Reduced secretion of neuronal growth regulator 1 contributes to impaired adipose-neuronal crosstalk in obesity

Figures

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  Fig. 1. Phenotypical characterisation and secretory profiling of different AT depots. a Experimental workflow showing anatomical localisation of AT depots and subsequent procedures performed on AT explants. b Upper panel: H&E stainings of tissue sections. Lower panel: immunofluorescence images of AT sections stained for perilipin and UCP1. Nuclei are counterstained with DAPI. Scale bars: 50 µm. Insets show higher magnifications of randomly selected areas. Representative pictures of 1 out of 2 replicates. c Western blot of representative AT lysates (2 out of 4 replicates) stained for perilipin and UCP1. A brain lysate was loaded as negative control for AT-specific proteins. d Western blot of representative AT lysates (1 out of 2 replicates) and corresponding supernatants stained for β-actin, adiponectin, perilipin and UCP1. e, f Unsupervised hierarchical clustering (heatmap, e) and principal component analysis (f) of the 363 consistently identified secreted proteins detected by MS in the conditioned media of different AT depots. Hierarchical clustering was performed using the complete agglomeration method, Z-score-scaled abundances are displayed. N = 6 biological replicates per group. AT…

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  Fig. 2. Identification of a perivascular secretory signature and characterisation of AT innervation. a Volcano plot showing proteins differentially secreted by perivascular (AR, TH and AB PVAT, N = 18) and non-perivascular (VI and SC WAT and BAT: non-PVAT, N = 18) AT depots (N = 6 biological replicates per depot). The limma package was used to compare different groups using the Ebayes algorithm (paired analysis) followed by Benjamini–Hochberg adjustment. Light-blue spots: proteins involved in lipid transport. Dark blue spots: serpin family members. Red spots: proteins involved in axon guidance and nervous system development. b Gene Ontology analysis showing Biological Processes categories enriched in non-PVAT and PVAT secretomes. Fisher’s Exact test p-values are shown. c Representative immunofluorescence images (from 1 out of 2 replicates) of TH PVAT sections stained for cell-adhesion molecules Ncam1, Chl1 and L1cam. Adipocytes are stained by perilipin. Nuclei are counterstained with DAPI. Insets show higher magnifications of white framed areas. Scale bars: 50 µm. d Representative immunofluorescence images (from 1 out of 2 replicates) showing the co-localisation of cell-adhesion…

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  Fig. 3. Obesity-induced alterations of PVAT phenotype and secretory profile. a Representative pictures of heart, aorta and PVAT dissected from wt and ob/ob mice. b H&E stainings (from 1 out of 3 replicates) on wt and ob/ob AT depots sections. Scale bar: 100 µm. c Principal Component Analysis of the 293 secreted proteins consistently detected by MS in the conditioned media of wt and ob/ob PVAT explants. d Volcano plot showing proteins differentially secreted by wt and ob/ob PVAT samples (N = 18 vs. 18, with N = 6 biological replicates per depot). The limma package was used to compare different groups using the Ebayes algorithm (unpaired analysis) followed by Benjamini–Hochberg adjustment. Green spots: well-established adipokines. Blue spots: proteins involved in lipid transport. Red spots: proteins involved in axonal guidance. e Heatmap showing the 107 secreted proteins significantly up- or downregulated in the secretomes of ob/ob PVAT explants with Gene Ontology analysis showing the Biological Processes categories enriched in the two groups. Fisher’s Exact test p-values are shown. Hierarchical clustering was performed using the complete agglomeration method, Z-score-scaled abundances are…

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  Fig. 4. Sympathetic innervation of murine and human AT depots in obesity. a, b Western blots showing TYR.H. abundances in wt and ob/ob TH (a) and AR (b) PVAT lysates and relative quantifications. N = 5 biological replicates per group, unpaired, two-tailed independent t-test (unequal distribution). Bar charts show mean ± SEM. c, d Representative immunofluorescence images showing TYR.H. distribution (in green and fire) in wt and ob/ob TH (c) and AR (d) PVAT sections. Adipocytes are labelled by perilipin (in red), nuclei are counterstained with DAPI. Insets show higher magnifications of framed areas. Scale bar: 50 µm. e Bar charts showing the average size of TYR.H. positive particles (mean ± SEM) obtained from 4 mice per condition, with 5 to 10 sections analysed in each mouse. N = 4, unpaired two-tailed independent t-test (unequal distribution). f Schematic cartoon showing the localisation of the human epicardial fat collected for this study. Created with BioRender.com. g Western blot showing TYR.H. and β3-TUB. staining on human epicardial fat lysates from 12 patients without (BMI ≤ 30, in black) and 8 patients with (BMI ≥ 30, in red) obesity. h Scatter dot plot showing the quantification…

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  Fig. 5. Local effect of AT-conditioned media on cultured sympathetic neurons. a Schematic cartoon showing the structure of microfluidic devices employed for sympathetic neuronal cultures. b Representative images of SCG sympathetic neurons seeded on microfluidic devices, with the axonal chamber incubated for 24 h with either wt or ob/ob fat explants conditioned media (C.M.) showing brightfield appearance (BF, left panel) and β3-tubulin staining (IF, right panel) of sympathetic neurites. Arrows point at axonal swellings. Scale bars: 20 µm. Bar chart shows the quantification of axonal swellings normalised on total axonal length. Data are shown as mean ± SEM of 39 and 35 random fields acquired from 3 independent experiments and analysed by unpaired two-tailed independent t-test (unequal distribution), p = 2.98E-06. c, d Western blots showing TYR.H. abundances in wt and ob/ob hearts (c) and PVAT-denuded aortas (d) lysates and relative quantifications. N = 5 biological replicates, unpaired two-tailed independent t-test (unequal distribution). Data are shown as mean ± SEM. SCG superior cervical ganglia, TH thoracic PVAT, AR aortic arch PVAT, TYR.H. tyrosine hydroxylase, M molecular weight…

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  Fig. 6. Negr1 levels and secretion are reduced in the PVAT of ob/ob mice. a MS-determined relative abundances of Negr1 in the secretomes of wt and ob/ob AR and TH PVAT. N = 6 biological replicates per group, unpaired, two-tailed t-test, mean ± SEM is shown. b Western blot on wt TH PVAT lysate (LYS.) and supernatants (SN.) pools (N = 6 biological replicates per pool) stained for Negr1. c Representative immunofluorescence images (1 out of 2 replicates) showing Negr1 localisation in wt TH PVAT. Adipocytes are labelled by perilipin. Nuclei are counterstained with DAPI. Insets show higher magnification of white framed areas. Scale bar: 50 µm. d Western blot showing Negr1 and perilipin levels in stromal vascular fraction (SVF) and adipocytes obtained after digestion and fractionation of VI and SC WAT and BAT explants from wt mice (1 out of 2 replicates). e Western blot on primary murine sympathetic neurons lysates from 3 biological replicates stained for TYR.H. and Negr1. f Western blot on supernatants from TH (top) and AR (bottom) PVAT explants from wt mice incubated for 24 h in absence (DMSO) or presence of the wide spectrum MMP inhibitor GM6001 and stained for Negr1 and PAI1. N = 4…

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  Fig. 7. Negr1 exerts a trophic effect on sympathetic neurons. a Immunofluoescence images showing Negr1 and TYR.H. distribution in wt and ob/ob TH PVAT sections (from 1 out of 2 replicates). Insets show higher magnification of framed areas. Nuclei are counterstained with DAPI. Scale bar: 50 µm. b Representative micrographs of SCG sympathetic neurons cultured at low density for 24 h in presence of 1 or 10 ng/ml NGF, with or without 500 ng/ml recombinant Negr1, and stained for β3-tubulin. Scale bar: 100 µm. c Quantifications of total axonal length, branching points and principal neurites on primary sympathetic neurons incubated for 24 h in medium supplemented with the indicated factors. Violin plots show median and quartiles of 80 < N < 120 neurons per condition from 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA followed by Tuckey’s multiple comparisons test. d Bar chart shows the quantification of axonal swellings normalised on total axonal length. Data are shown as mean ± SEM of 40 to 80 random fields from 3 independent experiments analysed by one-way ANOVA followed by Tuckey’s multiple comparison test. ****p < 0.0001. e Representative…