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: PLoS ONE

Article Title: Long-lived weight-reduced αMUPA mice show higher and longer maternal-dependent postnatal leptin surge

doi: 10.1371/journal.pone.0188658

Figure Lengend Snippet: Each time point represents the mean ± s.e.m. of 5 to 17 mice (see for details). Pups were sampled every four days, following four hours of chow deprivation (first experiment), and were weaned at day 24 (P24). (A) Postnatal circulating leptin level measured using ELISA kit (R&D Systems Inc.). Insert of A. Mean P4-P24 leptin level. Bars with different letters are significantly different ( P <0.05, based on two-ways ANOVA followed by a post-hoc test). (B) Leptin:FM ratio. *, P <0.05 by post hoc comparison following one-way ANOVA of all four mice groups.

Article Snippet: Leptin level in the serum, milk, and stomach content was measured using a mouse leptin ELISA kit (R&D Systems Inc.), as used previously for both strains [ , ].

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: PLoS ONE

Article Title: Long-lived weight-reduced αMUPA mice show higher and longer maternal-dependent postnatal leptin surge

doi: 10.1371/journal.pone.0188658

Figure Lengend Snippet: Multiple regression of leptin on strain, gender, age, fat mass, and fat-free mass.

Article Snippet: Leptin level in the serum, milk, and stomach content was measured using a mouse leptin ELISA kit (R&D Systems Inc.), as used previously for both strains [ , ].

Techniques:

Journal: PLoS ONE

Article Title: Long-lived weight-reduced αMUPA mice show higher and longer maternal-dependent postnatal leptin surge

doi: 10.1371/journal.pone.0188658

Figure Lengend Snippet: Multiple regression of leptin on age, fat mass, strain, and gender, carried out separately for each age group.

Article Snippet: Leptin level in the serum, milk, and stomach content was measured using a mouse leptin ELISA kit (R&D Systems Inc.), as used previously for both strains [ , ].

Techniques:

Journal: PLoS ONE

Article Title: Long-lived weight-reduced αMUPA mice show higher and longer maternal-dependent postnatal leptin surge

doi: 10.1371/journal.pone.0188658

Figure Lengend Snippet: Each time point represents the mean ± s.e.m. of 6 to 22 mice, females only. Pups were sampled every four days, following four hours of chow deprivation (first and second experiments) and dam deprivation (second experiment only). Leptin and corticosterone levels were measured using ELISA kits (R&D Systems Inc.). (A and B) Postnatal circulating leptin level of dam-deprived and non-deprived female WT (left) and αMUPA (right) mice. (C) Postnatal circulating leptin levels of dam-deprived female αMUPA and WT mice. (D) Postnatal body weight growth curves of dam-deprived and non-deprived female WT mice. (E) Postnatal body weight growth curves of dam-deprived and non-deprived female αMUPA mice. (F) Postnatal body weight growth curves of dam-deprived female αMUPA and WT mice. (G) Mean P4-P24 leptin level of dam-deprived and non-deprived female αMUPA and WT mice. (H) Corticosterone P12 level of dam-deprived and non-deprived female αMUPA and WT mice. Bars with different letters are significantly different ( P <0.05, based on two-ways ANOVA followed by a post-hoc test). *, P <0.05 by post hoc comparison following one-way ANOVA of all four mice groups.

Article Snippet: Leptin level in the serum, milk, and stomach content was measured using a mouse leptin ELISA kit (R&D Systems Inc.), as used previously for both strains [ , ].

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: PLoS ONE

Article Title: Long-lived weight-reduced αMUPA mice show higher and longer maternal-dependent postnatal leptin surge

doi: 10.1371/journal.pone.0188658

Figure Lengend Snippet: Bars represent the mean ± s.e.m. of 6 dams per strain (A) and their female pups (B), one female pup per dam, all sampled at P12. Dams' serum and milk samples (A) were obtained 4 hours after their separation from their pups. Milk secretion was encouraged by subcutaneous oxytocin injection (2 IU/kg) administered 15 min before sampling. Pups' serum and stomach content (B) were sampled 30 min after they were reunited with their dams. Leptin level was measured using ELISA kit (R&D Systems Inc.). *, P<0.05 between strains, by Mann-Whitney non-parametric test. #, P<0.05 within strains, by Wilcoxon non-parametric Test.

Article Snippet: Leptin level in the serum, milk, and stomach content was measured using a mouse leptin ELISA kit (R&D Systems Inc.), as used previously for both strains [ , ].

Techniques: Injection, Sampling, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Nutrients

Article Title: Stimulation of GHRH Neuron Axon Growth by Leptin and Impact of Nutrition during Suckling in Mice

doi: 10.3390/nu15051077

Figure Lengend Snippet: Primary and secondary antibodies used for immunohistochemistry experiments.

Article Snippet: Plasma leptin concentrations in 10-day-old male mice were determined individually with the Mouse/Rat Leptin (R&D systems) ELISA kit, according to the manufacturer’s instructions.

Techniques: Immunohistochemistry

Journal: Nutrients

Article Title: Stimulation of GHRH Neuron Axon Growth by Leptin and Impact of Nutrition during Suckling in Mice

doi: 10.3390/nu15051077

Figure Lengend Snippet: Underfeeding during suckling results in lower body weight and plasma leptin levels. ( A ) Increases in litter size from six (normally fed, open bars) to nine (underfed, blue bars) pups per dam are associated with a lower body weight in pups, by the age of seven days ( n = 32 normally fed pups and 41 underfed pups of both sexes). ( B ) Underfeeding during suckling was associated with lower plasma levels of leptin at 10 days of age, as determined by ELISA ( n = 8 per group). ( C ) Illustration (40X magnification obtained with a BX43 Olympus fluorescence microscope equipped with a DP73 CCD camera) of a growing axon from an arcuate nucleus explant cultured in vitro, showing that the GHRH+ axon in green (uppermost image) expresses the leptin receptor in red (middle image). A merged image is shown at the bottom (arrow). Scale bars represent 20 µm. Data are presented as the mean ± SEM. Comparisons were performed in Mann–Whitney tests, with ** p < 0.01 and *** p < 0.001.

Article Snippet: Plasma leptin concentrations in 10-day-old male mice were determined individually with the Mouse/Rat Leptin (R&D systems) ELISA kit, according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Fluorescence, Microscopy, Cell Culture, In Vitro, MANN-WHITNEY

Journal: Nutrients

Article Title: Stimulation of GHRH Neuron Axon Growth by Leptin and Impact of Nutrition during Suckling in Mice

doi: 10.3390/nu15051077

Figure Lengend Snippet: Leptin stimulates axon growth in GHRH neurons in arcuate nucleus explants from normally fed pups. ( A ) Illustrative IHC of AgRP neurons from arcuate nucleus explants micro-dissected from one-week-old normally fed pups, in control conditions (left panels), and after stimulation with leptin (middle panels) and with leptin/IGF-1 (right panels). ( B ) Illustrative images of dual IHC for the axons of total arcuate nucleus neurons labeled with NF (top panels in red) and GHRH neurons labeled with eGFP (bottom panels in green), in the same conditions. Scale bars represent 1000 µm for AgRP+ and NF+ IHC (4X magnification) and 200 µm for GHRH+ IHC (10X magnification), for images from a BX612 Olympus fluorescence microscope equipped with a DP71 CCD camera. ( C ) Quantification of the growth of AgRP axons after 24 h of stimulation with leptin or leptin/IGF-I relative to control conditions ( n = 4 experiments), and ( D ) of the growth of NF (plain bars) and GHRH (dashed bars) axons ( n = 5–7 experiments). Data are presented as the mean ± SEM. Results were compared in a one-way ANOVA with the Newman–Keuls post hoc test (c) or a two-way ANOVA with Bonferroni correction (d), with *: p < 0.05 and **: p < 0.01 vs. control conditions and #: p < 0.05 vs. leptin stimulation.

Article Snippet: Plasma leptin concentrations in 10-day-old male mice were determined individually with the Mouse/Rat Leptin (R&D systems) ELISA kit, according to the manufacturer’s instructions.

Techniques: Control, Labeling, Fluorescence, Microscopy

Journal: Nutrients

Article Title: Stimulation of GHRH Neuron Axon Growth by Leptin and Impact of Nutrition during Suckling in Mice

doi: 10.3390/nu15051077

Figure Lengend Snippet: Signaling pathways involved in the axon growth of arcuate neurons in explants from normally fed pups. ( A ) Illustrative triple IHC of arcuate nucleus explants from the hypothalamus micro-dissected from one-week-old normally fed pups under control conditions (first panel), and following stimulation with leptin (second panel), leptin/NSC (third panel), leptin/LY (fourth panel) and leptin/PD (fifth panel), with NF (top panels in green), GHRH (middle panels in red) and AgRP (bottom panels in blue). Scale bars represent 100 µm for NF+ IHC (4X magnification) and 200 µm for GHRH+ and AgRP+ IHC (10X magnification), on images obtained with an Olympus BX43 fluorescence microscope equipped with a DP73 CCD camera. Quantifications of the growth of ( B ) NF ( n = 5–9 experiments), ( C ) GHRH ( n = 4–10 experiments) and ( D ) AgRP ( n = 5–10 experiments) axons stimulated for 24 h with leptin alone, or in combination with one of the three inhibitors (NSC_33994, LY_294002 or PD_0325901). Data are presented as the mean ± SEM. Results were analyzed by two-way ANOVA with Bonferroni correction, with *: p < 0.05 and ***: p < 0.001 vs. control conditions and ###: p < 0.001 vs. leptin stimulation.

Article Snippet: Plasma leptin concentrations in 10-day-old male mice were determined individually with the Mouse/Rat Leptin (R&D systems) ELISA kit, according to the manufacturer’s instructions.

Techniques: Protein-Protein interactions, Control, Fluorescence, Microscopy

Journal: Nutrients

Article Title: Stimulation of GHRH Neuron Axon Growth by Leptin and Impact of Nutrition during Suckling in Mice

doi: 10.3390/nu15051077

Figure Lengend Snippet: GHRH neurons from underfed pups are resistant to leptin for the stimulation of axon growth. ( A ) Illustrative IHC of arcuate nucleus explants from the hypothalamus micro-dissected from one-week-old underfed pups in control conditions (left panel), and after stimulation with leptin alone (middle panel), or with leptin/IGF-1 (right panel), with AgRP axons labeled in orange. ( B ) Axons from total arcuate nucleus neurons and GHRH neurons labeled by dual-IHC for neurofilament (NF, in red) and eGFP (in green), respectively. Scale bars represent 1000 µm for AgRP+ and NF+ IHC (4X magnification) and 200 µm for GHRH+ IHC (10X magnification), on images from an Olympus BX612 fluorescence microscope equipped with a DP71 CCD camera. ( C ) Quantification of the growth of AgRP axons stimulated by incubation for 24 h with leptin or leptin/IGF-I, relative to control conditions ( n = 5 experiments), and ( D ) quantification of the growth of NF (plain bars) and GHRH (dashed bars) axons ( n = 6 experiments). Data are presented as the mean ± SEM. Results were compared in a one-way ANOVA with the Newman–Keuls post hoc test (c) or in a two-way ANOVA with Bonferroni correction (d), with *: p < 0.05.

Article Snippet: Plasma leptin concentrations in 10-day-old male mice were determined individually with the Mouse/Rat Leptin (R&D systems) ELISA kit, according to the manufacturer’s instructions.

Techniques: Control, Labeling, Fluorescence, Microscopy, Incubation

Journal: Nutrients

Article Title: Stimulation of GHRH Neuron Axon Growth by Leptin and Impact of Nutrition during Suckling in Mice

doi: 10.3390/nu15051077

Figure Lengend Snippet: Alterations to leptin-stimulated signaling pathways in arcuate nucleus explants from underfed pups. The activation of the three signaling pathways by the exposure of seven-day explants to leptin (+) for 15 min was different in explants from underfed pups (blue bars) and in explants from normally fed pups (open bars). Data are presented as the fold induction of phosphorylated protein/total protein ratios (normalized against actin) for stimulated relative to unstimulated conditions for ( A ) phosphorylated Jak2/Jak2/actin ( n = 5 per group), ( B ) phosphorylated Stat3/Stat3/actin ( n = 5–7 per group), ( C ) phosphorylated-AKT/AKT/actin ( n = 9 per group), ( D ) phosphorylated MEK1/MEK1/actin ( n = 5–6 per group), ( E ) phosphorylated ERK1/ERK1/actin (left panel) and phosphorylated-ERK2/ERK2/actin (right panel; n = 8 per group). Data are presented as the mean ± SEM, with a Mann–Whitney analysis, with *: p < 0.05, **: p < 0.01.

Article Snippet: Plasma leptin concentrations in 10-day-old male mice were determined individually with the Mouse/Rat Leptin (R&D systems) ELISA kit, according to the manufacturer’s instructions.

Techniques: Protein-Protein interactions, Activation Assay, MANN-WHITNEY

Journal: Molecular metabolism

Article Title: Adipose knockout of H-ferritin improves energy metabolism in mice.

doi: 10.1016/j.molmet.2024.101871

Figure Lengend Snippet: Figure 5: Fth AKO mice exhibited an altered adipose gene expression profile. (A,B) qRT-PCR analysis of Pparg, C/ebpa, Retn, Plin1, AdipoQ, Glut4, Scd1, Lep, Fabp4, Hsl, Atgl, and Mgl expression in iBAT and iWAT of Fth AKO and Fthfl/flmice (n ¼ 3e4, from two independent experiments). (C) Serum leptin of Fth AKO and Fthfl/flmice (n ¼ 3e4). (D) qRT-PCR analysis of Cpt1a and Cpt1b expression in iBAT and iWAT of Fth AKO and Fthfl/flmice (n ¼ 5e6, from two independent experiments). Data was analyzed by two-tailed unpaired t- tests. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Serum leptin Serum leptin concentration was measured by a mouse leptin ELISA kit (ProteinTech, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Heliyon

Article Title: Improved leptin sensitivity and increased soluble leptin receptor concentrations may underlie the additive effects of combining PYY [ [1] , [2] , [3] , [4] , [5] , [6] , [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] , [29] , [30] , [31] , [32] , [33] , [34] ] and exendin-4 on body weight lowering in diet-induced obese mice

doi: 10.1016/j.heliyon.2024.e32009

Figure Lengend Snippet: PYY(3 – 36) and Ex4 in combination increases leptin sensitivity and increases the concentration of circulating soluble leptin receptor. A-D) Accumulated food intake (kcal) and body weight change (g) 4h (A, B) and 20h (C, D) after leptin injection(5 mg/kg) at treatment day 10.E) Plasma leptin concentration (pM) at day 15, F: Plasma concentration of soluble leptin receptor (SLR) at day 15, and G: Hepatic Lepra expression at day 15. Data are expressed as means ± SEM, n = 12. Statistical significance was assessed by one-way ANOVA (E) or by two-way ANOVA followed (F and G). In both cases, ANOVA test's were followed Tukey's multiple comparisons test. In case of A-D, test of significance was restricted to comparing within group effects (vehicle and leptin treatment). *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Soluble leptin receptor was analysed by a mouse leptin receptor picokine ELISA kit (BOSTER biological technology, Cat # EK0440).

Techniques: Concentration Assay, Injection, Clinical Proteomics, Expressing