Journal: The FASEB Journal

Article Title: Lymphocyte Antigen 6G Mediates Vagotomy‐Associated Reduction in Body Weight

doi: 10.1096/fj.202600151RR

Figure Lengend Snippet: Vagotomy promoted Ly6G + cell infiltration into eWAT. Wild‐type mice were subjected to left cervical vagotomy (VX) or sham surgery and tissues were collected at 7 days following surgery. (A) The percentage of non‐adipocyte nuclei of total nuclei per section was quantified using ImageJ ( n = 4) and right panels show representative images of paraffin sections of eWAT stained with H&E in sham and VX animals. (B) CCL2 release from eWAT was analyzed by ELISA. The bar shows the CCL2 levels from sham ( n = 4) or VX ( n = 4) mice normalized to eWAT weight: ng/mL per g ± SEM (unpaired Student's t test). (C) eWAT was collected at 1 ( n = 3), 4 ( n = 4 sham, n = 5 VX), and 7 ( n = 15) days following VX or sham surgery and the eWAT SVCs were analyzed by flow cytometry. The bar shows the % ± SEM of CD11b + Ly6G + cells from CD45 + (one‐way ANOVA, Uncorrected Fisher's LSD). (D) Graphs show representative gating for CD11b + Ly6G + cells in sham and VX eWAT at 7 days (concatenated n = 5–6). (E) Representative immunostaining of Ly6G (red) and Perilipin1 (green) in paraffin sections of eWAT. (F–H) Bone marrow neutrophils after sham ( n = 9) or VX ( n = 5) surgery were isolated using negative magnetic beads and analyzed using bulk RNAseq (DESeq2). Heatmap (F), volcano plot (G) of differentially expressed genes, and GO (Gene Ontology) (H) enrichment bar plot. ns = not significant, * p < 0.05. VX, Vagotomy; eWAT, epididymal white adipose tissue; H&E, hematoxylin–eosin; SVCs, stromal vascular cells.

Article Snippet: In vitro release rate was calculated as mg NEFAs per mg eWAT tissue per hour. (2) CCL2 levels were quantified using a mouse CCL2 ELISA kit (R&D Systems, #DY497‐05), according to the manufacturer's instructions ( n = 1 experiment).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunostaining, Isolation, Magnetic Beads, RNA sequencing

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: Mouse recombinant leptin (R&D Systems, #498-OB) was reconstituted to 1 mg/ml in 20 mM Tris-HCl pH 8.0 (Invitrogen, #10434742).

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 , 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: Mouse recombinant leptin (R&D Systems, #498-OB) was reconstituted to 1 mg/ml in 20 mM Tris-HCl pH 8.0 (Invitrogen, #10434742).

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: Mouse recombinant leptin (R&D Systems, #498-OB) was reconstituted to 1 mg/ml in 20 mM Tris-HCl pH 8.0 (Invitrogen, #10434742).

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: Mouse recombinant leptin (R&D Systems, #498-OB) was reconstituted to 1 mg/ml in 20 mM Tris-HCl pH 8.0 (Invitrogen, #10434742).

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: Mouse recombinant leptin (R&D Systems, #498-OB) was reconstituted to 1 mg/ml in 20 mM Tris-HCl pH 8.0 (Invitrogen, #10434742).

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: Mouse recombinant leptin (R&D Systems, #498-OB) was reconstituted to 1 mg/ml in 20 mM Tris-HCl pH 8.0 (Invitrogen, #10434742).

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: Mouse recombinant leptin (R&D Systems, #498-OB) was reconstituted to 1 mg/ml in 20 mM Tris-HCl pH 8.0 (Invitrogen, #10434742).

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: Mouse recombinant leptin (R&D Systems, #498-OB) was reconstituted to 1 mg/ml in 20 mM Tris-HCl pH 8.0 (Invitrogen, #10434742).

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: Mouse recombinant leptin (R&D Systems, #498-OB) was reconstituted to 1 mg/ml in 20 mM Tris-HCl pH 8.0 (Invitrogen, #10434742).

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: Mouse recombinant leptin (R&D Systems, #498-OB) was reconstituted to 1 mg/ml in 20 mM Tris-HCl pH 8.0 (Invitrogen, #10434742).

Techniques: Control, Clinical Proteomics