Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) Left panel: Representative immunoblot of FABP4 expression in perirenal adipose tissue of WT, Adipo-KO, Endo-KO, and Total-KO mice. Right panel: Quantification of FABP4 relative to β-tubulin signal.***p<0.001, **p<0.01. ( B ) FABP4 immunostaining in perigonadal adipose tissue from WT, Adipo-KO, Endo-KO, and Total-KO mice. 40X magnification. ( C ) Left panel: Immunoblot of FABP4 expression in isolated PGWAT adipocytes of WT, Adipo-KO, Endo-KO, and Total-KO mice. Right panel: Quantification of FABP4 relative to β-tubulin signal.*p<0.05, **p<0.01. n=1 for Total-KO. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test and are presented as mean ± SEM. ND = No signal detected.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Western Blot, Expressing, Immunostaining, Isolation, Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) Left panel: Immunoblot of FABP4 expression in isolated liver endothelial cells of WT, Adipo-KO, and Endo-KO mice. Right panel: Quantification of FABP4 relative to β-tubulin signal. ( B ) FABP4 immunostaining in liver from WT, Adipo-KO, Endo-KO, and Myeloid-KO mice. 20X magnification. ND = No signal detected.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Western Blot, Expressing, Isolation, Immunostaining

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A) Top panel: Plasma FABP4 levels in WT, Adipo-KO, Endo-KO, and Total-KO lean male mice, after 6h daytime food withdrawal. Data are pooled from samples from 9 experiments. ****p<0.0001, **p<0.01. Bottom panel: Immunoblots of perirenal and mesenteric adipose FABP4 protein expression (same images as in and S2A), for comparison with plasma levels. ( B ) Plasma FABP4 levels in 6h WT vs. Myeloid-KO mice. Data are pooled from samples from 2 experiments. ( C ) Plasma FABP5 levels in WT, Adipo-KO, Endo-KO and Total-KO mice. *p<0.05. Data in 3A, 3C, and 3D were analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test. Data in 3B were analyzed by unpaired t-test. Data are presented as mean ± SEM. ND: No signal detected.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Clinical Proteomics, Western Blot, Expressing, Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) Plasma FABP4, ( B ) Non-esterified fatty acid (NEFA), and ( C ) glycerol responses to 10mg/kg isoproterenol-induced lipolysis in WT, Adipo-KO, Endo-KO, and Total-KO male mice. FABP4 responses are presented as induction over baseline. Data are pooled from 6 separate experiments. WT n=52, Adipo-KO n=39, Endo-KO n=35, Total-KO n=8 for , n=15 for , . ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 vs. WT; °°p<0.01 vs. Adipo-KO, °p<0.05 vs. Adipo-KO. ( D ) NEFA, ( E ) glycerol, and ( F ) FABP4 responses to forskolin (FSK)-induced lipolysis in perigonadal adipose explants from male WT, Adipo-KO, and Endo-KO mice. Data are normalized to amount of adipose tissue per culture well. n=4 mice/group, 3 replicates per mouse. ****p<0.0001, ***p<0.001 vs. Adipo-KO. Data were analyzed by repeated measures two-way ANOVA followed by Tukey’s multiple comparison test and are presented as mean ± SEM. ND: No signal detected.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Clinical Proteomics, Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) Plasma FABP4 responses to 10mg/kg isoproterenol-induced lipolysis in WT and Myeloid FABP4-KO mice, n=8/group, and ( B ) in WT vs. mice with deletion of FABP4 in both adipocytes and endothelial cells (Adipo Endo-KO). n=6/group. ****p<0.0001, **p<0.01. Data were analyzed by repeated measures two-way ANOVA followed by Tukey’s multiple comparison test and are presented as mean ± SEM.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Clinical Proteomics, Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) 4-hour conditioned media FABP4 levels from CD31-isolated liver, heart, and lung endothelial cells of WT, Adipo--KO, and Endo-KO mice. FABP4 levels are normalized to total cellular protein levels. *p<0.05. ( B ) FABP5 levels in CD31-isolated liver endothelial cell 24-hour conditioned media and (C ) lysates of WT and Endo-KO mice. Data in 6A were analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test and are presented as mean ± SEM. ND: No signal detected.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Isolation, Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) FABP4 in HUVEC lysates normalized to total cellular protein ( B ) and in 5-hour conditioned media normalized to total cellular protein at days 3 through 14 post-seeding. Pool of 2 experiments. *p<0.05, **p<0.01. ( C ) Time-course of cumulative FABP4 levels in media of day 7 HUVECs. n=4/time point. Inset: Media lactate dehydrogenase (LDH) levels during the same time course. n=4. ( D ) Effects of increasing doses of the ER-Golgi pathway inhibitor, brefeldin A (BFA) on FABP4 and endothelin-1 (ET-1) secretion from day 11 HUVECs. n=3 per BFA dose. *p<0.05. ( E ) Effects of increasing doses of forskolin (FSK) on FABP4 secretion in HUVECs vs. 3T3-L1 adipocytes. n=3 per FSK dose. *p<0.05, **p<0.01 vs. HUVEC. ( F ) Effects of increasing doses of FSK on FABP4 and Von Willibrand Factor (vWF) secretion from day 11 HUVECs. n=3 per FSK dose. *p<0.05, **p<0.01. 7A-C were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. 7D-F were analyzed by repeated measures 2-way ANOVA with Geisser-Greenhouse correction, followed by Dunnett’s (D,F) or Sidak’s (E) multiple comparison test. Data are presented as mean ± SEM.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) Plasma insulin responses to 10mg/kg isoproterenol-induced lipolysis in WT, Adipo-KO, Endo-KO, and Total-KO mice. Data are pooled from 6 experiments. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 vs. WT. °°°°p<0.0001, °°°p<0.001, °°p<0.01, °p<0.05 vs. Adipo-KO. WT, n=51; Adipo-KO, n=39; Endo-KO, n=34; Total-KO, n=15. ( B ) Plasma insulin responses to 10mg/kg isoproterenol-induced lipolysis in WT vs. Myeloid-KO mice. n=8/group. ( C ) Plasma FAPB4 and ( D ) insulin responses in WT and FABP4-KO mice injected with PBS or 7ug of FABP4 prior to induction of lipolysis with 10mg/kg isoproterenol. n=8/group. ***p<0.0005, **p<0.001, *p<0.05 vs. WT. Data were analyzed by repeated measures 2-way ANOVA with Geisser-Greenhouse correction, followed by Sidak’s (C) or Tukey’s (A,D) multiple comparison test. Data are presented as mean ± SEM. ND: No signal detected.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Clinical Proteomics, Injection, Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) Upper panel: FABP4 immunostaining of pancreas of WT, Adipo-KO, Endo-KO and Myeloid-KO mice. Islets are encircled by black dotted lines. 40X magnification. Lower panel: 2X enlargement of boxed area in upper panel. ( B ) Insulin immunostaining of pancreas from WT, Adipo-KO, Endo-KO, and Total-KO mice, 40X magnification, and ( C ) quantification of insulin-positive area. ( D ) Insulin secretion from isolated islets of WT, Adipo-KO, Endo-KO and Total-KO mice in response to low glucose (2.8mM), high glucose (16.7mM), high glucose + KCl (30mM). *p<0.05. ( E ) Fold-increase in insulin secretion induced by HG + FSK (10uM) over HG. Data in D were analyzed by unpaired t-test. Data in C and E were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Data are presented as mean ± SEM.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Immunostaining, Isolation, Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A) The mouse FABP4 gene was first converted to the the human FABP4 open reading frame by substituting the 11 amino acids differing between mouse and human FABP4 located in exons 2 and 3. ( B ) Exon 2 was flanked at the 3’ end by a FRT-neomycin-FRT-loxP cassette and by a single lox P site at the 5’ end. This distal lox P site was positioned upstream of exon 2 within the intron 1 sequences. The model was generated by homologous recombination in embryonic stem cells. The FRT-flanked selection cassette was removed in vivo by crossing with Flp-recombinase-expressing mice. Hatched rectangles represent FABP4 coding sequences. Gray rectangles indicate non-coding exon portions. Solid lines represent chromosome sequences. The 11 murine/human amino acid substitutions are represented by stars.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Generated, Homologous Recombination, Selection, In Vivo, Expressing

Journal: bioRxiv

Article Title: ABHD4 regulates adipocyte differentiation in vitro but does not affect adipose tissue lipid metabolism in mice

doi: 10.1101/2022.11.29.518076

Figure Lengend Snippet: Cellular RNA was extracted from wildtype (WT) and ABHD4 knockout (KO) 3T3-L1 cells (n=3/genotype) and reverse-transcribed into cDNA for real-time PCR quantification of Pparγ, Srebf1, Acc1, Fasn, Fabp4, Cd36, Agpat9, Agpat6, Agpat1, Agpat2, Dgat1, and Dgat2 normalized to 18s (endogenous control). Results are mean ± SEM, presented as the fold change compared to WT at Day 0, and analyzed using a two-way ANOVA with Sidak multiple comparisons.

Article Snippet: Real-time PCR was performed in duplicate on the 7500 Real-Time PCR Systems using TaqMan ® Fast Advanced Master Mix and TaqMan ® gene expression assays (ThermoFisher Scientific) including Abhd4 (Mm00506368), Pparg (Mm0040940_m1), Srebf1 (Mm00550338_m1), Acc1 (Mm01304257_m1), Fasn (Mm00662319_m1), Fabp4 (Mm00445878_m1), Cd36 (Mm00432403_m1), Agpat9 (Mm04211965), Agpat6 (Mm04211965), Agpat1 (Mm0047900_m1), Agpat2 (Mm00458880_m1), Dgat1 (Mm00515643_m1), and Dgat2 (Mm00499536_m1).

Techniques: Knock-Out, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: PLoS ONE

Article Title: Possible Involvement of Opa-Interacting Protein 5 in Adipose Proliferation and Obesity

doi: 10.1371/journal.pone.0087661

Figure Lengend Snippet: A, Images of EdU staining in the adenovirus-transfected Coxsackie-Adenovirus Receptor (CAR)-3T3-L1 adipocytes. CAR-3T3-L1 adipocytes were stained with 5-ethynyl-2′-deoxyuridine (EdU) and 4′,6-diamidino-2-phenylindole (DAPI) and analyzed by HS All-in-one Fluorescence Microscope BZ-9000. B, Number of EdU-positive CAR-3T3-L1 cells. Shown is the proportion of EdU-positive CAR-3T3-L1 cells to all nuclei. C, Images for EdU staining of CAR-3T3-L1 adipocytes analyzed by the differential interference contrast microscopy. D, CAR-3T3-L1 adipocytes co-staining with EdU and fatty acid binding protein 4 (FABP4). Images were obtained by confocal laser scanning microscopy. Oip5, Opa-interacting protein 5; βgal, β-galactosidase. Values are mean ± SE; n = 3. * P <0.05.

Article Snippet: CAR-3T3-L1 adipocytes were also stained with a rabbit anti-fatty acid binding proteins (FABP4) antibody (Catalog No. #3544, Cell signaling, Danvers, USA) as the 1 st -antibody and a goat anti-rabbit IgG conjugated Alexa 594 (Life Technologies, Gaithersburg, MD) as the 2 nd -antibody.

Techniques: Staining, Transfection, Fluorescence, Microscopy, Binding Assay, Confocal Laser Scanning Microscopy