: 10.1016/j.jlr.2023.100386

Figure Lengend Snippet: FABP4 secretion is dependent on lipolysis in vitro. A: FABP4 secretion from wild-type epididymal adipose tissue explants under unstimulated (basal) conditions or induction of lipolysis by FSK (1 μM) or ISO (10 μM) (N = 7–8/condition; one-way ANOVA). B: Plasma FABP4 in wild-type mice following injection of PBS or ISO (10 mg/kg) (N = 6/group; two-way ANOVA). FABP4 in conditioned media from (C) 3T3-L1 adipocytes (Western blot; FSK 10 μM) and (D) epididymal adipose tissue explants (ELISA quantification) following 2 h pretreatment with atglistatin (ATGLi; 10 μM) (N = 3/group; two-way ANOVA). E: Western blot of epididymal adipose tissue confirming ATGL deletion in ATGL AdpKO (N = 4/group). F: Quantification of FABP4 in adipose depots from ATGL fl/fl and ATGL AdpKO mice (N = 4/group; Student’s t -test). G: Plasma glycerol following induction of lipolysis in vivo (N = 14–16/group; two-way ANOVA). H: Body compo as determined by dual-energy X-ray absorptiometry (DEXA) (N = 7–9/group; Student’s t -test). I: Weight of liver, epididymal (Epi) adipose tissue, subcutaneous (SubQ) adipose tissue, heart, brown adipose tissue (BAT), and gastrocnemius muscle (N = 4/group; Student’s t -test). Glucose excursion curves in (J) TT and (K) intraperitoneal glucose tolerance test (ipGTT) (N = 9–10/group; two-way ANOVA). L: FABP4 secretion from epididymal adipose tissue explants under unstimulated (basal) conditions or induction of lipolysis by ISO (10 μM), NE (1 μM) or FSK (3 μM) (N = 4–6/condition; Student’s t -test). All mouse experiments performed in 8- to 10-week-old male mice. Data are presented as mean ± SEM.

Article Snippet: Proteins of interest were detected using 1:1,000 dilutions of ATGL antibody (CST; catalog no.: 2138), HRP-tagged anti-FABP4 antibody (clone 351.4.5E1.H3, generated in house for Hotamisligil laboratory by Dana Farber Antibody Core), or HRP-tagged anti-β-actin antibody (CST; catalog no.: 5125) as a loading control.

Techniques: In Vitro, Clinical Proteomics, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, In Vivo

Journal: Journal of Lipid Research

Article Title: Sympathetic tone dictates the impact of lipolysis on FABP4 secretion

doi: 10.1016/j.jlr.2023.100386

Figure Lengend Snippet: Adipocyte-specific ATGL KO mice have enhanced FABP4 secretion in vivo. Quantification of (A) plasma FABP4, (B) blood glucose, (C) body weight, and (D) plasma insulin in ATGL fl/fl (black bars) and ATGL AdpKO (red bars) mice under ad libitum-fed conditions, following 24 h food withdrawal, and 3.5 h after food was added back to cages (N = 15–17/group; two-way ANOVA). E: Plasma FABP4 in ATGL fl/fl and ATGL AdpKO following induction of lipolysis with ISO (10 mg/kg) (N = 15–16/group; two-way ANOVA). F: Plasma FABP4 levels in ATGL fl/fl and ATGL AdpKO mice injected with 10 μg recombinant FABP4 (N = 3–5/group; two-way ANOVA). G: Plasma insulin in ATGL fl/fl and ATGL AdpKO following induction of lipolysis with ISO (10 mg/kg) (N = 15–16/group; two-way ANOVA). All mouse experiments performed in 8- to 10-week-old male mice. Data are presented as mean ± SEM.

Article Snippet: Proteins of interest were detected using 1:1,000 dilutions of ATGL antibody (CST; catalog no.: 2138), HRP-tagged anti-FABP4 antibody (clone 351.4.5E1.H3, generated in house for Hotamisligil laboratory by Dana Farber Antibody Core), or HRP-tagged anti-β-actin antibody (CST; catalog no.: 5125) as a loading control.

Techniques: In Vivo, Clinical Proteomics, Injection, Recombinant

Journal: Journal of Lipid Research

Article Title: Sympathetic tone dictates the impact of lipolysis on FABP4 secretion

doi: 10.1016/j.jlr.2023.100386

Figure Lengend Snippet: Lipolysis-induced FABP4 secretion is from adipocytes in vivo. A: Western blot of epididymal adipose tissue confirming ATGL and FABP4 deletion in FABP4/ATGL AdpKO (DKO) mice (N = 4/group). B: Quantification of FABP4 in adipose depots from ATGL fl/fl FABP4 fl/fl and DKO mice (N = 4/group; Student’s t -test). C: Plasma glycerol following induction of lipolysis in vivo (N = 13–15/group; two-way ANOVA). D: Body composition as determined by dual-energy X-ray absorptiometry (DEXA) (N = 5–13/group; Student’s t -test). E: Weights of liver, epididymal (Epi) adipose tissue, subcutaneous (SubQ) adipose tissue, heart, brown adipose tissue (BAT), and gastrocnemius muscle (N = 4/group; Student’s t -test). F: Glucose excursion curves in ITT (N = 6–9; two-way ANOVA). G: Plasma FABP4 and (H) plasma insulin in ATGL fl/fl FABP4 fl/fl and DKO mice following induction of lipolysis with ISO (10 mg/kg) (N = 14–15/group; two-way ANOVA). All mouse experiments performed in 8- to 10-week-old male mice. Data are presented as mean ± SEM.

Article Snippet: Proteins of interest were detected using 1:1,000 dilutions of ATGL antibody (CST; catalog no.: 2138), HRP-tagged anti-FABP4 antibody (clone 351.4.5E1.H3, generated in house for Hotamisligil laboratory by Dana Farber Antibody Core), or HRP-tagged anti-β-actin antibody (CST; catalog no.: 5125) as a loading control.

Techniques: In Vivo, Western Blot, Clinical Proteomics

Journal: Journal of Lipid Research

Article Title: Sympathetic tone dictates the impact of lipolysis on FABP4 secretion

doi: 10.1016/j.jlr.2023.100386

Figure Lengend Snippet: Glucose bolus stimulates FABP4 secretion in vivo. A: Glucose excursion curve during OGTT (N = 9–10/group; two-way ANOVA). B: Corresponding plasma insulin levels during OGTT (N = 9–10/group; two-way ANOVA). C: Plasma FABP4 in ATGL fl/fl and ATGL AdpKO mice under ad libitum fed, 10 min following ISO injection (10 mg/kg), 16 h fasting, and 10 min following glucose injection (1 g/kg) (N = 10–21/group; Student’s t -test). D: Plasma glycerol and (E) plasma FABP4 levels in ATGL AdpKO and HSL AdpKO and respective floxed control mice injected with ISO (10 mg/kg) (N = 9–16/group; two-way ANOVA). F: Plasma glycerol and (G) plasma FABP4 levels in MGL fl/fl and MGL AdpKO mice injected with ISO (10 mg/kg) (N = 5/group; two-way ANOVA). H: Schematic illustration of the protocol for serum induction of lipolysis in 3T3-L1 cells. I: Plasma FABP4 levels in mice 10 min following injection of PBS or ISO (10 mg/kg) (N = 4–7/group; one-way ANOVA). J: Quantification of FABP4 in conditioned media from 3T3-L1 adipocytes treated with control, ISO (1 μM), or 10% serum from mice in (I) (N = 6–14/group; one-way ANOVA). All mouse experiments performed in 8- to 10-week-old male mice. Data are presented as mean ± SEM.

Article Snippet: Proteins of interest were detected using 1:1,000 dilutions of ATGL antibody (CST; catalog no.: 2138), HRP-tagged anti-FABP4 antibody (clone 351.4.5E1.H3, generated in house for Hotamisligil laboratory by Dana Farber Antibody Core), or HRP-tagged anti-β-actin antibody (CST; catalog no.: 5125) as a loading control.

Techniques: In Vivo, Clinical Proteomics, Injection, Control

Journal: Journal of Lipid Research

Article Title: Sympathetic tone dictates the impact of lipolysis on FABP4 secretion

doi: 10.1016/j.jlr.2023.100386

Figure Lengend Snippet: In vivo FABP4 secretion is blunted upon sympathetic nervous inhibition. A: Plasma corticosterone levels in 6 h fasted ATGL fl/fl and ATGL AdpKO mice (N = 5–7/group; Student’s t -test). B: Plasma FABP4 in ATGL fl/fl and ATGL AdpKO injected with 100 μl sterile PBS (N = 3–5/group; two-way ANOVA). C: Correlation of plasma FABP4 and plasma corticosterone levels in ATGL fl/fl and ATGL AdpKO mice (N = 20). D: Plasma FABP4, (E) NEFAs, and (F) insulin following administration of 20 mg/kg hexamethonium and 0.1 mg/kg CL-316,243 (N = 5–6/group; two-way ANOVA). Western blot and quantification of ATGL and FABP4 protein levels, normalized to total protein (Ponceau S stain), in (G) epididymal white adipose tissue (WAT) and (H) brown adipose tissue (BAT) from ATGL fl/fl and ATGL AdpKO housed at room temperature or 3 weeks at thermoneutrality (N = 4/group; one-way ANOVA). I: Plasma corticosterone levels in 6 h fasted ATGL fl/fl and ATGL AdpKO mice housed at room temperature or 2 week thermoneutrality and 15 min post ISO injection (10 mg/kg) (N = 6/group; two-way ANOVA). Plasma (J) FABP4, (K) NEFA, and (L) insulin following administration of ISO (N = 6/group; two-way ANOVA). All mouse experiments performed in 8- to 10-week-old male mice. Data are presented as mean ± SEM.

Article Snippet: Proteins of interest were detected using 1:1,000 dilutions of ATGL antibody (CST; catalog no.: 2138), HRP-tagged anti-FABP4 antibody (clone 351.4.5E1.H3, generated in house for Hotamisligil laboratory by Dana Farber Antibody Core), or HRP-tagged anti-β-actin antibody (CST; catalog no.: 5125) as a loading control.

Techniques: In Vivo, Inhibition, Clinical Proteomics, Injection, Sterility, Western Blot, Staining

Journal: Journal of Biological Chemistry

Article Title: Roles of Pofut1 and O-Fucose in Mammalian Notch Signaling

doi: 10.1074/jbc.m802027200

Figure Lengend Snippet: FIGURE5.EScellslackingPofut1aredeficientinNotchligandbinding.A,flowcytometricanalysisofsoluble Delta1-Fc (4 g/ml) binding to Pofut1/ (solid line) and Pofut1/ (dashed line) ES cells. The shaded profile is secondary antibody alone. B, Delta1-Fc binding to Pofut1/ (squares) and Pofut1/ (circles) ES cells in buffer containing1mMCaCl2(solid)or1mMCaCl2and5mMEDTA(open)analyzedbyflowcytometry.Dataareplotted as MFI minus MFI of secondary antibody alone. C, flow cytometric analysis of soluble Jagged1-Fc (2 g/ml) binding to Pofut1/ (solid line) and Pofut1/ (dashed line) ES cells as in A. D, soluble Jagged1 binding to Pofut1/ (squares) and Pofut1/ (circles) in buffer containing 1 mM CaCl2 (solid) or 1 mM CaCl2 and 5 mM EDTA (open) determined by flow cytometry as in B. E, Notch1, Notch2, and Notch3 in Pofut1/ ES cells. Western analysis of lysates from Pofut1/, Pofut1/, and Notch1/ 290-2 ES cells (100 g of protein) was performed using anti-Notch1 ECD antibody 8G10 (1:500). Pofut1/ and Pofut1/ cell lysates (50 g of protein) were probed with anti-Notch2 Ab sc-5545 (1:500). Pofut1/ and Pofut1/ cell lysates (44 g of protein) were probed with anti-Notch3 Ab 5E1 (1:500 culture medium). Molecular mass markers are in kDa. F–H, Pofut1/ and Pofut1/ ES cells were incubated with antibodies to the ECD of Notch1 (clone 8G10), Notch2 (sc-5545), or Notch3 (AF1308) and analyzed by flow cytometry. Secondary antibody alone is shaded in each profile.

Article Snippet: After centrifugation, cells were resuspended in 100 l of binding buffer or 100 l of binding buffer containing primary anti-Notch3 antibody (1:20 clone 5E1 (46) or 1:10 clone AF1308 (Santa Cruz Biotechnology)) and incubated for 60 min at room temperature.

Techniques: Binding Assay, Flow Cytometry, Western Blot, Incubation

Journal: Journal of Biological Chemistry

Article Title: Roles of Pofut1 and O-Fucose in Mammalian Notch Signaling

doi: 10.1074/jbc.m802027200

Figure Lengend Snippet: FIGURE 7. Intracellular Notch in Pofut1/ ES cells. A, Pofut1/ and Pofut1/ ES cells were fixed and analyzed by flow cytometry using anti-Notch3 antibody 5E1 (solid line) or secondary antibody alone (shaded profile). B, Pofut1/ and Pofut1/ ES cells were fixed or fixed and permeabilized and analyzed by flow cytom- etry using anti-Notch3 antibody AF1308 or secondary antibody as indicated. C, Pofut1/ and Pofut1/ ES cells treated with DMSO (vehicle) or 2 g/ml tunicamycin were incubated with anti-Notch3 ECD antibody AF1308 or secondary antibody, fixed, or fixed and permeabilized and subjected to flow cytometry. The per- centage of background-subtracted MFI for permeabilized compared with nonpermeabilized cells is plotted. Bars, the range of values in two experiments.

Article Snippet: After centrifugation, cells were resuspended in 100 l of binding buffer or 100 l of binding buffer containing primary anti-Notch3 antibody (1:20 clone 5E1 (46) or 1:10 clone AF1308 (Santa Cruz Biotechnology)) and incubated for 60 min at room temperature.

Techniques: Flow Cytometry, Incubation