Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: a Schematic diagram illustrating the experimental procedure for diet-induced obesity (DIO) mice model. 9-week-old Esrra fl/fl and Esrra AKO male mice were fed a normal chow diet (NCD) or high-fat diet (HFD) for 16 weeks. (Schematic created with BioRender.com. Agreement number: WP26KB8FER). b Representative pictures and body weights of NCD and DIO mice. c Representative images and weight analysis of white adipose tissue (WAT) depots, including gonadal WAT (gWAT), inguinal WAT (iWAT), and mesentery WAT (mWAT). d Plasma triglyceride (TG) and free fatty acid (FFA) levels. e Plasma leptin, IL-6 and TNFa levels. f Representative micro-CT images of the distal femoral trabecular bone. g Quantitative analysis of bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N) and trabecular separation (Tb. Sp). h H&E staining of femur sections (scale bar: 100 μm). Yellow arrows indicate the bone marrow adipocytes. Osteoblast surface to bone surface ratio (Ob.S/BS) and osteoblast number to bone surface ratio (Ob.N/BS) are shown on the right panel. i Calcein double labeling of trabecular bone (scale bar: 50 μm). Mineral apposition rate (MAR) and bone formation rate (BFR/BS) were determined as graphs. j TRAP staining of femur sections with quantitative analysis of Oc.S/BS and Oc.N/BS. TRAP‐positive purple spots indicate multinucleated osteoclasts (scale bar: 100 μm). Plasma P1NP ( k ) and CTX1 ( l ) levels. m PLIN1 positive marrow adipocytes (PLIN1 + , red) and SPP1 (green) immunofluorescence staining in femur sections (scale bar: 100 μm). The box in the upper showing the metaphysis region near growth plate is represented at higher magnification in the bottom (scale bar: 50 μm). n The number and area of adipocytes in the femur marrow per tissue area and the quantification of SPP1 fluorescence intensity were measured from femur sections in ( m ). Data are shown as mean ± SD ( n = 6 mice per group). * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis is performed using two-way ANOVA with Fisher’s LSD post hoc analysis. Source data are provided as a Source Data file.

Article Snippet: ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions.

Techniques: Clinical Proteomics, Micro-CT, Staining, Labeling, Immunofluorescence, Fluorescence

Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: a Schematic diagram illustrating the experimental procedure for ovariectomy (OVX)-induced osteoporosis mice model. 10-week-old Esrra fl/fl and Esrra AKO female mice underwent either sham or OVX operation for 8 weeks (schematic created with BioRender.com. Agreement number: II26KB8K2T). b Representative images and weights of adipose depots. c Representative images and adipocytes size analysis from H&E-stained gWAT sections (scale bar: 50 μm). d Plasma leptin levels. Micro-CT images of distal femurs in sham and OVX mice ( e ) with morphometric analysis of BV/TV, Tb.N, Tb.Th, and Tb.Sp ( f ). g Representative TRAP-stained images and quantification of Oc.S/BS and Oc.N/BS in distal femoral metaphysis regions from sham and OVX mice (scale bar: 100 μm). h Representative H&E-stained images and quantification of Ob.S/BS and Ob.N/BS (scale bar: 100 μm). Plasma P1NP ( i ) and CTX1 ( j ) levels. k Calcein double labeling with quantitative analysis of MAR and BFR/BS (scale bar: 50 μm). l , m Immunofluorescence co-staining and quantification of PLIN1 + bone marrow adipocytes (red) and SPP1 (green) of femur sections from sham and OVX mice. Scale bar: upper panel, 100 μm; lower panel, 50 μm. Data are shown as mean ± SD ( n = 7 mice per group). * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis is performed using two-way ANOVA with Fisher’s LSD post hoc analysis. Source data are provided as a Source Data file.

Article Snippet: ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions.

Techniques: Staining, Clinical Proteomics, Micro-CT, Labeling, Immunofluorescence

Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: a Protein expression levels of ESRRA were evaluated in BMSCs upon adipogenic induction for indicated days, comparing Esrra fl/fl mice (blue font) with Esrra AKO mice (red font). b Schematic representation of the experimental design. BMSCs were isolated from Esrra fl/fl and Esrra AKO mice and subsequently subjected to either adipogenic or osteogenic induction for indicated days (schematic created with BioRender.com. Agreement number: LW26KBAHRA). c Representative images and quantitative analyses of alizarin red S staining and oil red O staining following the indicated induction. n = 4 biologically independent experiments. Rosi, rosiglitazone. d Volcano plot of transcriptional profiling between BMSCs-derived BMAds lineage cells from Esrra fl/fl and Esrra AKO mice. Differentially expressed genes were identified using DESeq2 analysis ( p < 0.05). n = 4 biologically independent samples. Gene Ontology (GO) ( e ) and Kyoto Encyclopedia of Genes and Genomes (KEGG) ( f ) pathway enrichment analyses of all differentially expressed genes by RNA-seq (top 10 according to adjusted p value). g Heatmap depicting selected genes related to secreted factors ( p < 0.05). n = 4 biologically independent samples. h Boxplot showing the transcript expression value (FPKM) of Spp1 based on RNA-seq data. Data are represented as box and whiskers with bars representing maximum and minimum values and with median highlighted as a line. n = 4 biologically independent samples. Validation of SPP1 and leptin expression were performed by qRT-PCR ( i ) and western blotting analysis ( j ) in BMAds that were fully differentiated for 14 days. n = 6 biologically independent samples. All the data are shown as mean ± SD. ** p < 0.01 and *** p < 0.001. Statistical analysis is performed using unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.

Article Snippet: ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions.

Techniques: Expressing, Isolation, Staining, Derivative Assay, RNA Sequencing, Biomarker Discovery, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: a Plasma SPP1 levels of Esrra fl/fl and Esrra AKO mice at 8 weeks post-OVX or sham operation. n = 7 mice per group. b Representative images and analysis of SPP1 and leptin co-staining of gWAT from OVX mice studied in ( a ) (scale bar: 100 μm). n = 7 mice per group. c mRNA expression of Spp1 , Leptin , Adipoq , Pparg , Cebpa and Fabp4 of gWAT from OVX mice. n = 7 mice per group. d Protein levels of SPP1, leptin and ESRRA of gWAT from Esrra fl/fl and Esrra AKO mice at 4 and 8 weeks post-OVX or sham operation. e Schematic diagram displays the potential binding sites of ESR1 within the Spp1 promoter, including S1, S2 and S3. Fragments for ChIP assay shown as region 1 (R1) and region 2 (R2). f Luciferase reporter activities of the Spp1 promoter in adipogenesis induced 3T3-L1 cells transfected with Esrra or Esr1 expressing plasmids in the presence of E2 or not. n = 3 biologically independent experiments. The consensus sequence binding motifs for ESR1 response element (ERE) and ESRRA response element (ERRE) are presented. g Luciferase reporter activities of the Spp1 promoter regulated by E2/ESR1 in the presence of wild-type (WT) or DNA-binding domain-deleted ESRRA construct (ESRRA-ΔDBD). n = 4 biologically independent experiments. h ChIP assay with ESR1 antibody in BMSCs from Esrra fl/fl and Esrra AKO mice after adipogenic induction for 4 days along with or without E2. n = 3 biologically independent experiments. i Luciferase reporter activities of the R2 deleted- Spp1 promoter (ΔR2-luc) as compared to Spp1 promoter (WT-luc). n = 4 biologically independent experiments. j Enrichment of ESRRA in R2 of Spp1 promoter in adipogenesis induced 3T3-L1 cells with the indicated treatments. n = 3 biologically independent experiments. Spp1 mRNA in murine BMAds ( k ), matured 3T3-L1 adipocytes ( l ) or human BMSCs-derived BMAds ( m ) infected with adenovirus expressing ESRRA or GFP with E2 treatment for 2 days. n = 4, 6, 4 biologically independent experiments, respectively. n Diagram illustrating the mechanism of ESRRA-regulated repression of Spp1 transcriptional expression via interfering with E2/ESR1 signaling in adipocytes (schematic created with BioRender.com. Agreement number: BH26KF823M). Data are shown as mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis is performed using unpaired two-tailed Student’s t test ( c ), one-way ANOVA followed by Bonferroni’s post hoc tests ( k , l , m ). Source data are provided as a Source Data file.

Article Snippet: ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions.

Techniques: Clinical Proteomics, Staining, Expressing, Binding Assay, Luciferase, Transfection, Sequencing, Construct, Derivative Assay, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: a Schematic diagram showing the procedure of the conditioned medium (CM) preparation from cultured BMAds or minced gWAT; and wild-type BMSCs were differentiated in osteogenic/adipogenic mixed induction medium (OIM:AIM = 1:1) supplemented with the indicated CM (schematic created with BioRender.com. Agreement number: ZP26KB8SN4). b The concentrations of soluble leptin in gWAT-CM prepared from Esrra fl/fl and Esrra AKO OVX mice were measured by ELISA. n = 4 biologically independent samples. c mRNA levels of osteogenesis markers Runx2 , Sp7 , Bglap , as well as adipogenic markers Pparg , Cebpa , Fabp4 in wild-type BMSCs cultured with mixed induction medium and indicated gWAT-CM for 14 days. n = 4 biologically independent experiments. Representative images and quantification of alizarin red S staining ( d ) and oil red O staining ( e ) of BMSCs cultured as in ( c ) with an addition of gWAT-CM for 14 days, in the presence of rSPP1, SPP1 Nab, recombinant leptin (rLeptin), leptin receptor antagonist Allo-aca or IgG as indicated. n = 4 biologically independent experiments. Scale bar: 2 mm ( d ); scale bar: 100 μm ( e ). f The concentrations of soluble leptin in BMAds-CM as prepared from ( a ). n = 6 biologically independent samples. g mRNA levels of indicated genes in wild-type BMSCs cultured in mixed induction medium supplemented with the indicated BMAds-CM for 14 days. n = 6 biologically independent experiments. Representative images and quantification of alizarin red S staining ( h ) and oil red O staining ( i ) of BMSCs cultured as in ( g ) with the indicated treatments for 14 days. The experiments were conducted according to the procedure shown in ( a – e ). n = 4 biologically independent experiments. Scale bar: 2 mm ( h ); scale bar: 100 μm ( i ). Data are shown as mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis is performed using unpaired two-tailed Student’s t test ( b , c , f , g ) and two-way ANOVA with Fisher’s LSD post hoc analysis ( d , h , e , i ). Source data are provid e d as a Source Data file.

Article Snippet: ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Recombinant, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: a Experimental design. Wild-type BMSCs were isolated from C57BL/6 mice and differentiated into BMAds, and 3T3-L1 preadipocytes were cultured in adipogenic medium for 14 days. Mature adipocytes were subsequently treated with C29 for an additional 2 days (schematic created with BioRender.com. Agreement number: IH26KB8VKA). The mRNA and protein levels of leptin and SPP1 in mature 3T3-L1 adipocytes ( b , c ) or BMAds ( d , e ). In vitro experiments were repeated four times. f Schematic diagram showing the experimental design for pharmacological treatments in DIO mice. Seven-week-old C57BL/6 mice were fed either a NCD or HFD for 18 weeks, and received either vehicle or C29 (30 mg/kg/body weight) every day during the last 4 weeks (schematic created with BioRender.com. Agreement number: WH26KBA0SE). g Plasma leptin, TNFa and IL6 levels. h , i Representative micro-CT images and histomorphometry analysis of BV/TV, Tb.N, Tb.Th and Tb.Sp at the distal femurs. j Representative micro-CT images of middle-segment of cortical bone and histomorphometry analysis of cortical bone volume/tissue volume ratio (BV/TV) and cortical thickness (Ct.Th). k Representative images of TRAP-stained femoral sections (scale bar: 100 μm). Quantitative assessment of trabecular Oc.S/BS and Oc.N/BS based on TRAP-stained sections. Plasma CTX-1 ( l ) and P1NP ( m ) levels. n Representative images of H&E-stained femur sections (scale bar: 100 μm). Quantitative assessment of trabecular Ob.S/BS and Ob.N/BS based on H&E-stained sections. o Representative PLIN1 and SPP1 immunostaining in femoral sections. Scale bar: upper panel, 100 μm; lower panel, 50 μm. p Quantification of PLIN1 + adipocyte number and of SPP1 fluorescence intensity. Six mice per group were used in all animal experiments. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis is performed using one-way ANOVA followed by Bonferroni’s post hoc tests ( b – d ) and two-way ANOVA with Fisher’s LSD post hoc analysis ( g , i – n , p ). Source data are provided as a Source Data file.

Article Snippet: ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions.

Techniques: Isolation, Cell Culture, In Vitro, Clinical Proteomics, Micro-CT, Staining, Immunostaining, Fluorescence

Journal: Nature Communications

Article Title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

doi: 10.1038/s41467-024-48255-8

Figure Lengend Snippet: Estrogen deficiency or high-fat diet-induced obesity results in excessive bone marrow adipocytes and distorted type H vessel. Adipocyte ESRRA deficiency preserves bone formation and counteracts high marrow adiposity by decreased leptin and enhanced SPP1 secretion, dictating BMSCs fate commitment toward osteogenesis and promoting vessel formation (schematic created with BioRender.com. Agreement number: QL26KB8YYQ).

Article Snippet: ELISA was conducted to detect SPP1 (R&D systems #MOST00), leptin (R&D systems #MOB00B), CTX1 (USCN #CEA665Mu), PINP (NOVUS biologicals #NBP2-76466), TNFa (USCN #MEA133Mu) or IL6 (USCN #MEA079Mu) from plasma or CM based on manufacturer’s instructions.

Techniques:

Journal: Pediatric Reports

Article Title: Resistin in Urine and Breast Milk: Relation to Type of Feeding and Anthropometry at 1-Month

doi: 10.3390/pediatric14010013

Figure Lengend Snippet: Correlation between resistin concentration in the breast milk and ( a ) body weight; ( b ) age; ( c ) body mass index (BMI); and ( d ) leptin concentration in mothers’ breast milk at 1 month post-partum.

Article Snippet: We analyzed urinary and breast milk resistin levels (Human Resistin ELISA kit; Arigo biolaboratories, Hsinchu City, Taiwan) and breastmilk leptin (Human Leptin Quantikine ELISA Kit; R&D Systems, Minneapolis, MI, USA) in infants using an enzyme-linked immunosorbent assay kit.

Techniques: Concentration Assay

Journal:

Article Title: Leptin surge precedes onset of autoimmune encephalomyelitis and correlates with development of pathogenic T cell responses

doi: 10.1172/JCI200316721

Figure Lengend Snippet: Serum leptin increase precedes the acute onset of chronic-progressive EAE and correlates with disease susceptibility, body-weight loss, and food-intake inhibition in EAE-susceptible C57BL/6J wild-type mice but not in EAE-resistant leptin-deficient C57BL/6J ob/ob mice. (a) Mean clinical score (bars) and body weight (curves) of C57BL/6J wild-type littermate controls (black bars and triangles) and leptin-deficient C57BL/6J ob/ob mice (white bars and circles) after immunization with MOG35–55 peptide. Leptin-deficient mice are EAE resistant and do not lose weight after immunization, whereas wild-type controls are EAE susceptible and lose body weight. (b) Serum leptin (bars) increases before clinical onset of EAE only in wild-type controls and is undetectable in leptin-deficient mice; this increase correlates with food-intake inhibition, which is only present in wild-type animals. (c) Simple regression analysis showing a significant correlation (P = 0.0005, r = 0.89) between the change in serum leptin before and after immunization with MOG35–55 peptide (Δ indicates the increase in serum leptin) and the CDI, calculated as the sum of each daily clinical score of each single mouse (n = 10). A significant correlation was observed in wild-type control mice but not in leptin-deficient mice. One representative experiment out of two is shown. y, equation that defines this regression; R2, regression coefficient, R, correlation coefficient.

Article Snippet: All serum samples (dilution 1/20) were tested in a mouse leptin-specific ELISA (R&D Systems) according to the manufacturer’s instructions; the detection limit of the assay was typically less than 22 pg/ml, and the intra- and interassay variability were 4.3% and 7.6%, respectively.

Techniques: Inhibition

Journal:

Article Title: Leptin surge precedes onset of autoimmune encephalomyelitis and correlates with development of pathogenic T cell responses

doi: 10.1172/JCI200316721

Figure Lengend Snippet: Leptin levels and neurological impairment during EAE induction with MOG35–55 and PLP139–151 encephalitogenic peptides or after adoptive transfer of MOG35–55-specific CD4+ T cells

Article Snippet: All serum samples (dilution 1/20) were tested in a mouse leptin-specific ELISA (R&D Systems) according to the manufacturer’s instructions; the detection limit of the assay was typically less than 22 pg/ml, and the intra- and interassay variability were 4.3% and 7.6%, respectively.

Techniques: Adoptive Transfer Assay

Journal:

Article Title: Leptin surge precedes onset of autoimmune encephalomyelitis and correlates with development of pathogenic T cell responses

doi: 10.1172/JCI200316721

Figure Lengend Snippet: Serum leptin increase precedes acute onset of adoptively induced EAE and correlates with disease susceptibility, body-weight loss, and food-intake inhibition in EAE-susceptible C57BL/6J wild-type mice but not in EAE-resistant leptin-deficient C57BL/6J ob/ob mice. (a) Mean clinical score (bars) and body weight (curves) of C57BL/6J wild-type littermate controls (black bars and triangles) and leptin-deficient C57BL/6J ob/ob mice (white bars and circles) after adoptive transfer of 2.5 × 107 MOG35–55–specific CD4+ T cells. Leptin-deficient mice are EAE resistant and do not lose weight after adoptive transfer, whereas wild-type controls are EAE susceptible and lose body weight. (b) Serum leptin (bars) significantly increases before clinical onset of EAE in wild-type controls but increases very little in leptin-deficient mice; this increase correlates with food-intake inhibition, which is only present in wild-type animals. (c) Simple regression analysis showing a significant correlation (r = 0.97, P = 0.004) between the change in serum leptin before and after adoptive transfer of MOG35–55–specific CD4+ T cells (Δ indicates the increase in serum leptin) and the CDI, calculated as the sum of each daily clinical score of each single mouse (n = 5). A significant correlation was observed in wild-type control mice but not in leptin-deficient mice. One representative experiment of two is shown.

Article Snippet: All serum samples (dilution 1/20) were tested in a mouse leptin-specific ELISA (R&D Systems) according to the manufacturer’s instructions; the detection limit of the assay was typically less than 22 pg/ml, and the intra- and interassay variability were 4.3% and 7.6%, respectively.

Techniques: Inhibition, Adoptive Transfer Assay

Journal:

Article Title: Leptin surge precedes onset of autoimmune encephalomyelitis and correlates with development of pathogenic T cell responses

doi: 10.1172/JCI200316721

Figure Lengend Snippet: Serum leptin increase precedes the acute onset of relapsing-remitting EAE and correlates with disease susceptibility, body-weight loss, and food-intake inhibition in EAE-susceptible SJL/J female mice but not in EAE-resistant male mice. (a) Mean clinical score (bars) and body weight (curves) of SJL/J female mice (black bars and triangles) and male mice (white bars and circles) after immunization with PLP139–151 peptide. SJL/J male mice are EAE resistant and do not lose weight after immunization, whereas SJL/J female mice are EAE susceptible and lose body weight. (b) Serum leptin (bars) increases before clinical onset of EAE only in SJL/J female mice and is significantly lower in male mice in preimmune conditions (2080.0 ± 325.0 pg/ml in SJL/J female mice and 470.0 ± 100.0 pg/ml in SJL/J male mice, P < 0.01); the increase correlates with food-intake inhibition present only in female mice. (c) Simple regression analysis showing a significant correlation (r = 0.71, P = 0.02) between the difference in serum leptin before and after immunization with PLP139–155 peptide (Δ indicates the increase in serum leptin) and the CDI, calculated as the sum of each daily clinical score of each single mouse (n = 10). A significant correlation was observed in SJL/female mice but not in male mice. Data are accumulated and averaged from two independent experiments with similar results. y, equation that defines regression; R2, regression coefficient; R, correlation coefficient.

Article Snippet: All serum samples (dilution 1/20) were tested in a mouse leptin-specific ELISA (R&D Systems) according to the manufacturer’s instructions; the detection limit of the assay was typically less than 22 pg/ml, and the intra- and interassay variability were 4.3% and 7.6%, respectively.

Techniques: Inhibition

Journal:

Article Title: Leptin surge precedes onset of autoimmune encephalomyelitis and correlates with development of pathogenic T cell responses

doi: 10.1172/JCI200316721

Figure Lengend Snippet: Starvation at priming with the encephalitogenic peptide of SJL/J females reduces the clinical severity of EAE by inducing a Th2 cytokine switch, reversible by recombinant leptin administration. (a) Mean clinical score (bars) and body weight (curves) of SJL/J female mice starved for 48 hours (hatched bars and circles), ad libitum–fed controls (black bars and triangles), and leptin-treated female mice (gray bars and squares). Starvation delayed disease onset and reduced clinical score and body-weight loss during the acute phase of the disease. Leptin replacement during the 48-hour starvation reversed the starvation-induced blunting of the disease, so that the EAE course was similar to that observed in the ad libitum–fed group. (b) Proliferative response of lymph node–derived T cells against PLP139–151 is impaired after 48 hours of starvation when compared with the control group. (c and d) Starvation for 48 hours reduced IFN-γ secretion but increased IL-4 production. (e) Addition of recombinant leptin to T-cell cultures is able to partially restore the capacity of T cells from starved mice to secrete IFN-γ. One representative experiment out of two is shown.

Article Snippet: All serum samples (dilution 1/20) were tested in a mouse leptin-specific ELISA (R&D Systems) according to the manufacturer’s instructions; the detection limit of the assay was typically less than 22 pg/ml, and the intra- and interassay variability were 4.3% and 7.6%, respectively.

Techniques: Recombinant, Derivative Assay

Journal:

Article Title: Leptin surge precedes onset of autoimmune encephalomyelitis and correlates with development of pathogenic T cell responses

doi: 10.1172/JCI200316721

Figure Lengend Snippet: Lymph node and CNS expression of leptin during acute/active EAE. (a) Leptin expression in SJL/J female mouse adipose tissue used as positive control. (b and c) Expression of leptin in T cells and macrophages in a draining lymph node from SJL/J female mice after immunization with PLP139–151. (d) Leptin was not expressed in the brain of C57BL/6J ob/ob mice after immunization with MOG35–55 peptide (n = 4). (e and f) Expression of leptin in inflammatory infiltrates (white square) and in choroid plexus (arrow) during the acute phase of EAE in C57BL/J6 WT mice (n = 4). (g) Leptin was not expressed in the brain of SJL/J male mice after immunization with PLP139–151 peptide (n = 6). (h and i) Leptin expression in inflammatory lesions in the acute phase of EAE in SJL/J female mice (n = 6). (j) Cerebellum of SJL/J male mice did not express leptin after immunization with PLP139–151 peptide, whereas in k and l leptin was expressed in inflammatory infiltrates (white square) and choroid plexus (arrow) of SJL/J females. (m) Spinal cord C57BL/J6 ob/ob mice immunized with MOG35–55 peptide did not express leptin. (n and o) Expression of leptin in neurons (white square in n) and two inflammatory infiltrates around blood vessels (arrows in n) detectable during the acute phase of EAE in C57BL/6J WT mice spinal cord. (p–r) Leptin expression was revealed in T cells present in inflammatory infiltrates of the brain, cerebellum, and spinal cord (arrows) of C57BL/J6 WT mice after adoptive transfer, but it was not detectable in the CNS of C57BL/6J ob/ob mice after adoptive transfer (not shown). The white squares in b, e, h, k, and n represent the zone of higher magnification shown in c, f, i, l, and o, respectively. Magnifications, ×200 (b, d, e, g, h, j, k, and m); ×300 (a, l, and n); and ×400 (c, f, i, o, and p–r).

Article Snippet: All serum samples (dilution 1/20) were tested in a mouse leptin-specific ELISA (R&D Systems) according to the manufacturer’s instructions; the detection limit of the assay was typically less than 22 pg/ml, and the intra- and interassay variability were 4.3% and 7.6%, respectively.

Techniques: Expressing, Positive Control, Adoptive Transfer Assay

Journal:

Article Title: Leptin surge precedes onset of autoimmune encephalomyelitis and correlates with development of pathogenic T cell responses

doi: 10.1172/JCI200316721

Figure Lengend Snippet: Simple regression analysis between the number of leptin-positive cells in active-EAE lesions and the inflammatory score in EAE-susceptible mice, and in vitro leptin secretion by activated CD4+ T cells. (a and b) In both chronic-progressive and relapsing-remitting EAE, there is a statistically significant positive correlation between the number of leptin-positive cells in active EAE lesions and the CNS inflammatory score. (c) PLP139–151–specific CD4+ T cells, derived from immunized mice (see Methods), secrete on PLP139–151 72-hour in vitro stimulation a small but consistent amount of immunoreactive leptin in the supernatants, when compared to unstimulated T cells (387.5 ± 96.01 pg/ml vs. 32.5 ± 5.6 pg/ml, respectively; P = 0.01).

Article Snippet: All serum samples (dilution 1/20) were tested in a mouse leptin-specific ELISA (R&D Systems) according to the manufacturer’s instructions; the detection limit of the assay was typically less than 22 pg/ml, and the intra- and interassay variability were 4.3% and 7.6%, respectively.

Techniques: In Vitro, Derivative Assay

Journal:

Article Title: Leptin surge precedes onset of autoimmune encephalomyelitis and correlates with development of pathogenic T cell responses

doi: 10.1172/JCI200316721

Figure Lengend Snippet: Antileptin receptor (anti-ObR) blocking antibodies inhibit antigen-specific and non-antigen-specific T-cell activation. (a) The anti-PLP139–151 proliferative response of lymph node–derived T lymphocytes from immunized SJL/J female mice is completely inhibited by the addition to cell cultures of anti-ObR. (b–d) Anti-ObR partially inhibits the proliferative response of T cells from immunized SJL/J female mice toward polyclonal mitogenic stimuli such as concanavalin A, anti-CD3ε, and MLR against irradiated allogeneic splenocytes from C57BL/6J mice (see Methods). (e and f) The addition of anti-ObR antibody to concanavalin A– or anti-CD3ε–activated spleen-derived T cells, obtained from leptin receptor mutant C57BL/Ks db/db mice, is not able to affect their proliferative response as compared with the control antibody. One representative experiment out of four is shown.

Article Snippet: All serum samples (dilution 1/20) were tested in a mouse leptin-specific ELISA (R&D Systems) according to the manufacturer’s instructions; the detection limit of the assay was typically less than 22 pg/ml, and the intra- and interassay variability were 4.3% and 7.6%, respectively.

Techniques: Blocking Assay, Activation Assay, Derivative Assay, Irradiation, Mutagenesis

Journal:

Article Title: Leptin directly acts within the hypothalamus to stimulate gonadotropin-releasing hormone secretion in vivo in rats

doi: 10.1113/jphysiol.2002.023895

Figure Lengend Snippet: Plasma concentrations of leptin, oestradiol and progesterone in the six experimental groups examined in this study

Article Snippet: All the MPOA, ME-ARC and AHA groups were perfused with 1.0, 3.0 or 10 ng ml −1 of the recombinant rat leptin (R & D Systems, Inc., Minneapolis, MN, USA) during the period of 14:00-17:20 h. The rat leptin was dissolved in the ACSF immediately before use.

Techniques: Clinical Proteomics

Journal:

Article Title: Leptin directly acts within the hypothalamus to stimulate gonadotropin-releasing hormone secretion in vivo in rats

doi: 10.1113/jphysiol.2002.023895

Figure Lengend Snippet: In this and subsequent figures (2, ​,44 and ​and5):5): (1) data from only the highest concentration of leptin (10 ng ml−1) are shown; (2) the horizontal bar indicates the period during which leptin (▴) or vehicle (ACSF, ○) was infused; (3) the time of perfusate collection in the upper three graphs is shifted 10 min ahead of the actual time of perfusion because the dead space of the pull system (150 μl) corresponds to a 10 min period of perfusion (flow rate, 15 μl min−1); (4) measurements of α-MSH, NPY and GnRH in the perfusates are expressed as point values at the centre of their collection periods; and (5) dotted lines in the graphs for α-MSH, NPY and GnRH indicate the limits of detection for each peptide. Number of rats in each subgroup = 8-10.

Article Snippet: All the MPOA, ME-ARC and AHA groups were perfused with 1.0, 3.0 or 10 ng ml −1 of the recombinant rat leptin (R & D Systems, Inc., Minneapolis, MN, USA) during the period of 14:00-17:20 h. The rat leptin was dissolved in the ACSF immediately before use.

Techniques: Concentration Assay

Journal:

Article Title: Leptin directly acts within the hypothalamus to stimulate gonadotropin-releasing hormone secretion in vivo in rats

doi: 10.1113/jphysiol.2002.023895

Figure Lengend Snippet: Open bars, ACSF (control); bars with horizontal lines, leptin (1.0 ng ml−1); bars with vertical lines, leptin (3.0 ng ml−1); filled bars, leptin (10 ng ml−1).* Statistically significant vs. the ‘before’ values of the respective groups. † Statistically significant vs. the other three groups. ‡ Statistically significant vs. the leptin (1.0 ng ml−1) group.

Article Snippet: All the MPOA, ME-ARC and AHA groups were perfused with 1.0, 3.0 or 10 ng ml −1 of the recombinant rat leptin (R & D Systems, Inc., Minneapolis, MN, USA) during the period of 14:00-17:20 h. The rat leptin was dissolved in the ACSF immediately before use.

Techniques: Control

Journal:

Article Title: Leptin directly acts within the hypothalamus to stimulate gonadotropin-releasing hormone secretion in vivo in rats

doi: 10.1113/jphysiol.2002.023895

Figure Lengend Snippet: Open bars, ACSF (control); bars with horizontal lines, leptin (1.0 ng ml−1); bars with vertical lines, leptin (3.0 ng ml−1); filled bars, leptin (10 ng ml−1). *Statistically significant vs. the ‘before’ values of the respective groups. † Statistically significant vs. the other three groups. ‡ Statistically significant vs. the leptin (1.0 ng ml−1) group. ** Statistically significant vs. the ACSF and leptin (1.0 ng ml−1) groups.

Article Snippet: All the MPOA, ME-ARC and AHA groups were perfused with 1.0, 3.0 or 10 ng ml −1 of the recombinant rat leptin (R & D Systems, Inc., Minneapolis, MN, USA) during the period of 14:00-17:20 h. The rat leptin was dissolved in the ACSF immediately before use.

Techniques: Control

Journal: PLoS Genetics

Article Title: Congenital lipodystrophy induces severe osteosclerosis

doi: 10.1371/journal.pgen.1008244

Figure Lengend Snippet: A) Transplanted fat depots 3 months after surgery. B) Fat depot weight 3 months after transplantation. C) Serum leptin and adiponectin of FF mice 3 months after fat depot transplantation. WT and non-transplanted FF mice serve as control. D) μCT analysis of trabecular bone volume and bone mineral density of distal femurs of FF mice 3 months after sham operation or transplantation of fat derived from WT or adipokine-deficient mice. Data are presented as mean ± SD. **p<0.01; *** p<0.001 as determined by ANOVA with Holm-Sidak's post hoc analysis for multiple comparisons test. D) comparison with FF Sham except where detailed.

Article Snippet: The primary antibody cocktail contained rat anti-mouse CD45-BUV395 (BD Horizon, clone 30-F11, final dilution factor 1:200), rat anti-mouse TER-119-APC (BioLegend, clone TER-119, 1:200), rat anti-mouse CD41-BV421 (BioLegend, clone MWReg30, 1:300), rat anti-mouse/human CD11b (BioLegend, clone M1/70, 1:400), and rat-anti mouse Leptin receptor (LepR)-biotin (R&D Systems, polyclonal, 1:50) in Brilliant Stain Buffer (BD Biosciences) containing 10 μg/mL FcBlock.

Techniques: Transplantation Assay, Control, Derivative Assay, Comparison