lepr  (R&D Systems)

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Continuous intraosseous administration of SCS prevents glucocorticoid-induced bone degeneration. ( A ) Schematic illustration of the glucocorticoid (GC; MPS)-induced bone deterioration and intraosseous SCS treatment. ( B-D ) Representative H&E staining images of the femur at 6 weeks (B). Magnified views of the cortical bone and trabecular bone in the marrow cavity are shown on the right. Solid arrows indicate normal osteocytes, while hollow arrows indicate empty osteocyte lacunae. Quantification of empty lacunae ratios in cortical bone (C) and trabecular bone (D). n = 6 biological replicates. (Scale bars, 500 μm and 25 μm) ( E-H ) Representative immunofluorescence staining of OPN + mature osteoblasts, osteolectin + osteoprogenitors, and VE-cadherin + endothelial cells (ECs) in femur at 6 weeks (E), and corresponding quantifications (F–H). n = 6 biological replicates. (Scale bars, 100 μm and 20 μm) ( I and J ) Representative flow cytometry plots of capillary subtypes in the femur (I), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (J). n = 6 biological replicates. ( K and L ) Flow cytometry plots showing Sca-1 hi CD31 hi arteriolar ECs (K), and corresponding quantification (L). n = 6 biological replicates. ( M and N ) Representative micro-CT 3D images of the femur (M). Quantitative analysis of percent bone volume (BV/TV) (N). n = 6 biological replicates. (Scale bars, 1.5 mm, 600 μm and 545 μm) ( O and P ) ELISA analysis of VEGF (O) and PDGF-BB (P) levels in bone marrow supernatant and peripheral serum from PBS- and SCS-treated groups at week 6. n = 6 biological replicates. ( Q ) ELISA quantification of the osteogenic factor osteocalcin in peripheral serum at week 6. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, F, G, H, J, L, N, O, P and Q ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Staining, Immunofluorescence, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS attenuates full-blown bone marrow senescence during GC-induced skeletal degeneration. ( A ) Schematic illustration of the experimental design for assessing bone marrow senescence at 4 weeks after combined SCS and MPS treatment. ( B ) Representative images of SA-β-Gal–positive cells (green) in femur after MPS treatment. BM indicates bone marrow; TBM indicates trabecular bone matrix. (Scale bars, 100 μm and 25 μm) ( C – E ) Representative immunofluorescence images at week 4 showing Emcn + sinusoidal ECs, ALP + osteoblasts, and p16 + senescent cells (C), with corresponding quantification of Emcn + p16 + (D) and ALP + p16 + cells (E). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) ( F – H ) Flow cytometry analysis of CD45 − Ter119 − CD31 + arteriolar ECs in the femur after PBS or SCS treatment (F). Ki-67 + proliferative status was further analyzed within this population (G), and corresponding double-positive cell quantification is shown in (H). n = 6 biological replicates. ( I – K ) Representative flow cytometry plots of CD45 − Ter119 − CD31 − leptin receptor + (LepR + ) mesenchymal stem cells (MSCs) in the bone marrow at 4 weeks (I), with analysis of the proportion of SA-β-Gal–positive cells (J) and corresponding quantification (K). n = 6 biological replicates. ( L ) Representative flow cytometry plots of CD45 − Ter119 − CD144 + cells (including endothelial cells and endothelial progenitors) in the bone marrow at week 4 post-MPS treatment. ( M and N ) Gating and analysis of CD45 − Ter119 − CD144 + HMGB1 + ECs by flow cytometry (M), and corresponding quantification (N). n = 6 biological replicates. ( O and P ) Representative immunofluorescence images showing OPN + osteoblasts and γ-H2A.X + DNA damage marker–positive cells in the femur at 4 weeks (O), with quantification of senescent osteoblasts (P). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) Data are presented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Statistical significance was determined using an unpaired two-tailed Student's t -test ( D, E, H, K, N and P ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Immunofluorescence, Flow Cytometry, Marker, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS suppresses senescence cascade amplification by attenuating secondary spread from GC-induced primary senescent adipocytes. ( A ) Schematic illustration of SCS intervention exclusively during the fully developed senescent phase of MPS-induced bone marrow. ( B ) qPCR analysis of senescence-associated markers ( Cdkn1b , Cdkn1a , and Cdkn2c ) in bone tissues at 4 weeks following combined SCS and MPS treatment. n = 3 biological replicates. ( C ) ELISA analysis of bone marrow senescence-associated factors (IL-1β, IL-18, TNF-α, IL-6, CXCL1, and CCL3) after 4 weeks of combined treatment with SCS and MPS. n = 4 biological replicates. ( D ) Quantification of the maximal compressive load of the isolated distal femur and femoral diaphysis. n = 6 biological replicates. ( E ) Schematic diagram depicting isolation of bone marrow adipocytes from mice treated with SCS and MPS for 14 days using mature adipocyte-specific fast centrifugation and construction of a senescence propagation model in vitro . ( F and G ) Representative flow cytometry plots (D) and quantification (E) of EdU-positive (proliferating) CD45 − Ter119 − CD31 − LepR + MSCs cultured for 3 days with adipocyte conditioned medium (CM). n = 6 biological replicates. ( H and I ) Representative ALP staining images (F) and corresponding quantification of ALP activity (G) in CD45 − Ter119 − CD31 − LepR + MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 30 μm) ( J and K ) Representative Oil Red O staining (H) and quantification (I) of adipogenic differentiation in MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) ( L and M ) Representative images (J) and quantification (K) of crystal violet-stained fibroblast colony-forming units (CFU-F) in MSCs cultured with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 400 μm) ( N ) qPCR analysis of senescence-related markers ( Cdkn2a and Cdkn1a ) in MSCs treated with different adipocyte CMs. n = 3 biological replicates. ( O and P ) Representative immunofluorescence-FISH images (M) and quantification (N) showing colocalization of γ-H2A.X with telomere-associated foci (TAF) in MSCs cultured with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 7 μm and 1 μm) ( Q and R ) Representative images (O) and quantification (P) of 2D tube formation assays in HUVECs cultured for 3 days with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( S and T ) Representative images (Q) and quantification (R) of SA-β-Gal–positive HUVECs (green) following 3-day treatment with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( U ) qPCR analysis of the senescence-related gene LMNB1 in HUVECs treated with various adipocyte CMs. n = 3 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B, C, D, G, I, K, M, N, R, T and U ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Amplification, Enzyme-linked Immunosorbent Assay, Isolation, Centrifugation, In Vitro, Flow Cytometry, Cell Culture, Staining, Activity Assay, Immunofluorescence, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS reprograms the lineage commitment of MSCs after GC treatment and inhibits the generation of primary senescent adipocytes. ( A ) Schematic illustration of the in vitro investigation of SCS targeting the prostaglandin/PPARγ/INK positive feedback loop in MPS-induced primary senescent adipocytes. ( B ) Representative flow cytometry plot showing p16 + senescent cells in adipocytes derived from bone marrow after 14 days of in vivo MPS induction and subsequently treated with SCS in vitro . ( C ) qPCR analysis of 12 senescence-associated markers in primary senescent adipocytes after in vitro SCS treatment. n = 3 biological replicates. ( D ) ELISA analysis of IL-1β levels in adipocyte supernatant following in vitro SCS treatment. n = 6 biological replicates. ( E ) ELISA analysis of secreted prostaglandins PGD2 and PGE2 in adipocytes under different treatment conditions. D-PBS: bone marrow adipocytes isolated from mice treated in vivo with the solvent control DMSO, followed by in vitro treatment with PBS; M-PBS: bone marrow adipocytes isolated from mice treated in vivo with MPS, followed by in vitro treatment with PBS. M-SCS: bone marrow adipocytes isolated from mice treated in vivo with MPS, followed by in vitro treatment with SCS. ( F ) Western blot analysis of intracellular COX-2 protein levels in adipocytes across the three treatment conditions. ( G ) Schematic illustration of competitive osteogenic–adipogenic differentiation of CD45 − Ter119 − CD31 − LepR + MSCs after 7 days of in vivo SCS and MPS co-treatment. ( H ) qPCR analysis of pan-adipocyte markers ( Fabp4 , Adipoq , Plin1 , Cd36 , and Lep ) in CD45 − Ter119 − CD31 − LepR + MSCs after 14 days of in vitro competitive lineage differentiation. n = 3 biological replicates. ( I and J ) Representative immunofluorescence images (I) and quantification (J) of perilipin + adipocytes and osteopontin + mature osteoblasts derived from lineage-committed MSCs. n = 6 biological replicates. (Scale bars, 30 μm, 15 μm and 15 μm). ( K ) Western blot analysis of adipogenesis-related markers C/EBPα, PPARγ, and C/EBPβ in the lineage-mixed cells after in vitro competitive differentiation of CD45 − Ter119 − CD31 − LepR + MSCs. ( L ) qPCR analysis of lipogenesis-related markers Fasn , Scd1 , Srebf1 , Acaca , and Acacb . n = 3 biological replicates. ( M and N ) Representative H&E staining images (M) of the femurs at day 14 following SCS and MPS co-treatment. Yellow arrows indicate bone marrow adipocytes. Magnified images show hypertrophic adipocyte morphology, with quantification of adipocyte diameter (N). n = 19 biological replicates. (Scale bars, 200 μm, 50 μm and 20 μm). Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( C, D, H, J, L and N ), or one-way ANOVA with Tukey's post hoc test ( E ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: In Vitro, Flow Cytometry, Derivative Assay, In Vivo, Enzyme-linked Immunosorbent Assay, Isolation, Solvent, Control, Western Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Gene expression profiles of bone marrow-derived LepR + MSCs after 7-day in vivo co-treatment with SCS and MPS. ( A ) Heatmap showing DEGs in CD45 − Ter119 − CD31 − LepR + MSCs sorted from bone marrow at day 7 post-treatment with SCS versus PBS ( P < 0.05, |log fold change| > 1.5). n = 3 biological replicates. ( B ) Representative GO biological process enrichment analysis of downregulated DEGs. ( C ) Top 20 enriched KEGG pathways of downregulated DEGs in SCS versus PBS. ( D ) GSEA plots of biological processes positively enriched in the SCS group (|NES| > 1, nominal P < 0.05, FDR <0.25). ( E ) Representative downregulated DEGs associated with adipogenesis and lipogenesis identified through KEGG pathway analysis. n = 3 biological replicates. ( F ) Top 20 enriched KEGG pathways of upregulated DEGs in SCS versus PBS. ( G ) Representative GO biological process enrichment analysis of upregulated DEGs. ( H ) Representative upregulated DEGs identified through biological process enrichment analysis. n = 3 biological replicates. ( I and J ) GSEA plots of KEGG pathways negatively enriched in the SCS group (|NES| > 1, nominal P < 0.05, FDR <0.25).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Gene Expression, Derivative Assay, In Vivo

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS targets downstream senescent lineage commitment of bone marrow MSCs to mitigate GC-induced bone deterioration. ( A ) Schematic diagram illustrating the experimental design: CD45 − Ter119 − CD31 − LepR + MSCs isolated from mice co-treated with SCS and MPS for 7 days were subjected to in vitro lineage-competitive differentiation, followed by DEX-induced senescence in lineage-mixed cells. These cells were then adoptively transplanted into healthy bone marrow cavity to assess bone deterioration development. ( B ) Representative H&E-stained images of the femur 12 weeks after adoptive transfer. PBS-DEX group: LepR + MSCs from PBS and MPS co-treated mice subjected to in vitro lineage differentiation and DEX-induced senescence, followed by transplantation. SCS-DEX group: LepR + MSCs from SCS and MPS co-treated mice processed similarly. PBS group: solvent control without cell transplantation. Solid arrows indicate intact osteocytes; hollow arrows indicate empty lacunae. (Scale bars, 250 μm and 25 μm) ( C – E ) Quantitative analysis of marrow hypertrophic adipocyte diameter (C), proportion of empty osteocyte lacunae in trabecular bone (D), and adipocyte number (E) in the metaphysis 12 weeks post-transplantation. n = 19 biological replicates (C), n = 6 biological replicates (D), n = 8 biological replicates (E). ( F ) Quantification of empty lacunae in epiphysis at 12 weeks post-transplantation. n = 6 biological replicates. ( G – I ) Representative flow cytometry plots of capillary ECs subtypes in the femur at 12 weeks (G), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (H) and CD45 − Ter119 − CD31 lo Emcn lo ECs (I). n = 6 biological replicates. ( J and K ) Representative flow cytometry plots (J) and corresponding quantification (K) of CD45 − Ter119 − Sca-1 hi CD31 hi arteriolar ECs in the femur at 12 weeks post-transplantation. n = 6 biological replicates. ( L ) Representative micro-CT images of the femur at 12 weeks post-transplantation across different treatment groups. (Scale bars, 1.5 mm and 500 μm) ( M – P ) Quantitative analysis of bone parameters in the metaphysis: bone mineral density (BMD) (M), percent bone volume (BV/TV) (N), trabecular separation (Tb.Sp) (O), and trabecular number (Tb.N) (P). n = 6 biological replicates. ( Q ) Serum ELISA analysis of the osteogenic marker osteocalcin at 12 weeks post-transplantation. n = 6 biological replicates. ( R and S ) ELISA analysis of PDGF-BB (R) and VEGF (S) in both bone marrow supernatant and peripheral serum at 12 weeks post-transplantation. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, E, F, H, I, K, M, N, O, P, Q, R and S ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Isolation, In Vitro, Staining, Adoptive Transfer Assay, Transplantation Assay, Solvent, Control, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay, Marker

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS modulates mesenchymal stem cell lineage bias via activation of the IGF-1/PI3K/Akt/mTOR signaling pathway. ( A ) Quantitative analysis of osteocyte morphology in the trabecular bone matrix of the bone marrow at week 6 after MPS treatment with or without SCS, in the presence of various neutralizing antibodies (NAbs) and antagonistic proteins. ( B ) ELISA analysis of IGF-1 and BMP-2 levels in the femoral bone marrow and peripheral serum at day 7 following SCS treatment under MPS conditions. ( C and D ) Western blot analysis of phospho-PI3K, phospho-Akt, and phospho-mTOR (C), as well as phospho-Smad1/5/8, phospho-ERK, and phospho-p38 (D), in CD45 − Ter119 − CD31 − LepR + MSCs after 15-min stimulation with conditioned medium (CM) derived from bone marrow fluid at day 7 following SCS treatment. ( E – G ) Representative flow cytometry plots (E, F) and quantitative analysis (G) of CD45 − CD31 − Sca-1 + CD24 − adipocyte progenitor cells (APCs), CD45 − CD31 − Sca-1 + CD24 + MSCs (E), and CD45 − CD31 − Sca-1 − PDGFRα + (Pα + ) osteoprogenitor cells (OPCs) (F) from femoral bone marrow at day 14 post-MPS induction with or without combined treatment using SCS and IGF-1 NAb or Noggin. ( H and I ) Representative SA-β-Gal staining images (green) of the femur (H), and corresponding quantification (I), at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. Insets show magnified views of bone marrow (BM) and trabecular bone matrix (TBM) regions. (Scale bars, 100 μm and 25 μm) ( J ) qPCR analysis of 12 senescence-associated markers in ex vivo femoral bone tissues at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. ( K ) Representative Oil Red O staining images of CD45 − Ter119 − CD31 − LepR + MSCs sorted from femurs at day 7 following MPS treatment with SCS in combination with LY294002 or LDN-193189, after in vitro adipogenic induction. (Scale bars, 50 μm and 25 μm) ( L and M ) γ-H2A.X and telomere-associated DNA damage foci (TAFs) co-localization analysis (L), and corresponding quantification (M), in CD45 − Ter119 − CD31 + arteriolar ECs sorted from femurs at day 28 following MPS treatment with SCS in combination with rapamycin or LDN-193189, using immuno-FISH staining. (Scale bars, 7 μm and 1 μm) ( N and O ) Sequential fluorescent labeling using calcein (N) and quantification of mineral apposition rate (O) in femurs treated with SCS and MPS for 4 weeks, with or without LY294002 and/or GW9662. (Scale bars, 50 μm) ( P ) ELISA analysis of five senescence-associated cytokines in femoral bone marrow at day 28 following MPS treatment with SCS in combination with rapamycin and/or T0070907. ( Q and R ) Representative t-distributed stochastic neighbor embedding (t-SNE) plots (Q) from flow cytometric analysis of CD45 − CD31 − Sca-1 + CD24 − APCs, CD45 − CD31 − Sca-1 + CD24 + MSCs, CD45 − CD31 − Sca-1 − Pα + OPCs, CD45 − Ter119 − CD31 + arteriolar ECs, and CD45 − Ter119 − Emcn + sinusoidal ECs at day 14 following MPS treatment with SCS in combination with IGF-1 and/or rosiglitazone, and quantitative analysis of APCs (R) ( S ) Heatmap showing the fluorescent intensity distribution of Lamin-B1 expression across five cellular subpopulations as identified in the t-SNE clustering plot. ∗ P < 0.05 vs. IgG (empty lacunae); # P < 0.05 vs. IgG (filled lacunae). ∗ P < 0.05 vs. SCS; # P < 0.05 vs. SCS + IGF-1 NAb. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B ), or one-way ANOVA with Tukey's post hoc test ( A, G, I, J, O, P and R ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Flow Cytometry, Staining, Ex Vivo, In Vitro, Labeling, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: A uniquely leptin sensitive hypothalamic neuron population limits hyperphagia and weight gain in diet-induced obesity

doi: 10.64898/2026.03.26.714161

Figure Lengend Snippet: a, Schematic of the combined Xenium spatial transcriptomics and pSTAT3 immunofluorescence workflow used to quantify leptin signaling across hypothalamic cell types in chow-fed and diet-induced obese (DIO) mice. b , UMAP embedding of neurons (17 populations; n=6 mice total) across all spatially profiled sections, colored by transcriptionally defined Lepr cell clusters. c , Marker gene expression across Lepr neuron subtypes, highlighting canonical cell type markers. d , UMAP embedding showing pSTAT3-positive Lepr neurons (green). e, Numbers of pSTAT3-positive cells across Lepr neuron subtypes in chow-fed and HFD-fed mice (n=3 mice per group; 2–3 sections per mouse). DIO increased pSTAT3 positivity nearly 10-fold overall (4.45±0.65% vs. 0.47±0.2%, P <0.001; generalized linear mixed-effects model with binomial distribution, animal as random effect). This was driven predominantly by Lepr/Glp1r neurons, which showed a >20-fold increase (21.1±1.1% vs. 0.33±0.2%, P <1×10⁻¹⁰) and accounted for >60% of all pSTAT3-positive Lepr neurons in DIO. P -values adjusted by Benjamini-Hochberg. f , Representative distribution of the each population of Lepr neurons across a single coronal section, color-coded as in b, c. Right panels show representative Xenium and immunofluorescence images showing spatial colocalization of Lepr, Glp1r, Trh , and pSTAT3 in the boxed areas of the larger panel. g , DIO-induced transcriptional changes in Lepr/Glp1r neurons, including increased expression of canonical leptin target genes ( Socs3, Nlrc5, Sbno2, Atf3; red) and immediate-early genes (e.g., Junb, Vgf ), indicating robust and sustained leptin signaling.

Article Snippet: A Glp1r -specific knockout of Lepr (Lepr Glp1r KO mice) was produced by crossing the Lepr flox mice with Glp1r-ires-Cre mice (Jackson Laboratory, #029283).

Techniques: Spatial Transcriptomics, Immunofluorescence, Marker, Gene Expression, Expressing

Journal: bioRxiv

Article Title: A uniquely leptin sensitive hypothalamic neuron population limits hyperphagia and weight gain in diet-induced obesity

doi: 10.64898/2026.03.26.714161

Figure Lengend Snippet: a , UMAP embedding of all cells from Xenium in situ sequencing annotated by seurat cluster numbers. Colors illustrate clusters. b , Representative images from Xenium in spatial context. Colors match clusters in panel a. Top image from Xenium slide 1 and bottom image from Xenium slide 2. c , UMAP embeddings of all cells from Xenium in situ sequencing annotated by animal (left panel) or by diet; 15 weeks HFD (DIO) and chow (right panel). d , UMAP embeddings of all cells from Xenium in situ sequencing colored by expression of Slc17a6 (glutamatergic neurons), Gad1 (GABAergic neurons), Rax (tanycytes) and Lepr . e , UMAP embedding of all neurons from Xenium in situ sequencing colored by seurat cluster (left panel), by Campbell et al. label transfer annotations (middle panel), and by Rupp et al. label transfer annotations (right panel).

Article Snippet: A Glp1r -specific knockout of Lepr (Lepr Glp1r KO mice) was produced by crossing the Lepr flox mice with Glp1r-ires-Cre mice (Jackson Laboratory, #029283).

Techniques: In Situ, Sequencing, Expressing

Journal: bioRxiv

Article Title: A uniquely leptin sensitive hypothalamic neuron population limits hyperphagia and weight gain in diet-induced obesity

doi: 10.64898/2026.03.26.714161

Figure Lengend Snippet: a , Experimental design for snRNA-seq profiling of hypothalamic neurons across metabolic states: chow-fed (Chow; n=7), overnight-fasted (Fasting; n=3), fasted and refed (90 min) (Refeed; n=4), and DIO mice (HFD; n=8). b , UMAP embedding of MBH neurons (>40,000 nuclei; mean 12,401 transcripts per nucleus), with Lepr -expressing neurons labelled by subtype. c , Effects of nutritional manipulations on the total transcriptome of each neuronal population. Expression distance estimate in response to fasting (x-axis) and DIO (y-axis). Data for Lepr neurons is highlighted in cell-type specific colors. Solid diagonal line indicates matched effects between perturbations. d , Leptin gene signature (LGS) expression in for each nutritional manipulations across the indicated subsets of Lepr neurons; HFD (red), refeed (light green), chow (yellow), fasting (dark green)). e , Hierarchical clustering and grouping of differentially expressed genes identified in Lepr/Glp1r neurons. Neuronal activity genes and GABA receptor subunits highlighted.

Article Snippet: A Glp1r -specific knockout of Lepr (Lepr Glp1r KO mice) was produced by crossing the Lepr flox mice with Glp1r-ires-Cre mice (Jackson Laboratory, #029283).

Techniques: Expressing, Activity Assay

Journal: bioRxiv

Article Title: A uniquely leptin sensitive hypothalamic neuron population limits hyperphagia and weight gain in diet-induced obesity

doi: 10.64898/2026.03.26.714161

Figure Lengend Snippet: a,b , UMAP embedding of mediobasal hypothalamic neurons colored by individual mouse (a) and sequencing batch (b). c , Distribution of UMI counts (top) and genes detected (bottom) per cell across all samples (chow n=7, DIO n=8, fasted n=3, refed n=4). d , UMAP colored by predicted Campbell neuron subtype. e , Mapping confidence scores, with highest confidence in the Lepr/Glp1r population. f , Proportion of cells assigned to each neuronal cluster (n01-n34) across individual samples, showing consistent cluster composition across mice and conditions. g , MINT sPLS-DA projection of three independent leptin treatment transcriptomic datasets used to derive the leptin gene signature (LGS). Fasted/control (blue) and leptin-treated (orange) samples separate along the first two components. h , Gene ontology enrichment of LGS genes, highlighting response to peptide hormone and JAK-STAT signaling among the top terms. i , Model weights for individual LGS genes. j , LGS expression across nutritional states in Tbx19 and Irx3 neurons (***P<0.001). k , Correlation between fasting-induced (y-axis) and DIO-induced (x-axis) log₂ fold changes in AgRP, Lepr/Glp1r, and POMC neurons.

Article Snippet: A Glp1r -specific knockout of Lepr (Lepr Glp1r KO mice) was produced by crossing the Lepr flox mice with Glp1r-ires-Cre mice (Jackson Laboratory, #029283).

Techniques: Sequencing, Control, Expressing

Journal: bioRxiv

Article Title: A uniquely leptin sensitive hypothalamic neuron population limits hyperphagia and weight gain in diet-induced obesity

doi: 10.64898/2026.03.26.714161

Figure Lengend Snippet: a , Experimental design of wild-type (WT) mice treated with 3 mg/kg of leptin of saline and sacrificed 1, 3, 6, or 24 hours after injection. MBH nuclei were collected for snRNA-seq. b, Transcriptional distance between cells from leptin treated and control mice. c, LGS changes across Lepr/Glp1r , Pomc and Agrp neurons . d, Overlap between genes induced by acute leptin treatment and those upregulated in DIO in Lepr/Glp1r neurons (93 shared genes; odds ratio=185.3, P =6.28×10⁻²⁴, Fisher’s exact test), indicating that direct leptin action recapitulates a substantial portion of the DIO transcriptional program.

Article Snippet: A Glp1r -specific knockout of Lepr (Lepr Glp1r KO mice) was produced by crossing the Lepr flox mice with Glp1r-ires-Cre mice (Jackson Laboratory, #029283).

Techniques: Saline, Injection, Control

Journal: bioRxiv

Article Title: A uniquely leptin sensitive hypothalamic neuron population limits hyperphagia and weight gain in diet-induced obesity

doi: 10.64898/2026.03.26.714161

Figure Lengend Snippet: a-c , UMAP embedding of mediobasal hypothalamic neurons from lean mice treated with leptin (3 mg/kg) or saline and harvested at 1, 3, 6, or 24 hours post-injection (n=4-6 per timepoint per group), colored by treatment (a), hours post-injection (b), and sequencing batch (c). d , UMAP colored by neuronal cell type identity, with key Lepr-expressing populations labeled. e , Number of differentially expressed genes (leptin vs saline; adjusted P<0.05) per neuronal population. Red, upregulated; blue, downregulated. f , Volcano plot of leptin-induced differential expression in Lepr/Glp1r neurons. Highlighted genes include canonical leptin targets (blue), immediate early/neuronal activity genes (red), and GABA receptor subunits (green). Dashed line, adjusted P =0.05.

Article Snippet: A Glp1r -specific knockout of Lepr (Lepr Glp1r KO mice) was produced by crossing the Lepr flox mice with Glp1r-ires-Cre mice (Jackson Laboratory, #029283).

Techniques: Saline, Injection, Sequencing, Expressing, Labeling, Quantitative Proteomics, Activity Assay

Journal: bioRxiv

Article Title: A uniquely leptin sensitive hypothalamic neuron population limits hyperphagia and weight gain in diet-induced obesity

doi: 10.64898/2026.03.26.714161

Figure Lengend Snippet: a , Lepr/Glp1r neurons constitute ∼78% of all Lepr-expressing GABAergic (Gad1+) input to AgRP (Npy+) neurons, based on reanalysis of published rabies-traced AgRP neuron afferents. Feature plots show expression of Gad1, Glp1r, Npy, and Lepr across traced populations; circled clusters indicate the Lepr/Glp1r population. b,c , UMAP embedding of mediobasal hypothalamic neurons from Glp1r Lepr KO and control mice (35,538 nuclei total), colored by genotype (b) and diet (c) (chow vs DIO; n=6-7 per group). d , UMAP colored by predicted cell type identity (41 clusters) based on label transfer from the nutritional perturbation reference atlas. e , Change in leptin gene signature (LGS) expression in Glp1r Lepr KO relative to control mice across Lepr neuron subtypes (open circles denote P <0.05; linear mixed-effects model).

Article Snippet: A Glp1r -specific knockout of Lepr (Lepr Glp1r KO mice) was produced by crossing the Lepr flox mice with Glp1r-ires-Cre mice (Jackson Laboratory, #029283).

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: A uniquely leptin sensitive hypothalamic neuron population limits hyperphagia and weight gain in diet-induced obesity

doi: 10.64898/2026.03.26.714161

Figure Lengend Snippet: a , Schematic of retrograde rabies tracing from ARC Npy neurons in Lepr Cre-sun1Gfp ;Npy Flp mice. Starter cells (ARC) express TVA and rabies-G via Npy Flp ; rabies-labeled cells are magenta, Lepr -expressing cells green, and co-labeled cells ( Lepr -expressing afferents) show both signals. b, c, Representative images of tissue sections from experiments as in (a). *Indicates viral hit site. Afferent DMH Lepr cells indicated by white arrows. d , Schematic of retrograde rabies tracing from Agrp neurons in Agrp Cre; Glp1r Flp-TDT mice. Rabies-labeled cells are green, Glp1r -expressing cells red, and co-labeled cells ( Glp1r -expressing afferents) show both signals. e, f , Representative images of tissue sections from experiments as in (d); *indicates viral hit site. Rabies-labeled Glp1r afferent cells indicated with white arrows. Scale bars: 200 µm (main images), 100 µm (insets). g , h , Experimental paradigm: Lepr was ablated in Glp1r expressing neurons and animals were fed chow diet ( Agrp , n=7; Glp1r , n=6 Lepr KO and n=6 control) and either sacrificed at 4-5 weeks of age switched onto a HFD for 15 weeks ( Agrp n=2; Glp1r , n=7 Lepr KO and n=6 control) until sacrifice. Mediobasal hypothalami were collected for snRNA-seq. i, Leptin gene signature (LGS) expression in Lepr/Glp1r neurons from for lean (Chow) or DIO Lepr Glp1r KO and control (WT) mice. LGS was significantly reduced in KO neurons (β=−0.087, P <1.0×10⁻⁹; linear mixed-effects model), with a significant genotype × diet interaction ( P =2×10⁻⁹). j, PCA projection of Lepr/Glp1r neurons from DIO Lepr Glp1r KO (red) and Control (blue) mice onto the nutritional perturbation embedding. Centroids for DIO (orange) and fasted (teal) conditions shown as large circles. Lepr Glp1r KO neurons cluster with fasted wild-type neurons (PC1 permutation test, P=0.001), indicating LepR signaling is required to adopt the DIO transcriptional state. k, Volcano plot of differentially expressed genes in Lepr/Glp1r neurons (KO versus WT, DIO). Loss of Lepr abolished the DIO-associated induction of immediate early genes ( Fos , log₂FC=−2.14, adj. P =0.018; Vgf , log₂FC=−1.76, adj. P =1.3×10⁻⁴; Homer1 , log₂FC=−0.66, adj. P =0.027) and reversed the downregulation of GABA receptor subunits ( Gabra3, Gabra4, Gabra5, Gabrb1–b3 ). Dashed line, adjusted P =0.05. See Supplementary Table 7 for full results. l, PCA projection of Agrp neurons from DIO Lepr Glp1r KO (red) and Control (blue) mice; centroids for DIO (orange) and fasted (teal) conditions shown as large circles, as in ( j ). Agrp neurons shift toward the fasted transcriptional state in Lepr Glp1r KO mice (PC1 permutation test, P=0.001), indicating propagation of the transcriptional effect from Lepr/Glp1r neurons. m, Volcano plot of differentially expressed genes in Agrp neurons (KO versus WT, DIO; 128 genes). Genes colored by their response to fasting: orange, fasting-upregulated; blue, fasting-downregulated; grey, neither. Fasting-upregulated genes were enriched among genes increased in KO (OR=8.56, P =1.05×10⁻⁸, Fisher’s exact test), and fasting-downregulated genes were enriched among decreased genes (OR=22.67, P =6.76×10⁻¹⁹), confirming a fasting-like transcriptional state despite obesity.

Article Snippet: A Glp1r -specific knockout of Lepr (Lepr Glp1r KO mice) was produced by crossing the Lepr flox mice with Glp1r-ires-Cre mice (Jackson Laboratory, #029283).

Techniques: Labeling, Expressing, Control

Journal: bioRxiv

Article Title: A uniquely leptin sensitive hypothalamic neuron population limits hyperphagia and weight gain in diet-induced obesity

doi: 10.64898/2026.03.26.714161

Figure Lengend Snippet: a , Schematic and representative histology of bilateral AAV-FLEX-FREX-hM3Dq injection into the caudal ARC/ventral DMH of Glp1r-ires-Cre;Trh-p2a-Dre mice. Scale bar: 200 µm. b , Spatial transcriptomics reference map showing the Lepr/Glp1r neuron population (red) targeted by the intersectional DREADD strategy. c , Summary of the DREADD cohort (main text ). d , Baseline characteristics of chow-fed Glp1r Lepr KO (n=20) and control (n=14) mice used for leptin-dependent feeding suppression experiments (main text ). e , Baseline chow-period body weight and DEXA body composition of Glp1r Lepr KO (n=9) and control (n=10) mice prior to HFD exposure . Data are mean ± SD.

Article Snippet: A Glp1r -specific knockout of Lepr (Lepr Glp1r KO mice) was produced by crossing the Lepr flox mice with Glp1r-ires-Cre mice (Jackson Laboratory, #029283).

Techniques: Injection, Spatial Transcriptomics, Control

Journal: bioRxiv

Article Title: A uniquely leptin sensitive hypothalamic neuron population limits hyperphagia and weight gain in diet-induced obesity

doi: 10.64898/2026.03.26.714161

Figure Lengend Snippet: Mice containing activating DREADDs in Glp1r/Trh neurons were treated with saline or CNO at the onset of the dark cycle (a), during refeeding following an overnight fast (b), or prior to ghrelin treatment (c). a , Cumulative dark-cycle food intake at 0, 1, 2, and 3 hours following IP CNO or saline in a within-subject crossover design (n=5).. b , Cumulative post-fast food intake at 0, 1, 2, 4, 6, and 8 hours following IP CNO or saline (n=5). c , Cumulative food intake following IP ghrelin with CNO or saline pre-treatment at 0, 1, 2, 4, 6, and 8 hours (n=4). d, Effect of leptin (dark teal) versus saline (light teal) pre-treatment on ghrelin-induced 24-hour food intake in lean Lepr Glp1r KO (KO) and control (WT) mice. Lines connect within-subject measurements (crossover design). e, Cumulative post-fast food intake following leptin (dark teal) or saline (light teal) administration in WT (left) and KO (right) mice. f, g , Body weight (f) and daily food intake (g) before and 7 days after HFD exposure in Lepr Glp1r KO (n=9) and Control (n=10) mice. h , Food intake from (g) separated by dark (top) and light (bottom) cycle. The excess intake in Lepr Glp1r KO mice was concentrated in the dark cycle (genotype × time: χ²(9)=38.33, P =1.52×10⁻⁵). Dashed line indicates HFD switch. All panels: * P <0.05, **P<0.01, ***P<0.001. A-c, e-h: Plotted points represent mean values. Error bars (a-c) and shaded regions (e-h) denote SEM.

Article Snippet: A Glp1r -specific knockout of Lepr (Lepr Glp1r KO mice) was produced by crossing the Lepr flox mice with Glp1r-ires-Cre mice (Jackson Laboratory, #029283).

Techniques: Saline, Control

Journal: bioRxiv

Article Title: A uniquely leptin sensitive hypothalamic neuron population limits hyperphagia and weight gain in diet-induced obesity

doi: 10.64898/2026.03.26.714161

Figure Lengend Snippet: a,b, Baseline characteristics of Glp1r Lepr KO and control mice used in the chow vs DIO fasting-refeeding experiment (main text ). Chow-fed and DIO cohorts are independent groups of animals. c-e, Baseline characteristics of DIO Glp1r Lepr KO (n=12) and control (n=11) mice used in the ghrelin feeding experiment. c , Summary table including plasma leptin for a subset of mice (KO n=4, WT n=3). d , Individual body weights and ( e ) plasma leptin by genotype, confirming hyperleptinemia in both genotypes. Data are mean ± SD (tables) or mean ± SEM (dot plots) with individual animals shown.

Article Snippet: A Glp1r -specific knockout of Lepr (Lepr Glp1r KO mice) was produced by crossing the Lepr flox mice with Glp1r-ires-Cre mice (Jackson Laboratory, #029283).

Techniques: Control, Clinical Proteomics

Journal: bioRxiv

Article Title: A uniquely leptin sensitive hypothalamic neuron population limits hyperphagia and weight gain in diet-induced obesity

doi: 10.64898/2026.03.26.714161

Figure Lengend Snippet: a , Cumulative post-fast food intake (kcal) at 0, 1, 2, 4, and 8 hours in Control (WT, left) and Lepr Glp1r KO (right) mice on chow (light) or HFD (dark). * P <0.05, *** P <0.001. Shaded regions denote SEM. b , Five-hour food intake following IP ghrelin (dark) or saline (light) in DIO WT (n=11) and Lepr Glp1r KO (n=12) mice. Lines connect within-subject measurements (crossover design). ** P <0.01, error bars denote SEM. c , Representative FOS immunofluorescence in the mbARC of DIO Control and Lepr Glp1r KO mice following saline (top) or ghrelin (bottom) injection. 3V, third ventricle; ME, median eminence. d , Quantification of FOS-positive ARC neurons after saline (top; P=0.15) or ghrelin (bottom; P =0.013). Shown are mean-/+ SEM, along with individual data points. e , Left: schematic indicating the mbARC region sampled. Right: representative IBA1 immunofluorescence in DIO WT and Lepr Glp1r KO (KO) mice. f , Quantification of IBA1-positive microglia in the ARC of control (ctrl) and KO mice. Shown are mean-/+ SEM, along with individual data points.

Article Snippet: A Glp1r -specific knockout of Lepr (Lepr Glp1r KO mice) was produced by crossing the Lepr flox mice with Glp1r-ires-Cre mice (Jackson Laboratory, #029283).

Techniques: Control, Saline, Immunofluorescence, Injection

Journal: International Journal of Molecular Sciences

Article Title: MyD88 Inhibition Ameliorates Diabetes-Induced Hepatic Inflammation and Gluconeogenesis Through Adipose IL-10 Induction

doi: 10.3390/ijms27062883

Figure Lengend Snippet: MyD88 deficiency in Lepr db / db mice enhances IL−10 in adipose tissue and liver and decreased circulating adiponectin and DPP4 activity. Adipose tissue, liver, and blood were collected from Lepr +/+ , Lepr db / db , and Lepr db / db MyD88 −/− mice to examine the effects of MyD88 signaling deletion. ( A ) Foxp3 protein expression in stromal vascular fractions (SVFs) isolated from adipose tissue was assessed by Western blotting. ( B ) Densitometric quantification of Foxp3 expression illustrated in ( A ). ( C , D ) IL−10 protein levels in adipose tissue ( C ) and liver ( D ) were measured by ELISA. ( E ) Plasma dipeptidyl peptidase-4 (DPP4) enzymatic activity was determined using a commercial DPP4 activity assay kit (BioVision, Milpitas, CA, USA, # K779-100). ( F ) Circulating adiponectin levels were quantified by ELISA. Data are presented as mean ± SEM. n = 5 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001. Foxp3, forkhead box P3.

Article Snippet: Lepr db/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and bred to generate diabetic Lepr db/db and non-diabetic Lepr +/+ mice littermates.

Techniques: Activity Assay, Expressing, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: MyD88 Inhibition Ameliorates Diabetes-Induced Hepatic Inflammation and Gluconeogenesis Through Adipose IL-10 Induction

doi: 10.3390/ijms27062883

Figure Lengend Snippet: MyD88 depletion suppresses hepatic inflammatory and gluconeogenic gene expression in Lepr db / db mice. Liver tissues from Lepr +/+ , Lepr db / db , and Lepr db / db MyD88 −/− mice were harvested for quantitative PCR (Q-PCR) analysis. Hepatic mRNA expression levels of inflammatory markers ( ICAM , IL − 1β , TNF − α , IL − 6 , iNOS ), metabolic regulators ( DPP4 , FGF21 ), and gluconeogenic enzymes ( G6pc and Pck1 ) were determined. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Lepr db/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and bred to generate diabetic Lepr db/db and non-diabetic Lepr +/+ mice littermates.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: International Journal of Molecular Sciences

Article Title: MyD88 Inhibition Ameliorates Diabetes-Induced Hepatic Inflammation and Gluconeogenesis Through Adipose IL-10 Induction

doi: 10.3390/ijms27062883

Figure Lengend Snippet: MyD88 deficiency reduces inflammatory gene expression in Kupffer cells and attenuates liver injury in Lepr db / db mice. Kupffer cells and blood samples were isolated from Lepr +/+ , Lepr db / db , and Lepr db / db MyD88 −/− mice. ( A ) mRNA expression of inflammatory mediators ( IL − 1β , TNF − α , IL − 6 , iNOS , and DPP4 ) in Kupffer cells was quantified by Q-PCR. ( B ) Serum alanine aminotransferase (ALT) levels were measured as an indicator of hepatic injury. Data are presented as mean ± SEM. n = 5 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001. ALT alanine aminotransferase, AST aspartate transaminase.

Article Snippet: Lepr db/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and bred to generate diabetic Lepr db/db and non-diabetic Lepr +/+ mice littermates.

Techniques: Gene Expression, Isolation, Expressing

Journal: International Journal of Molecular Sciences

Article Title: MyD88 Inhibition Ameliorates Diabetes-Induced Hepatic Inflammation and Gluconeogenesis Through Adipose IL-10 Induction

doi: 10.3390/ijms27062883

Figure Lengend Snippet: Altered cytokine expression in adipose SVFs from Lepr db/db MyD88 −/− mice and modulation by IL−10 treatment SVFs were harvested from the adipose tissue of Lepr db/db and Lepr db/db MyD88 −/− mice and purified for mRNA expression of different cytokines by Q-PCR analysis to examine IL-6 , IL − 33 , Foxp3 , CCL2 , IL − 1β , TNF − α , and PDGFα cytokine mRNA expression in adipose SVFs and MyD88 involvement. Furthermore, SVFs (2 × 10 7 cells) purified from the adipose tissue of Lepr db/db and Lepr db/db MyD88 −/− mice were treated with PBS or 10 and 100 ng of IL−10 for 3.5 h followed by Q-PCR analysis of IL − 6 , IL − 33 , Foxp3 , CCL2 , IL − 1β , TNF − α , and PDGFα mRNA expression. n = 5/group. ** p < 0.01, *** p < 0.001. SVF, stromal vascular fraction; forkhead box p3, Foxp3; PDGF, platelet-derived growth factor.

Article Snippet: Lepr db/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and bred to generate diabetic Lepr db/db and non-diabetic Lepr +/+ mice littermates.

Techniques: Expressing, Purification, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: MyD88 Inhibition Ameliorates Diabetes-Induced Hepatic Inflammation and Gluconeogenesis Through Adipose IL-10 Induction

doi: 10.3390/ijms27062883

Figure Lengend Snippet: Local IL−10 administration induces Foxp3 expression and suppresses inflammatory signaling pathways in adipose SVFs of Lepr db/db mice. PBS or IL−10 (10, 50, or 100 ng) was injected directly into adipose tissue of Lepr db/db mice. SVFs were isolated 7 days after injection. ( A ) mRNA expression of inflammatory cytokines ( IL − 6 , IL-33 , CCL2 , IL − 1β , TNF − α ) and Foxp3 was quantified by Q-PCR. ( B ) Protein expression of phosphorylated and total JNK and NF-κB was evaluated by Western blotting to assess inflammatory signaling activity. Data are presented as mean ± SEM. n = 5 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Lepr db/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and bred to generate diabetic Lepr db/db and non-diabetic Lepr +/+ mice littermates.

Techniques: Expressing, Protein-Protein interactions, Injection, Isolation, Western Blot, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: MyD88 Inhibition Ameliorates Diabetes-Induced Hepatic Inflammation and Gluconeogenesis Through Adipose IL-10 Induction

doi: 10.3390/ijms27062883

Figure Lengend Snippet: IL−10 injection increases regulatory T cell accumulation in adipose tissue of Lepr db/db mice. PBS or IL−10 (10, 50, or 100 ng) was injected into adipose tissue of Lepr db/db mice. SVFs were isolated 7 days after injection and analyzed by flow cytometry. ( A ) Representative flow cytometry plots showing CD4 + regulatory T cells (Tregs). ( B ) Quantification of the frequency and absolute number of CD4 + Tregs in adipose tissue. Data are presented as mean ± SEM. N = 5 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Lepr db/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and bred to generate diabetic Lepr db/db and non-diabetic Lepr +/+ mice littermates.

Techniques: Injection, Isolation, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: MyD88 Inhibition Ameliorates Diabetes-Induced Hepatic Inflammation and Gluconeogenesis Through Adipose IL-10 Induction

doi: 10.3390/ijms27062883

Figure Lengend Snippet: IL−10 administration decreased blood adiponectin levels and DPP4 activity in Lepr db/db mice. PBS or IL−10 (10, 50, or 100 ng) was injected into adipose tissue of Lepr db/db mice, and tissues were harvested 7 days later. ( A ) IL-10 protein levels in adipose tissue were measured by ELISA. ( B ) Plasma adiponectin levels were determined by ELISA. ( C ) Plasma DPP4 enzymatic activity was assessed using a DPP4 activity assay kit (BioVision, Milpitas, CA, USA, # K779-100). Data are presented as mean ± SEM. n = 5 mice per group. * p < 0.05, ** p < 0.01.

Article Snippet: Lepr db/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and bred to generate diabetic Lepr db/db and non-diabetic Lepr +/+ mice littermates.

Techniques: Activity Assay, Injection, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: MyD88 Inhibition Ameliorates Diabetes-Induced Hepatic Inflammation and Gluconeogenesis Through Adipose IL-10 Induction

doi: 10.3390/ijms27062883

Figure Lengend Snippet: IL−10 administration suppresses hepatic ICAM , TNF − α , IL − 6 , DPP4 , and iNOS mRNA expression and activates insulin-related signaling pathways in Lepr db/db mice. PBS or IL-10 (10, 50, or 100 ng) was injected into adipose tissue of Lepr db/db mice, and livers were harvested 7 days later. ( A ) Hepatic mRNA expression of ICAM , FGF21 , IL − 1β , TNF − α , DPP4 , and iNOS was determined by Q-PCR. ( B ) Protein expression of phosphorylated and total Akt, STAT3, and ERK was assessed by Western blotting. ( C ) Quantification of the pAkt/Akt and pSTAT3/STAT3 ratio. Data are presented as mean ± SEM. n = 4 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Lepr db/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and bred to generate diabetic Lepr db/db and non-diabetic Lepr +/+ mice littermates.

Techniques: Expressing, Protein-Protein interactions, Injection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: MyD88 Inhibition Ameliorates Diabetes-Induced Hepatic Inflammation and Gluconeogenesis Through Adipose IL-10 Induction

doi: 10.3390/ijms27062883

Figure Lengend Snippet: IL−10 injection reduces inflammatory gene expression in Kupffer cells and improves liver injury markers in Lepr db/db mice. PBS or IL−10 (10, 50, or 100 ng) was injected into adipose tissue of Lepr db/db mice. Kupffer cells and blood were collected 7 days after injection. ( A ) Kupffer cell mRNA expression of IL- − 1β , TNF − α , IL − 6 , iNOS , and DPP4 was quantified by Q-PCR analysis. ( B ) Serum ALT levels were measured to assess hepatic injury. Data are presented as mean ± SEM. n = 6 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001. ALT alanine aminotransferase, AST aspartate transaminase.

Article Snippet: Lepr db/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and bred to generate diabetic Lepr db/db and non-diabetic Lepr +/+ mice littermates.

Techniques: Injection, Gene Expression, Expressing

Journal: International Journal of Molecular Sciences

Article Title: MyD88 Inhibition Ameliorates Diabetes-Induced Hepatic Inflammation and Gluconeogenesis Through Adipose IL-10 Induction

doi: 10.3390/ijms27062883

Figure Lengend Snippet: IL−10 administration reduces hepatic gluconeogenic gene expression, enhances Akt activation, and improves glucose tolerance in Lepr db/db mice. PBS or IL-10 (10 or 50 ng) was injected into inguinal white adipose tissue (WAT) of Lepr db/db mice, the liver was harvested 7 days after injection and subjected to Q-PCR analysis to determine the expression of G6pc and Pck1 mRNA ( A ). ( B ) One week after injection, mice were treated with insulin (1.25 mU/g body weight) for 20 min, followed by isolation of SVFs and Western blot analysis of phosphorylated and total Akt. ( C ) Quantification of the pAkt/Akt ratio. ( D ) Glucose tolerance tests were performed by intraperitoneal glucose administration (1 g/kg body weight), with blood glucose measured at baseline and at 15 min intervals for 2 h. Data are presented as mean ± SEM. n = 4 mice per group. * p < 0.05, ** p < 0.01; # p < 0.05 compared with PBS-treated Lepr db/db mice.

Article Snippet: Lepr db/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and bred to generate diabetic Lepr db/db and non-diabetic Lepr +/+ mice littermates.

Techniques: Gene Expression, Activation Assay, Injection, Expressing, Isolation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: MyD88 Inhibition Ameliorates Diabetes-Induced Hepatic Inflammation and Gluconeogenesis Through Adipose IL-10 Induction

doi: 10.3390/ijms27062883

Figure Lengend Snippet: ( A ) For in vitro treatment, SVFs (2 × 10 7 cells) were harvested from the adipose tissue of Lepr db/db and Lepr db/db MyD88 −/− mice and treated with recombinant mouse IL-10 (10 or 100 ng) at 37 °C for 3 h. ( B ) For in vivo treatment, IL-10 (10, 50, or 100 ng) or PBS was injected into inguinal adipose tissue. After 7 days, mice were sacrificed, and liver, adipose tissue, and blood samples were collected for further analysis.

Article Snippet: Lepr db/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and bred to generate diabetic Lepr db/db and non-diabetic Lepr +/+ mice littermates.

Techniques: In Vitro, Recombinant, In Vivo, Injection