human recombinant fabp4  (Millipore)

Journal: JCI insight

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

doi: 10.1172/jci.insight.164642

Figure Lengend Snippet: Figure 1. Validation of tissue-specific deletion of FABP4 from adipocytes. (A) Representative immunoblots (n = 2) and quantification of FABP4 protein relative to β-tubulin loading control in perirenal adipose tissue of WT, Adipo-KO, Endo-KO, and Total-KO mice. WT, Total-KO: n = 5/group; Adipo-KO, Endo-KO: n = 6/group. (B) Immunoblots and quantification of FABP4 protein relative to β-tubulin loading control in perirenal adipose tissue of WT and Myeloid-KO mice. n = 5/group. (C) Representative FABP4 immunostaining in perigonadal adipose tissue from WT, Adipo-KO, Endo-KO, and Total-KO mice. Magnification, 400×. (D) Immunoblots and quantification of FABP4 protein relative to β-tubulin loading control in isolated perigonadal adipocytes of WT, Adipo-KO, Endo-KO, and Total-KO mice. WT, Adipo-KO, Endo-KO: n = 5/group; Total-KO: n = 1. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA, followed by Tukey’s multiple-comparison test (A and D), or by t test (B). 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, as described previously (20) (clone 351.4.2E12.H1.F12 for capture, HRP-tagged clone 351.4.5E1.H3 for detection), and recombinant human FABP4 as a standard (R&D Systems).

Techniques: Biomarker Discovery, Western Blot, Control, Immunostaining, Isolation, Comparison

Journal: JCI insight

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

doi: 10.1172/jci.insight.164642

Figure Lengend Snippet: Figure 2. Validation of tissue-specific deletion of FABP4 from endothelial cells. (A) Immunoblots and quantification of FABP4 protein relative to β-tubulin loading control in CD31-isolated endothelial cells from liver, spleen, heart, and lungs of WT, Adipo-KO, and Endo-KO mice. n = 2/group. (B) Representative FABP4 immunostaining in livers of WT, Adipo-KO, Endo-KO, and Myeloid-KO mice; magnification, 200×. 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, as described previously (20) (clone 351.4.2E12.H1.F12 for capture, HRP-tagged clone 351.4.5E1.H3 for detection), and recombinant human FABP4 as a standard (R&D Systems).

Techniques: Biomarker Discovery, Western Blot, Control, Isolation, Immunostaining

Journal: JCI insight

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

doi: 10.1172/jci.insight.164642

Figure Lengend Snippet: Figure 3. Endothelial cells contribute to approximately 87% of basal circulating FABP4 during 6-hour daytime fasting. (A) Plasma FABP4 levels in lean WT (n = 43), Adipo-KO (n = 29), Endo-KO (n = 29), and Total-KO (n = 23) male mice. Data are pooled from 5 experiments. Average age of mice is 13 weeks old. Immunoblots of perirenal and mesenteric adipose FABP4 protein expression, for comparison with plasma levels. (B) Plasma FABP4 levels in WT versus Myeloid-KO mice. Data are pooled from 2 experiments, n = 8/group/experiment. (C) Plasma FABP5 levels in WT, Adipo-KO, Endo-KO, and Total-KO mice. n = 8/group. All samples were collected from male mice after 6-hour daytime food withdrawal. *P < 0.05, ****P < 0.0001 by unpaired t test (B) or by 1-way ANOVA, followed by Tukey’s multiple-comparison test (A and C).

Article Snippet: For in-house FABP4 ELISA, we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core, as described previously (20) (clone 351.4.2E12.H1.F12 for capture, HRP-tagged clone 351.4.5E1.H3 for detection), and recombinant human FABP4 as a standard (R&D Systems).

Techniques: Clinical Proteomics, Western Blot, Expressing, Comparison

Journal: JCI insight

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

doi: 10.1172/jci.insight.164642

Figure Lengend Snippet: Figure 4. Lipolysis-driven FABP4 secretion is primarily from adipocytes. (A–E) Plasma FABP4 levels, baseline-corrected plasma FABP4, AUC of baseline-corrected plasma FABP4, nonesterified fatty acid (NEFA), and glycerol responses to 10 mg/kg isoproterenol-induced lipolysis in ~13-week- old WT (n = 54), Adipo-KO (n = 40), Endo-KO (n = 34), and Total-KO (n = 8 for FABP4, n = 15 for NEFA, glycerol) mice. Data for A–E are pooled from 6 experiments. (F–H) NEFA, glycerol, and FABP4 responses to FSK-induced lipolysis in perigonadal adipose explants from WT, Adipo-KO, and Endo-KO mice; n = 4/group. Data are normalized to amount of adipose tissue per culture well. (I) Plasma FABP4 responses to 10 mg/kg isoproterenol-induced lipolysis in WT and Myeloid-KO mice; n = 8/group. (J) WT versus Adipo Endo-KO mice with deletion of FABP4 in both adipocytes and endothelial cells; n = 6/group. All experiments were in male mice. ****P < 0.0001, ***P < 0.001, ** P <0.01, *P < 0.05 versus WT; °°°°P < 0.0001, °°°P < 0.001, °°P < 0.01, °P < 0.05 versus Endo-KO; •P < 0.05 versus Adipo-KO, by mixed-effects analysis followed by Tukey’s multiple-comparison test (A, B, D, and E), or 2-way ANOVA followed by Tukey’s (F, G, and H) or Sidak’s (I and J) multiple-comparison test. ****P < 0.0001, ** P <0.01 by 1-way ANOVA followed by Tukey’s multiple-comparison test (C). 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, as described previously (20) (clone 351.4.2E12.H1.F12 for capture, HRP-tagged clone 351.4.5E1.H3 for detection), and recombinant human FABP4 as a standard (R&D Systems).

Techniques: Clinical Proteomics, Comparison

Journal: JCI insight

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

doi: 10.1172/jci.insight.164642

Figure Lengend Snippet: Figure 5. Endothelial and adipocyte FABP4 secretion are differentially regulated. (A) Twelve-hour conditioned media FABP4 levels from CD31-isolated endothelial cells from liver, heart, and lungs of WT, Adipo-KO, and Endo-KO mice, normalized to total cellular protein; n = 3 mice/group. (B and C) FABP4 levels in HUVEC lysates and 5-hour conditioned media, normalized to total cellular protein at days 3 through 14 after seeding. Pool of 2 exper- iments; n = 6/time point. (D) Time-course of cumulative FABP4 levels in media of day 7 HUVECs; n = 4/time point. Inset: Media lactate dehydroge- nase (LDH) levels during the same time course. °°°°P < 0.0001, °°°P < 0.001 versus 0 hours by 1-way ANOVA followed by Dunnett’s multiple-com- parison test. (E) Effects of the ER-Golgi pathway inhibitor, brefeldin A (BFA), on FABP4 and endothelin-1 (ET-1) secretion from day 11 HUVECs; n = 3/ BFA dose. (F) Effects of forskolin (FSK) on FABP4 secretion in HUVECs versus 3T3-L1 adipocytes. n = 3/FSK dose. *P < 0.05 versus HUVEC by 2-way ANOVA followed by Sidak’s multiple-comparison test. (G) Effects of FSK on FABP4 and von Willebrand Factor (vWF) secretion from day 11 HUVECs. FABP4, n = 4/FSK dose; vWF, n = 3/FSK dose. (H) Total FABP4 measured by ELISA in HUVEC cell lysates (CL), conditioned media (CM), and exosomes (Exos) isolated from CM; n = 3. (I) Western blots of FABP4, exosome markers CD-63 and ALiX, and double-membrane protein β-actin in HUVEC cell lysates, conditioned media, and exosomes. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 1-way ANOVA followed by Sidak’s (A), Tukey’s (B, C, and H), or Dunnett’s (E and G) multiple-comparison test.

Article Snippet: For in-house FABP4 ELISA, we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core, as described previously (20) (clone 351.4.2E12.H1.F12 for capture, HRP-tagged clone 351.4.5E1.H3 for detection), and recombinant human FABP4 as a standard (R&D Systems).

Techniques: Isolation, Comparison, Enzyme-linked Immunosorbent Assay, Western Blot, Membrane

Journal: JCI insight

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

doi: 10.1172/jci.insight.164642

Figure Lengend Snippet: Figure 6. Lipolysis-driven insulin secretion is blunted in mice lacking endothelial but not adipocyte FABP4. (A) Plasma insulin responses to 10 mg/ kg isoproterenol-induced lipolysis in WT (n = 52), Adipo-KO (n = 40), Endo-KO (n = 34), and Total-KO (n = 15) mice. Data are pooled from 6 experiments. (B) Plasma insulin responses to 10 mg/kg isoproterenol-induced lipolysis in WT versus Myeloid-KO mice; n = 8/group. (C and D) Blood glucose levels in response to 10 mg/kg isoproterenol-induced lipolysis in WT versus Endo-KO mice (n = 10/group) and in WT (n = 7) versus Adipo-KO (n = 10) mice. (E and F) Plasma FAPB4 and insulin responses in WT and Fabp4–/– mice injected with PBS or 7 μg of FABP4 prior to induction of lipolysis with 10 mg/kg isoprotere- nol; n = 8/group. All experiments were in male mice. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 versus WT; °°°°P < 0.0001, °°°P < 0.001, °P < 0.05 versus Adipo-KO, by mixed-effects analysis, followed by Tukey’s (A and F) or Sidak’s (C and E) multiple-comparison test, or 2-way ANOVA followed by Tukey’s multiple-comparison test (B and D). 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, as described previously (20) (clone 351.4.2E12.H1.F12 for capture, HRP-tagged clone 351.4.5E1.H3 for detection), and recombinant human FABP4 as a standard (R&D Systems).

Techniques: Clinical Proteomics, Injection, Comparison

Journal: JCI insight

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

doi: 10.1172/jci.insight.164642

Figure Lengend Snippet: Figure 7. Pancreas endothelial cells express FABP4, and Endo-KO islets show altered regulation of insulin secretion. (A) Upper panel: Representative FABP4 immunostaining of pancreas of WT, Adipo-KO, Endo-KO, and Myeloid-KO mice. Islets are encircled by black dotted lines. Magnification, 400×. Low- er panel: 2× enlargement of boxed area in upper panel. (B) Representative insulin immunostaining of pancreas from WT, Adipo-KO, Endo-KO, and Total-KO mice; magnification, 80×. (C) Quantification of insulin-positive area. Pool of 2 experiments. WT: n = 5; Adipo-KO, Endo-KO, Total-KO: n = 6/group. (D) Insulin secretion from isolated islets of WT, Adipo-KO, Endo-KO, and Total-KO mice in response to low glucose (LG, 2.8 mM), high glucose (HG, 16.7 mM), HG + forskolin (FSK, 10 μM), and HG + KCl (30 mM). Insulin secretion is normalized to cellular DNA. LG, HG: n = 4/group. HG + FSK or KCl: n = 3/group. (E) Fold-increase in insulin secretion induced by HG + FSK (10 μM) over HG from 7D. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA followed by Tukey’s (C and E) or Dunnett’s (D) multiple-comparison test. HG Endo-KO is P < 0.05 by t test (D).

Article Snippet: For in-house FABP4 ELISA, we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core, as described previously (20) (clone 351.4.2E12.H1.F12 for capture, HRP-tagged clone 351.4.5E1.H3 for detection), and recombinant human FABP4 as a standard (R&D Systems).

Techniques: Immunostaining, Isolation, Comparison

Journal: International Journal of Molecular Sciences

Article Title: FABP4 Controls Fat Mass Expandability (Adipocyte Size and Number) through Inhibition of CD36/SR-B2 Signalling

doi: 10.3390/ijms24021032

Figure Lengend Snippet: Differential effects of FAT/CD36 inhibition in 3T3-L1 cell cultures versus adipose tissue explants. ( A ) Rat adipose tissue explants were treated during 48 h in low LG (1 g/L) versus high glucose (HG 4.5 g/L) media either alone or in presence of FAT/CD36 inhibitor AP5258 (1 μM). An example of rat adipose tissue explant labeled with AdipoRed (red) and Hoechst 33258 (blue) is presented, the scale bar represents 1 mm. Triglyceride contents were quantified using AdipoRed (AR) uptake after 40 min, fluorescence intensity was normalized at the time the marker was added (T0). Inhibition of FAT/CD36 induced a reduction of lipid content increase observed in HG media by comparison to LG media. Data are presented as mean fold changes AR after 40 min to T0 +/− SEM (n = 8), different letters represent significant differences (ANOVA test, p < 0.05). ( B ) Analysis of droplet size regulation 3 days after treatment with oleic acid (10 μM) in HG culture media in 3T3-L1 adipocytes. In presence of either lipolysis inhibitor ATGListatin (1 μM), AP5258 (1 μM), or cell-permeable FABP4 inhibitor (FABP4i 20 μM), lipid droplet sizes were increased. PPARA or PPARG (inhibited respectively by GW6471 and GW9662, 10 μM each) did not affect both cell lines. Insulin (0.05 U/mL) was used as a control of the inhibitory effect on droplet size increase. Results were obtained in independent experiments and normalized to corresponding control media (n = 8 biological replicates), and significant Student t -test p -values ( p < 0.05) are indicated.

Article Snippet: Human recombinant FABP4 (Sigma Aldrich) was labeled using Lightning-Link Rapid Alexa Fluor Atto488 Labeling Kit (Ozyme) and loaded onto 3T3-MTX cells plated onto Ibidi dishes (Clinisciences, Nanterre, France) at 100 ng/mL then rinsed in PBS, fixed with formalin 3% then stained with TRITC Phalloïdin 10 nM (Sigma Aldrich) and Dapi 10 ng/mL (Cayman Chemical, Montigny-le-Bretonneux, France) in PBS 0.1% Triton before analysis using the 3D Nanolive microscope (Lausanne, Switzerland).

Techniques: Inhibition, Labeling, Fluorescence, Marker

Journal: International Journal of Molecular Sciences

Article Title: FABP4 Controls Fat Mass Expandability (Adipocyte Size and Number) through Inhibition of CD36/SR-B2 Signalling

doi: 10.3390/ijms24021032

Figure Lengend Snippet: Transcriptional regulation pathways commonly regulated in FAT/CD36 knock-out mice and by fatty acids. Pathways over-represented in adipose cell lineage are reported as ASC (adipose stem cells), AT (adipose tissue), and dA (in vitro differentiated adipocytes). Pathways significantly over-represented in the gene dataset regulated by intracellular FABP4i are indicated in green, in that of extracellular FABP4 in red. Only significantly over-represented pathways in comparison to the genome dataset are reported (z-score confidence levels > 95%).

Article Snippet: Human recombinant FABP4 (Sigma Aldrich) was labeled using Lightning-Link Rapid Alexa Fluor Atto488 Labeling Kit (Ozyme) and loaded onto 3T3-MTX cells plated onto Ibidi dishes (Clinisciences, Nanterre, France) at 100 ng/mL then rinsed in PBS, fixed with formalin 3% then stained with TRITC Phalloïdin 10 nM (Sigma Aldrich) and Dapi 10 ng/mL (Cayman Chemical, Montigny-le-Bretonneux, France) in PBS 0.1% Triton before analysis using the 3D Nanolive microscope (Lausanne, Switzerland).

Techniques: Knock-Out, In Vitro

Journal: International Journal of Molecular Sciences

Article Title: FABP4 Controls Fat Mass Expandability (Adipocyte Size and Number) through Inhibition of CD36/SR-B2 Signalling

doi: 10.3390/ijms24021032

Figure Lengend Snippet: FABP4 is required to induce droplet size increase. ( A ). 3T3-MBX adipocytes treated during 24 h in low glucose (LG, 1 g/L), high glucose (HG, 4.5 g/L) with or without OA (10 μM) and or inhibitor of FABP4 (FABP4i 20 μM) then analyzed by image quantification of Adipored and Hoechst (n = 8 biological replicates). ( B ) Multisizer cell size distribution validates the inhibitory effect of FABP4i on cell size increase (n = at least 5000 cells). Significant differences are represented by letters (ANOVA test p -values p < 0.05). ( C ) Real-time imaging in high glucose media then merged AdipoRed (red) and Hoechst 33258 (blue), left panel, and representative images at ×4 and ×20 (right panel).

Article Snippet: Human recombinant FABP4 (Sigma Aldrich) was labeled using Lightning-Link Rapid Alexa Fluor Atto488 Labeling Kit (Ozyme) and loaded onto 3T3-MTX cells plated onto Ibidi dishes (Clinisciences, Nanterre, France) at 100 ng/mL then rinsed in PBS, fixed with formalin 3% then stained with TRITC Phalloïdin 10 nM (Sigma Aldrich) and Dapi 10 ng/mL (Cayman Chemical, Montigny-le-Bretonneux, France) in PBS 0.1% Triton before analysis using the 3D Nanolive microscope (Lausanne, Switzerland).

Techniques: Imaging

Journal: International Journal of Molecular Sciences

Article Title: FABP4 Controls Fat Mass Expandability (Adipocyte Size and Number) through Inhibition of CD36/SR-B2 Signalling

doi: 10.3390/ijms24021032

Figure Lengend Snippet: FAT/CD36 expression and activity in adipocytes. ( A – D ) The active form of FAT/CD36 was detected using an APC-coupled antibody directed against the extracellular part of the protein (blue color). Lipid droplets were labeled with AdipoRed (red color) and analyses were performed on 3T3-L1 adipocytes 3 days after treatment with oleic acid 10 μM: ( A ) Distribution of extracellular FAT/CD36 co-localized with lipid droplets (env. 5000 counts) is inversely regulated with droplet size. ( B ) Inhibitors of FAT/CD36 (AP5258 1 μM) and FABP4 (FABP4i 20 μM) did not affect droplet number (upper left panel), inhibition of FAT/CD36 promoted the increase of droplet size (upper right panel) although inhibition of FABP4 reduced the frequency of droplets with size up to 50 μm (lower left panel) and increased extracellular FAT/CD36 (lower right panel). ( C ) AdipoRed and APC-FAT/CD36 antibody detection on poorly vs. highly differentiated adipocytes and ( D ) in OA-treated adipocytes. ( E ) Gene transcription analysis of 3T3-MBX adipocytes treated over 3 days with oleic acid 5 μM in order to induce adipogenic genes, then 48 h, with either recombinant FABP4r (20 ng/mL), an inhibitor of FABP4 (FABP4i 20 μM), AMPK activator (AICAR 1 mM) or PPARG activator (rosiglitazone (20 μM). The effects on gene transcription for CIDEC , FABP4 , and FAT /CD36 were normalized to the standard HPRT gene. Results are presented as mean +/− SEM: n = 8 biological replicates with 3 images at objective ×4 per sample in ( B , F ), mean +/− SD in (E) (n = 4 replicates). ( F ) Then their capacity to increase droplet size (left panel) without significant effect on cell number (right panel) was assessed 3 days after treatment with oleic acid (OA 10 μM). ( E ) Results are presented as mean +/− SEM (n = 8 biological replicates). Different letters represent significant differences (Anova, p < 0.05).

Article Snippet: Human recombinant FABP4 (Sigma Aldrich) was labeled using Lightning-Link Rapid Alexa Fluor Atto488 Labeling Kit (Ozyme) and loaded onto 3T3-MTX cells plated onto Ibidi dishes (Clinisciences, Nanterre, France) at 100 ng/mL then rinsed in PBS, fixed with formalin 3% then stained with TRITC Phalloïdin 10 nM (Sigma Aldrich) and Dapi 10 ng/mL (Cayman Chemical, Montigny-le-Bretonneux, France) in PBS 0.1% Triton before analysis using the 3D Nanolive microscope (Lausanne, Switzerland).

Techniques: Expressing, Activity Assay, Labeling, Inhibition, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: FABP4 Controls Fat Mass Expandability (Adipocyte Size and Number) through Inhibition of CD36/SR-B2 Signalling

doi: 10.3390/ijms24021032

Figure Lengend Snippet: Lipolytic adipocytes inhibit the induction of adipogenesis. ( A ) Human adipocyte maturation was induced by oleic acid 10 μM over 48 h. FABP4 accumulation in the culture media was significantly detected after 48 h. Data are presented as mean FABP4 concentrations +/− SEM (n = 3 biological replicates in 6 wells plates). Different letters represent significant differences (ANOVA test, p -values < 0.05). ( B – D ) The 3T3-MBX adipocytes were differentiated in either 96-wells ( B ) or 6-wells inserts ( C ), for 4 days then full maturation was induced by treatment with oleic acid 10 μM over 3 days. They were then co-cultured with approx. 80% confluent 3T3-L1 fibroblasts prepared in low glucose media. Co-cultures were performed over 3 days in either low (LG) or high glucose (HG) culture media to induce basal lipolysis in adipocytes. ( B ) Micrographs at ×4 magnification of 96 wells insert adipocytes labeled with AdipoRed (red) and Hoechst (blue). ( C ) Micrographs at objectives ×4 (upper panel) and ×20 (lower panel) of adipocytes grown in 6-well inserts. ( D ) Lipid content in 3T3-L1 fibroblasts was measured as fluorescence intensity fold change of Adipored (AR) normalized to Hoechst (H) in co-cultures vs. alone in 96-wells plates. Data are presented as mean fold changes +/− SEM (n = 7 replicates) with Student’s t -test p -values p < 0.05 in HG vs. LG. ( E ) Transcriptional regulation of adipogenic markers in 3T3-L1 fibroblasts co-cultured with 3T3-MBX adipocytes during 3 days in high vs. low glucose media. Data are presented as mean fold changes mRNA (normalized to standard HPRT gene) of HG versus LG condition +/− SD of 3 replicates (6-wells inserts) with significant Student’s t -test p -values p < 0.05.

Article Snippet: Human recombinant FABP4 (Sigma Aldrich) was labeled using Lightning-Link Rapid Alexa Fluor Atto488 Labeling Kit (Ozyme) and loaded onto 3T3-MTX cells plated onto Ibidi dishes (Clinisciences, Nanterre, France) at 100 ng/mL then rinsed in PBS, fixed with formalin 3% then stained with TRITC Phalloïdin 10 nM (Sigma Aldrich) and Dapi 10 ng/mL (Cayman Chemical, Montigny-le-Bretonneux, France) in PBS 0.1% Triton before analysis using the 3D Nanolive microscope (Lausanne, Switzerland).

Techniques: Cell Culture, Labeling, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: FABP4 Controls Fat Mass Expandability (Adipocyte Size and Number) through Inhibition of CD36/SR-B2 Signalling

doi: 10.3390/ijms24021032

Figure Lengend Snippet: Extracellular Fatty acid binding protein 4 (FABP4) inhibits oleic acid (OA)-induced adipogenesis in human adipose stem cells (ASCs). At time (T) human ASCs were exposed during proliferation to OA complexed to human albumin (hSA) ( A , B ), recombinant FABP4 at several doses ( C , D ), or hSA 1%, OA 10 μM and/or FABP4 20 ng/mL ( E – G ), then differentiation was induced (D1 and D2). The effect of OA on differentiation was revealed by the dose-dependent effect on cell adhesion force monitored as a cell index normalized at the time cells were loaded. ( A ) Real-time monitoring of cell index (representing adhesion force) shows the dose-dependent promoting effect of oleic acid on adipogenesis, i.e., reduction of cell adhesion force after induction of differentiation for OA doses up to 0.5 μM, ( B ) validated by quantification at the end of the experiment of the mean droplet cell size (droplet count normalized to nuclei counts) on ×4 images. ( C ) Real-time monitoring of adipogenesis in presence of FABP4 at several doses shows no significant effect. ( D ) TG accumulation (AdipoRed fluorescence intensity normalized to cell number) due to OA uptake is independent of OA complexation to hSA versus FABP4. ( E ) Real-time monitoring of adipogenesis shows that although OA complexation to hSA improved adipogenesis (reduction of cell index after induction of differentiation), its complexation to FABP4 inhibited adipogenesis (an increase of cell adhesion after induction of differentiation), ( F ) without affecting accumulation of TG detected as described in ( D ). ( G ) Representative micrographs of merged Adipored (red) and Hoechst 33258 (blue) human cells at the end of experiments. Data are presented as mean +/− SEM and letters represent significant differences (n = 8 biological replicates, ANOVA test, p < 0.05) in ( F ).

Article Snippet: Human recombinant FABP4 (Sigma Aldrich) was labeled using Lightning-Link Rapid Alexa Fluor Atto488 Labeling Kit (Ozyme) and loaded onto 3T3-MTX cells plated onto Ibidi dishes (Clinisciences, Nanterre, France) at 100 ng/mL then rinsed in PBS, fixed with formalin 3% then stained with TRITC Phalloïdin 10 nM (Sigma Aldrich) and Dapi 10 ng/mL (Cayman Chemical, Montigny-le-Bretonneux, France) in PBS 0.1% Triton before analysis using the 3D Nanolive microscope (Lausanne, Switzerland).

Techniques: Binding Assay, Recombinant, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: FABP4 Controls Fat Mass Expandability (Adipocyte Size and Number) through Inhibition of CD36/SR-B2 Signalling

doi: 10.3390/ijms24021032

Figure Lengend Snippet: Fatty acid binding protein 4 (FABP4) uptake and regulation of active FAT/CD36 form in 3T3-MBX cells ( A ) Nanolive imaging after 3 h treatment with Atto488-FABP4 (FABP4r 100 ng/mL) and quantification of max Atto488 intensity per cell. ( B ) Atto488-FABP4 quantification by cytometry. ( C ) Effect of FABP4r on extracellular FAT/CD36 detected with APC-coupled anti-FAT/CD36 antibody after 4 h and after 3 days. Data are presented as mean values +/− SEM (n = 10 cells in A, 8 biological replicates in ( B , C )) with significant differences indicated by Student t -test p -values in ( A , B ), and different letters in ( C ) (Anova test p -values < 0.05).

Article Snippet: Human recombinant FABP4 (Sigma Aldrich) was labeled using Lightning-Link Rapid Alexa Fluor Atto488 Labeling Kit (Ozyme) and loaded onto 3T3-MTX cells plated onto Ibidi dishes (Clinisciences, Nanterre, France) at 100 ng/mL then rinsed in PBS, fixed with formalin 3% then stained with TRITC Phalloïdin 10 nM (Sigma Aldrich) and Dapi 10 ng/mL (Cayman Chemical, Montigny-le-Bretonneux, France) in PBS 0.1% Triton before analysis using the 3D Nanolive microscope (Lausanne, Switzerland).

Techniques: Binding Assay, Imaging, Cytometry

Journal: International Journal of Molecular Sciences

Article Title: FABP4 Controls Fat Mass Expandability (Adipocyte Size and Number) through Inhibition of CD36/SR-B2 Signalling

doi: 10.3390/ijms24021032

Figure Lengend Snippet: Fatty acid binding protein 4 (FABP4) inhibits the pro-adipogenic programme mediated by fatty acid/FAT/CD36 pathways in 3T3-L1 fibroblasts. Treatments were performed in DMEM high glucose media over 24 h with oleic acid (OA 0.5 μM), recombinant FABP4 (FABP4 20 ng/mL), FAT/CD36 inhibitor (AP5258, 1 μM) or rosiglitazone (Rosi 10 μM). ( A ) Gene transcription analyses by qRT-PCR, normalized to HPRT standard, show the inhibitory effect of FABP4 and FAT/CD36 inhibition on their own induction by OA. ( B ) Phycoerythrin (PE)-coupled anti CD36 antibody was analyzed by cytometry and showed that both FABP4r and FAT/CD36 inhibitor reversed the induction of extracellular FAT/CD36 by OA. Data are presented as mean values +/− SEM (( A ): n = 8 biological replicates, ( B ): 4 replicates; at least 5000 cells/sample) with significant differences represented by letters (ANOVA p -values < 0.05).

Article Snippet: Human recombinant FABP4 (Sigma Aldrich) was labeled using Lightning-Link Rapid Alexa Fluor Atto488 Labeling Kit (Ozyme) and loaded onto 3T3-MTX cells plated onto Ibidi dishes (Clinisciences, Nanterre, France) at 100 ng/mL then rinsed in PBS, fixed with formalin 3% then stained with TRITC Phalloïdin 10 nM (Sigma Aldrich) and Dapi 10 ng/mL (Cayman Chemical, Montigny-le-Bretonneux, France) in PBS 0.1% Triton before analysis using the 3D Nanolive microscope (Lausanne, Switzerland).

Techniques: Binding Assay, Recombinant, Quantitative RT-PCR, Inhibition, Cytometry

Journal: International Journal of Molecular Sciences

Article Title: FABP4 Controls Fat Mass Expandability (Adipocyte Size and Number) through Inhibition of CD36/SR-B2 Signalling

doi: 10.3390/ijms24021032

Figure Lengend Snippet: Putative mechanism of Fatty acid binding protein 4 (FABP4) role in the regulation of fat mass. Fat mass regulation proceeds through pro-adipogenic processes allowing lipid storage (red pathways) requiring fatty acid-induced PPARG gene transcription, lipid uptake, and inhibition of lipolysis. Fatty acid interaction with FAT/CD36 controls both AMPK anti-lipolytic activity and PPARG mediated gene transcription, including those of FAT/CD36 and FABP4 . FAT/CD36 increases during adipogenesis together with FABP4. Increased accumulation of FABP4 in absence of lipolysis in mature adipocytes is a limiting factor inducing reduction of FAT/CD36 through PPARG inhibition (negative feedback), which in turn alters its antilipolytic activity. Finally, FABP4 exerts a limitation to a fatty acid-induced increase of adipocyte size through the restoration of basal lipolysis (green pathways), then promotes small or immature adipocytes to reach the optimal size but inhibits the recruitment of new adipocytes. The model suggests that under normal conditions, lipid storage is then optimized to obtain a homogenous population of adipocytes (in a range of 50–100 μm) allowing to monitor the metabolic response to diet uptake versus energy expenditure.

Article Snippet: Human recombinant FABP4 (Sigma Aldrich) was labeled using Lightning-Link Rapid Alexa Fluor Atto488 Labeling Kit (Ozyme) and loaded onto 3T3-MTX cells plated onto Ibidi dishes (Clinisciences, Nanterre, France) at 100 ng/mL then rinsed in PBS, fixed with formalin 3% then stained with TRITC Phalloïdin 10 nM (Sigma Aldrich) and Dapi 10 ng/mL (Cayman Chemical, Montigny-le-Bretonneux, France) in PBS 0.1% Triton before analysis using the 3D Nanolive microscope (Lausanne, Switzerland).

Techniques: Binding Assay, Inhibition, Activity Assay