Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine

doi:10.1038/s41586-022-05041-0

. 2022 Sep;609(7926):361-368.

doi: 10.1038/s41586-022-05041-0. Epub 2022 Jul 5.

Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine

Birte Niemann^#¹, Saskia Haufs-Brusberg^#², Laura Puetz², Martin Feickert², Michelle Y Jaeckstein³, Anne Hoffmann⁴, Jelena Zurkovic², Markus Heine³, Eva-Maria Trautmann⁵, Christa E Müller^{6

7}, Anke Tönjes⁸, Christian Schlein⁹, Azin Jafari¹⁰, Holger K Eltzschig¹¹, Thorsten Gnad², Matthias Blüher^{4

8}, Natalie Krahmer^{5

12}, Peter Kovacs⁸, Joerg Heeren³, Alexander Pfeifer^{13

14}

Affiliations

¹ Institute of Pharmacology and Toxicology, University Hospital, University of Bonn, Bonn, Germany. Bini@uni-bonn.de.
² Institute of Pharmacology and Toxicology, University Hospital, University of Bonn, Bonn, Germany.
³ Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
⁴ Helmholtz Institute for Metabolic Obesity and Vascular Research (HI-MAG) of the Helmholtz Zentrum München at the University of Leipzig and University Hospital Leipzig, Leipzig, Germany.
⁵ Institute for Diabetes and Obesity, Helmholtz Center Munich, Neuherberg, Germany.
⁶ Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, Bonn, Germany.
⁷ PharmaCenter Bonn, University of Bonn, Bonn, Germany.
⁸ Medical Department III - Endocrinology, Nephrology, Rheumatology, University of Leipzig Medical Center, Leipzig, Germany.
⁹ Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
¹⁰ Clinic and Polyclinic for General, Visceral, Thoracic and Vascular Surgery, University Hospital, University of Bonn, Bonn, Germany.
¹¹ Department of Anesthesiology, University of Texas Health Science Center at Houston, McGovern Medical School, Houston, Texas, USA.
¹² German Center for Diabetes Research (DZD), Neuherberg, Germany.
¹³ Institute of Pharmacology and Toxicology, University Hospital, University of Bonn, Bonn, Germany. Alexander.Pfeifer@uni-bonn.de.
¹⁴ PharmaCenter Bonn, University of Bonn, Bonn, Germany. Alexander.Pfeifer@uni-bonn.de.

^# Contributed equally.

PMID: 35790189
PMCID: PMC9452294
DOI: 10.1038/s41586-022-05041-0

Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine

Birte Niemann et al. Nature. 2022 Sep.

. 2022 Sep;609(7926):361-368.

doi: 10.1038/s41586-022-05041-0. Epub 2022 Jul 5.

Authors

Affiliations

¹ Institute of Pharmacology and Toxicology, University Hospital, University of Bonn, Bonn, Germany. Bini@uni-bonn.de.
² Institute of Pharmacology and Toxicology, University Hospital, University of Bonn, Bonn, Germany.
³ Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
⁴ Helmholtz Institute for Metabolic Obesity and Vascular Research (HI-MAG) of the Helmholtz Zentrum München at the University of Leipzig and University Hospital Leipzig, Leipzig, Germany.
⁵ Institute for Diabetes and Obesity, Helmholtz Center Munich, Neuherberg, Germany.
⁶ Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, Bonn, Germany.
⁷ PharmaCenter Bonn, University of Bonn, Bonn, Germany.
⁸ Medical Department III - Endocrinology, Nephrology, Rheumatology, University of Leipzig Medical Center, Leipzig, Germany.
⁹ Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
¹⁰ Clinic and Polyclinic for General, Visceral, Thoracic and Vascular Surgery, University Hospital, University of Bonn, Bonn, Germany.
¹¹ Department of Anesthesiology, University of Texas Health Science Center at Houston, McGovern Medical School, Houston, Texas, USA.
¹² German Center for Diabetes Research (DZD), Neuherberg, Germany.
¹³ Institute of Pharmacology and Toxicology, University Hospital, University of Bonn, Bonn, Germany. Alexander.Pfeifer@uni-bonn.de.
¹⁴ PharmaCenter Bonn, University of Bonn, Bonn, Germany. Alexander.Pfeifer@uni-bonn.de.

^# Contributed equally.

PMID: 35790189
PMCID: PMC9452294
DOI: 10.1038/s41586-022-05041-0

Abstract

Brown adipose tissue (BAT) dissipates energy^1,2 and promotes cardiometabolic health³. Loss of BAT during obesity and ageing is a principal hurdle for BAT-centred obesity therapies, but not much is known about BAT apoptosis. Here, untargeted metabolomics demonstrated that apoptotic brown adipocytes release a specific pattern of metabolites with purine metabolites being highly enriched. This apoptotic secretome enhances expression of the thermogenic programme in healthy adipocytes. This effect is mediated by the purine inosine that stimulates energy expenditure in brown adipocytes by the cyclic adenosine monophosphate-protein kinase A signalling pathway. Treatment of mice with inosine increased BAT-dependent energy expenditure and induced 'browning' of white adipose tissue. Mechanistically, the equilibrative nucleoside transporter 1 (ENT1, SLC29A1) regulates inosine levels in BAT: ENT1-deficiency increases extracellular inosine levels and consequently enhances thermogenic adipocyte differentiation. In mice, pharmacological inhibition of ENT1 as well as global and adipose-specific ablation enhanced BAT activity and counteracted diet-induced obesity, respectively. In human brown adipocytes, knockdown or blockade of ENT1 increased extracellular inosine, which enhanced thermogenic capacity. Conversely, high ENT1 levels correlated with lower expression of the thermogenic marker UCP1 in human adipose tissues. Finally, the Ile216Thr loss of function mutation in human ENT1 was associated with significantly lower body mass index and 59% lower odds of obesity for individuals carrying the Thr variant. Our data identify inosine as a metabolite released during apoptosis with a 'replace me' signalling function that regulates thermogenic fat and counteracts obesity.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. Stimulatory effects of inosine on BAT metabolism.**
a, Left, representative image of BAT after thermoneutrality: whole mount staining of lipids (LipidTOX, red), apoptotic nuclei (TUNEL-stain, green) and nuclei (DAPI, blue). Right, quantification of TUNEL-positive nuclei in BAT at 22 or 30 °C for 3 or 7 days (n = 3, 10 images per BAT). Scale bar, 20 µm. b,c, Untargeted metabolomics of murine brown adipocytes after nutlin-3 treatment (n = 6): b, Volcano plot representing statistically increased (red) or reduced (blue) metabolites. Sig., significant. c, Qualitative enrichment analysis of metabolic pathways (also see Supplementary Fig. 1). d, Extracellular purinergic molecules after UV irradiation of brown adipocytes (n = 9). ADP, adenosine diphosphate; Ado, adenosine; AMP, adenosine monophosphate; Hypo, hypoxanthine; Ino, inosine. e, Expression of *Ucp1* in brown adipocytes after incubation with supernatants described in d (n = 6). f, Intracellular cAMP levels of murine brown adipocytes treated with indicated compounds (n = 3–6). g, Hierarchical clustering of P38 signalling regulated by inosine (INO) and FORSK. Regulatory sites: *Activating sites, red; inhibitory sites, blue. h,i, Expression of *Ucp1* in murine brown adipocytes (h) (n = 7) and white adipose (i) (n = 3) after inosine treatment. j,k, Basal oxygen consumption (n = 6) (j) and lipolysis (n = 16) (k) of BAT explants after inosine treatment. l, Oxygen consumption of mice after inosine injection (n = 5). m–r, Inosine administration via micro-osmotic pumps and HFD for 28 days. m, Body weight (n = 9–10). n, Analysis of covariance (ANCOVA) (non-linear fit) area under the curve (AUC) of oxygen consumption/body weight at 23 °C (n = 5). o, Oxygen consumption at 4 °C (n = 5). p,q, *Ucp1* expression in BAT (n = 8) (p) and WATi (n = 9) (q). r, Representative haematoxylin and eosin (HE) and UCP1 staining of WATi (HE n = 8, UCP1 n = 3, Scale bar, 100 µm; upper right, fourfold magnification). s,t, Inosine injections in obese mice (vehicle: n = 8, inosine: n = 7). s, Body weight. t, Body composition at days −1 and 25. For all: *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001. For exact P values, see source data. Data are represented as mean ± s.e.m. Two-tailed t-test, d,e,**h–m**,o–q; one-way ANOVA with Tukey’s post hoc test, a–c,f. Source data

**Fig. 2. Role of ENT1 in adipose tissue metabolism.**
a–d, Analysis of WT and ENT1^−/− (KO) murine brown adipocytes. a, ³H-inosine uptake (n = 3). b, Extracellular inosine concentrations (n = 12). c, Representative Oil Red O staining. d, Expression of *Ucp1*, *Ppargc1a* and *Pparg* (n = 9). e, Oxygen consumption of WT and ENT1-KO BAT (n = 3–5). f, Lipid uptake (¹⁴C-triolein) in indicated organs of WT and ENT1-KO mice (n = 5, multiple t-tests). g, Body weight of WT and ENT1-KO mice during control diet (CD) or HFD (n = 7) (summarized P values of HFD groups are shown), h–p, Twelve-week HFD in WT and ENT1-KO mice. h, Body composition (n = 7). i, Glucose tolerance test (n = 7). j, ANCOVA of oxygen consumption/body weight (n = 7). k, Mean oxygen consumption over 24 h at 23 °C (n = 7). l, Oxygen consumption at 4 °C (n = 7). m, Expression of *Ucp1* and *Elovl3* in BAT (n = 6–7). n, Expression of *Ucp1* and *Elovl3* in WATi (n = 5–6). o, Representative haematoxylin and eosin and UCP1 staining of WATi (HE n = 7, UCP1 n = 3, scale bar, 100 µm). p, Mean adipocyte area of WATi (n = 7). q–u, Analysis of ENT1-floxed-AdiponectinCre mice. q, Oxygen consumption of 8-week old male mice (n = 6). r, ANCOVA of AUC of oxygen consumption/body weight after 12 weeks of HFD (13 control and 11 ENT1-A-KO mice were analysed). s, Glucose tolerance after 12 weeks of HFD or CD (HFD 13 control and ten ENT1-A-KO mice, CD n = 6) (summarized P values of the HFD groups are shown). t,u, Thermogenic marker expression in (t) BAT (13 control and nine ENT1-A-KO mice). u, WATi (12 control and eight ENT1-A-KO mice) after 12 weeks of HFD. v, *Slc29a1* expression in PDGFRα-positive and -negative stromal vascular fraction cells of WATi (n = 8–9). For all: *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001. For exact P values see source data. Data are represented as mean ± s.e.m. Two-tailed t-test was applied except for j,r (ANCOVA, non-linear fit) and g,s (one-way ANOVA with Tukey’s post hoc test). Source data

**Fig. 3. Regulation of human adipocytes by inosine and SLC29A1.**
a, Concentrations of purinergic molecules in the supernatant of human brown adipocytes after UV irradiation (n = 6). b, Expression of thermogenic (*UCP1, TFAM*) and adipogenic (*PPARG, FABP4*) marker genes in human brown adipocytes treated with and without inosine (n = 6). c, ³H-Inosine uptake in human brown adipocytes with (ENT1-CRISPR) and without (NTC2B) ENT1 knockdown treated with and without dipyridamole (Dip) (1 µM) (n = 3). d, Lipolysis of human beige (hMADS) and human white adipoctyes (hWA) treated with and without inosine (300 nM) (n = 5). e, UCP1 expression of hWA with (ENT1-CRISPR) and without (NTC2B) ENT1 knockdown (n = 6). f, Oxygen consumption rate (OCR) of hWA with (ENT1-CRISPR) and without (NTC2B) ENT1 knockdown (n = 4). g,h, Linear regression between SLC29A1 and UCP1 expression in human subcutaneous WAT (subcutaneous, SC) (g) (n = 1,476; ρ = −0.72) and human visceral WAT (visceral, VIS) (h) (n = 1,583; ρ = −0.61), P values were corrected for multiple inference using the Holm method. i, ³H-inosine uptake of HEK293T cells overexpressing either the ENT1-WT or Ile216Thr variant (n = 3). j, BMI of Ile216Thr variant carriers (Ile/Thr and Thr/Thr) compared to the Ile/Ile homozygous participants (n(Ile/Thr) = 72, n(Thr/Thr) = 822). For all: *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001. For exact P values see source data. Data are represented as mean ± s.e.m. A two-tailed t-test was applied for a,b,d,e,i and one-way ANOVA with Tukey’s post hoc test for c. Source data

**Extended Data Fig. 1. Secreted factors of apoptotic murine brown adipocytes.**
**a–c**, Mature adipocytes, CD11b+ myeloid cells and CD31+ endothelial cells were isolated from BAT of mice housed at 22 °C or at 30 °C (thermoneutrality) for 3 days. Gene expression of apoptotic marker genes in (a) mature adipocytes, (b) CD11b+ myeloid cells, (c) CD31+ endothelial cells (n = 3). **d–h**, Untargeted metabolomics of apoptotic BA. d, Heatmap of top 50 secreted metabolites accumulated in the supernatant after nutlin-3 treatment, upregulated metabolites: red, downregulated metabolites: blue (n = 6). e, Heatmap of top 50 secreted metabolites after UV irradiation (n = 6). f, Qualitative enrichment analysis of metabolic pathways of secreted metabolites of BA after UV irradiation based on the KEGG metabolic pathways, g, Volcano plot of secreted metabolites after UV treatment of BA (n = 6). h, Venn diagram and shared metabolite list identified across all highly significantly (p < 0.01) up- or down-regulated metabolites after UV or nutlin-3 treatment. i, Extracellular concentrations of purinergic molecules of BA after nutlin-3 treatment (n = 6). j, Expression of *Ppargc1a, Pparg* and *Fabp4* in BA after treatment with the apoptotic supernatant described in Fig. 1d (n = 6). k, cAMP dose-response curve of inosine (n = 3). l, Extracellular inosine concentrations of murine mesenchymal endothelial cells (EC) and murine BAT-derived fibroblasts (Fibros) after UV irradiation (n = 5-6). m, Expression of the ATP-degrading enzymes *Entpd1, Nt5e, Ada, Pnp, Xo* in murine brown (BA) and white (WA) adipocytes (n = 6). For all: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. For exact p-values see Source Data. Data are represented as mean ± s.e.m. Two-tailed t-test was applied for **a–c**, i, j, l, m; One-way ANOVA with Tukey’s post hoc test for g, h. Source data

**Extended Data Fig. 2. Inosine stimulates thermogenesis.**
a, Venn-Diagram of significantly changed phosphorylation sites upon FORSK or INO treatments (FDR < 0.05) and Venn-diagram indicating significantly regulated sites upon FORSK and INO treatments and total number of PKA target sites with more than 3 valid values in at least one condition. **b–d**, Hierarchical clustering of significantly regulated sites of (b) the Sik2-Creb1 axis, (c) the Mtorc1 complex and regulatory MTOR target sites and (d) regulatory sites of MAPK/ERKs and MAPK target sites, significantly regulated by INO and FORSK. Regulatory sites: *. Activating sites: red, inhibitory sites: blue. **e–g**, Representative Western Blots and quantification of (e) P-p38 MAP kinase, (f) P-ATF-2 and (g) P-Creb in response to 30 min of inosine (300 nM) in murine BA (n = 3). Calnexin serves as a loading control. For gel source data see Supplementary Information. h, Expression of *Pparg* mRNA in murine BA inosine treatment (n = 5). i, Expression of *Ppargc1a* in murine WA after inosine treatment (n = 3). j, Expression of *Necdin* in premature WA after inosine-treatment (n = 5). k, UCP1-mediated oxygen consumption of BAT explants after inosine-treatment (n = 6). l, Lipolysis of murine BA after 120 min of incubation with solvent control, inosine (100 nM), norepinephrine (NE, 1 µM) with or without pre-treatment with the A_2A-antagonist MSX (150nM) or the A_2B-antagonist PSB603 (150 nM) (n = 3-4). m, n, Oxygen consumption at 23 °C after i.p injection of either Vehicle (Veh) or inosine (Ino) and respective AUCs of measured V(O2) over 60 min of 8-week-old, male WT and (m) A_2A-KO mice (n = 5-6) and (n) A_2B-KO mice (n = 4-7). For all: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. For exact p-values see Source Data. Data are represented as mean ± s.e.m. Two-tailed t-test was applied for **e–k**. One-way ANOVA with Tukey’s post hoc test for **l–n**. Source data

**Extended Data Fig. 3. Inosine-mediated effects *in vivo*.**
**a–i**, Inosine administration via micro-osmotic pumps and HFD for 28 days in C57Bl6/J mice: a, Food intake (n = 5), b Motility (n = 5), c, Oxygen consumption at 23 °C (n = 5), d, Representative Western Blot and quantification of UCP1 expression in BAT (n = 10). Calnexin serves as a loading control. For gel source data see Supplementary Information. e, Expression of *Ndufa* and *Nd5* in inosine treated and control BAT (n = 8), f, Representative macroscopic pictures of BAT after 28 days of treatment and high fat diet (HFD), g, Representative haematoxylin/eosin (HE) and UCP1 staining of BAT sections (n = 3). Scale bar refers to 100 µm, 4-fold magnification of the picture in the upper right corner. h, Expression of *Ppargc1a* and *Prdm16* in WATi after inosine treatment (n = 9), i, Mean cell area of WATi (n = 8). **j–m** Mice were fed a HFD for 12 weeks, followed by daily injections (s.c.) with either vehicle (0.9% NaCl) or inosine (1 mg/kg) for 26 days during HFD (Vehicle n = 8, Inosine: n = 7): j, Weight loss (after 26 days). k, Food intake (5 measurements per mouse). **l–m**, Blood glucose concentrations at day -1 (l) and day 25 (m). For all: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. For exact p-values see Source Data. Data are represented as mean ± s.e.m. Two-tailed t-test was applied for **a–e**, h, i. Source data

**Extended Data Fig. 4. Global knockout of ENT1.**
a, RNA-sequencing analysis of *Slc29a1* and *Slc29a2* expression in adipocytes isolated from BAT, WATi, and WATg (n = 4). b, Expression of *Adipoq* and *Fabp4* in WT and ENT1 KO murine brown adipocytes (n = 9). c, Oxygen consumption of murine WT and ENT1-KO brown adipocytes (n = 7-10). d, Lipolysis of WT and ENT1-KO murine brown adipocytes stimulated with and without NE (1 µM) (n = 6). **e,f**, Analysis of WT and ENT1-KO BAT explants: UCP1-dependent respiration (n = 3-5) (e) and lipolysis (n = 7-8) (f). g, ³H-deoxyglucose (DOG) uptake into indicated organs of WT and KO mice (n = 5). h, Energy content of feces from WT and ENT1-KO mice (n = 3). i, Lipid droplet accumulation of WT and KO murine white adipocytes (n = 3). **j–l**, Analysis of WT and ENT1-KO murine white adipocytes: thermogenic and adipogenic marker expression (n = 3) (j), Lipolysis (n = 6) (k), Time course and statistical analyses of oxygen consumption (n = 9-10, for basal OCR all 4 groups and for uncoupling efficiency the 2 control groups were calculated) (l). m,n, Lipolysis (n = 7) (m) and oxygen consumption (n = 6-7) (n) of WT and ENT1-KO WATi explants. **o–v**, Twelve weeks of HFD in WT and ENT1-KO mice: Weight gain (n = 7) (o), Adipose tissue weight (n = 7) (p), AUC of glucose concentration (n = 7) (q), whole-body oxygen consumption at 23 °C (n = 7) (r), motility (n = 7) (s), food intake (n = 7) (t), *Ppargc1a* expression in BAT (n = 7) (u) and WAT (n = 6) (v). For all: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. For exact p-values see Source Data. Data are represented as mean ± s.e.m. Two-tailed t-test was applied except for a, d and k (One-way ANOVA with Tukey’s post hoc test). Source data

**Extended Data Fig. 5. Adipocyte-specific deletion of ENT1.**
a, Expression of *Slc29a1* in BAT and WATi of ENT1-A-KO mice (6 control and 11-12 ENT1 A-KO mice were analyzed). b,c, Expression of adipogenic and thermogenic markers in control and ENT1-A-KO murine BA (n = 3-4) (b) and white adipocytes (WA) (n = 8) (c). d,e, *Ex vivo* oxygen consumption of control or ENT1-A-KO BAT (n = 7) (d) or WATi (n = 7) (e). f, Mitochondrial respiration of control or ENT1-A-KO WA (n = 14-15, for basal OCR all 4 groups for delta OCR the 2 CL groups and for uncoupling efficiency the 2 control groups were calculated). g, *Ex vivo* lipolysis of control or ENT1-A-KO BAT(n = 12) and WATi (n = 14). **h–m**, Characterization of 8-week-old male ENT1-A-KO mice (chow diet): whole-body oxygen consumption at 23 °C (n = 6) (h), food intake (6 mice were analyzed, 2 measurements per mouse) (i), energy content of feces (n = 6) (j), motility (n = 6) (k), thermogenic marker expression in BAT (n = 5-6) (l) and WATi (n = 6) (m). n, Weight gain of control and ENT1-A-KO mice on CD or HFD (13 control and 11 ENT1-A-KO mice on HFD and 6 mice per genotype on CD were analyzed). **o–q**, Analysis of control and ENT1-A-KO mice after HFD: motility (n = 11-13) (o), food intake (13 control and 11 ENT1-A-KO mice were analyzed, 1-3 measurements per mouse) (p) and energy content of feces (n = 9-13) (q). For all: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. For exact p-values see Source Data. Data are represented as mean ± s.e.m. Two-tailed t-test was applied except for n (One-way ANOVA with Tukey’s post hoc test). Source data

**Extended Data Fig. 6. Effects of ENT1-inhibition by dipyridamole on adipose tissue.**
a, Extracellular inosine concentrations of murine BA treated with and without dipyridamole (Dip) (n = 6). b, ³H-Inosine uptake in WT and ENT1 KO murine BA treated with and without dipyridamole (Dip) (n = 3-6). c, Lipolysis of murine BA treated with and without dipyridamole (1 µM) in presence and absence of NE (1 µM) (n = 5). d, Extracellular inosine levels of murine BA treated with and without NE (n = 9). e, Tissue inosine concentrations of BAT dialysates from mice housed at 23 °C treated with and without NE, or from cold exposed mice (4 °C) (n = 4-7). f, Oxygen consumption at 23 °C of C57Bl6/J mice after injection of vehicle, the β3-adrenergic agonist CL316,243 (0.3 µg/kg) in the presence or absence of dipyridamole (1 mg/kg) (n = 7-8) (left graph: timecourse, right graph: AUC). g, Oxygen consumption at 4 °C in WT and ENT1-KO mice after injection of vehicle or dipyridamole (1 mg/kg) (Dip) (n = 3-6) (left graph: timecourse, right graph: V02 max.). **h–j**, Analysis of C57Bl6/J mice after 7 days of cold exposure (4 °C) with and without daily dipyridamole injection (1 mg/kg): h, Oxygen consumption (n = 7), i, Weights of adipose tissues (n = 7), j, Representative HE and UCP1 staining of WATi sections (n = 3). For all: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. For exact p-values see Source Data. Data are represented as mean ± s.e.m. Two-tailed t-test was applied for a, d, h, i, One-way ANOVA with Tukey’s post hoc test for b, c, e, f, g. Source data

**Extended Data Fig. 7. Inosine- and ENT1-mediated effects on human adipocytes.**
a, b Heatmap of top 25 secreted metabolites of untargeted metabolomics of human BA: (a) treated with and without nutlin-3 (b) irradiated with UV, upregulated metabolites are shown in red, downregulated metabolites are shown in blue (n = 6). c, Concentrations of purinergic molecules in the supernatant of human BA treated with and without nutlin-3 (n = 6). d, Extracellular inosine concentrations of human BA treated with and without NE (n = 6). e, Representative Western Blot and quantification for ENT1 knockdown in human BA induced by different lentiviral Crispr-Cas9 vectors (CRISPR1, 2, 3) in comparison to a control vector (NTC2B) and to not-transduced (Control) cells (n = 3). Calnexin serves as a loading control. For gel source data see Supplementary Information. f, extracellular inosine concentrations in human BA with (ENT1-Crispr) and without (NTC2B) ENT1 knockdown treated with and without Dipyridamole (Dip) (1µM) (n = 4-6). g, mRNA expression of adipogenic (*PPARG)* and thermogenic (*PRDM16, TFAM, UCP1, PPARGC1A*) marker genes in human BA with (ENT1-Crispr) and without (NTC2B) ENT1 knockdown (n = 5-6). h, Expression of the purinergic enzymes *ENTPD1, NT5E, ADA, PNP* and XO in human brown (hBA) and white (hWA) adipocytes (n = 5-6). i, Basal oxygen consumption rate (OCR) of hWA with (ENT1-Crispr) and without (NTC2B) ENT1 knockdown (n = 4). j, k Linear regression of *SLC29A1* and (j) *PPARGC1A* expression (n = 1,476; ρSpearman = −0.71) and *PRDM16* expression (n = 1,476; ρSpearman = −0.64) in human subcutaneous WAT (SC) and (k) *PPARGC1A* expression (n = 1,583; ρSpearman = −0.6) and *PRDM16* expression (n = 1,583; ρSpearman = −0.63) in human visceral WAT (VIS), p-values were corrected for multiple inference using the Holm method. For all: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. For exact p-values see Source Data. Data are represented as mean ± s.e.m. Two-tailed t-test was applied for c, d, g, h, i, One-way ANOVA with Tukey’s post hoc test for a, b, e, f.

**Extended Data Fig. 8. Schematic representation of inosine production, clearance and action in thermogenic adipocytes.**
ATP and ADP are degraded by ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) to AMP, which in turn is metabolized to adenosine by ecto 5’-nucleotidase (NT5E). Inosine is generated by adenosine deaminase (ADA) or is released into the interstitial space via ENT1. Inosine signaling is terminated by either uptake of inosine into the cell by ENT1 or degradation by purine nucleoside phosphorylase (PNP). Extracellular inosine exerts its effects via the purinergic P1 receptors A_2A and A_2B resulting in G_s protein-induced cAMP production. Parts of the figure were drawn by using pictures from Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license (https://creativecommons.org/licenses/by/3.0/).

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Comment in

A 'replace me' signal from dying brown fat fires up weight loss.
De Bock K, Wolfrum C. De Bock K, et al. Nature. 2022 Sep;609(7926):252-253. doi: 10.1038/d41586-022-01826-5. Nature. 2022. PMID: 35821410 No abstract available.
Inosine boosts fat burning.
Crunkhorn S. Crunkhorn S. Nat Rev Drug Discov. 2022 Sep;21(9):633. doi: 10.1038/d41573-022-00126-x. Nat Rev Drug Discov. 2022. PMID: 35859060 No abstract available.
A purinergic call to arms from dying brown adipocytes.
Hankir MK. Hankir MK. Cell Stress. 2022 Jul 25;6(7):68-71. doi: 10.15698/cst2022.07.269. eCollection 2022 Jul. Cell Stress. 2022. PMID: 35975107 Free PMC article.
AcroBATics: how dying brown adipocytes trigger browning.
Zinngrebe J, Fischer-Posovszky P. Zinngrebe J, et al. Nat Rev Endocrinol. 2022 Nov;18(11):661-662. doi: 10.1038/s41574-022-00743-x. Nat Rev Endocrinol. 2022. PMID: 36064975 No abstract available.
Fire up the pyre: inosine thermogenic signaling for obesity therapy.
Willemsen N, Kotschi S, Bartelt A. Willemsen N, et al. Signal Transduct Target Ther. 2022 Nov 11;7(1):375. doi: 10.1038/s41392-022-01218-1. Signal Transduct Target Ther. 2022. PMID: 36357366 Free PMC article. No abstract available.

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References

1. Kajimura S, Spiegelman BM, Seale P. Brown and beige fat: physiological roles beyond heat generation. Cell Metab. 2015;22:546–559. - PMC - PubMed
1. Scheele C, Wolfrum C. Brown adipose crosstalk in tissue plasticity and human metabolism. Endocr. Rev. 2020;41:53–65. - PMC - PubMed
1. Becher T, et al. Brown adipose tissue is associated with cardiometabolic health. Nat. Med. 2021;27:58–65. - PMC - PubMed
1. Pellegrinelli V, Carobbio S, Vidal-Puig A. Adipose tissue plasticity: how fat depots respond differently to pathophysiological cues. Diabetologia. 2016;59:1075–1088. - PMC - PubMed
1. Graja A, Gohlke S, Schulz TJ. Aging of brown and beige/brite adipose tissue. Handb. Exp. Pharmacol. 2019;251:55–72. - PubMed

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[1] Kajimura S, Spiegelman BM, Seale P. Brown and beige fat: physiological roles beyond heat generation. Cell Metab. 2015;22:546–559. - PMC - PubMed

[2] Kajimura S, Spiegelman BM, Seale P. Brown and beige fat: physiological roles beyond heat generation. Cell Metab. 2015;22:546–559. - PMC - PubMed

[3] Scheele C, Wolfrum C. Brown adipose crosstalk in tissue plasticity and human metabolism. Endocr. Rev. 2020;41:53–65. - PMC - PubMed

[4] Scheele C, Wolfrum C. Brown adipose crosstalk in tissue plasticity and human metabolism. Endocr. Rev. 2020;41:53–65. - PMC - PubMed

[5] Becher T, et al. Brown adipose tissue is associated with cardiometabolic health. Nat. Med. 2021;27:58–65. - PMC - PubMed

[6] Becher T, et al. Brown adipose tissue is associated with cardiometabolic health. Nat. Med. 2021;27:58–65. - PMC - PubMed

[7] Pellegrinelli V, Carobbio S, Vidal-Puig A. Adipose tissue plasticity: how fat depots respond differently to pathophysiological cues. Diabetologia. 2016;59:1075–1088. - PMC - PubMed

[8] Pellegrinelli V, Carobbio S, Vidal-Puig A. Adipose tissue plasticity: how fat depots respond differently to pathophysiological cues. Diabetologia. 2016;59:1075–1088. - PMC - PubMed

[9] Graja A, Gohlke S, Schulz TJ. Aging of brown and beige/brite adipose tissue. Handb. Exp. Pharmacol. 2019;251:55–72. - PubMed

[10] Graja A, Gohlke S, Schulz TJ. Aging of brown and beige/brite adipose tissue. Handb. Exp. Pharmacol. 2019;251:55–72. - PubMed

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Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine

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Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine

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