DOI: 10.5061/dryad.bk3j9kdp3
title: Adipocyte release of nucleosides contributes to obesity-associated inflammation

description: Obese adipose tissue drives inflammation through several mechanisms.
 Extracellular nucleosides can induce macrophage inflammation through the
 activation of toll-like and purinergic receptors. The present study
 quantified nucleoside release from adipocytes and adipose tissue. Cultured
 mouse adipocytes released many nucleosides used in RNA/DNA. Adipose tissue
 from obese mice released more nucleosides than that from control non-obese
 mice, resulting in higher interstitial fluid concentrations. Consistent
 with the mouse study, human adipose tissue also showed significant release
 of adenosine/deoxyadenosine, guanosine/deoxyguanosine, and uridine ex
 vivo. Adipocytes release nucleosides in part through the equilibrative
 nucleoside transporter 1 (ENT1), and this release induces macrophage TNFα
 and IL-1β expression. Treatment of mice with the ENT1 inhibitor in vivo
 increased adipose tissue interstitial nucleoside concentrations and
 cytokine expression. These data uncover a previously unknown phenomenon of
 adipocyte release of nucleosides, which contribute to adipose tissue
 inflammation in obesity.
description: # Adipocyte release of nucleosides contributes to obesity-associated
 inflammation Dataset DOI:
 [10.5061/dryad.bk3j9kdp3](https://doi.org/10.5061/dryad.bk3j9kdp3) ##
 Description of the data and file structure Cells were cultured in various
 conditions, and nucleoside release was quantified by LC-MS/MS-MRM. Mouse
 and human adipose tissue explants were cultured in a PDSM flow chamber,
 and nucleoside release was quantified in the effluent. Expression of
 nucleoside transporter ENT1 was quantified by qPCR and Western blot in
 human adipose tissue. Mice were treated with the ENT1 inhibitor, and
 adipose tissue interstitial fluid collected for measure of nucleoside
 concentration. Adipose tissue was also collected for cytokine expression.
 Murine macrophages were treated with adipocyte conditioned media or
 guanosine and cytokine expression quantified with qPCR ### Files and
 variables #### File: Fig_1A_3T3.csv **Description:** 3T3-L1 fibroblasts
 and adipocytes were cultured and media release of nucleosides quantified
 over 48 hours ##### Variables * Cell counts: Number of cells in control
 well for normalization * Condition (48hrs): media, adipocytes, or
 fibroblasts * Measure: concentration, net release (subtracting media
 alone), net release per 10^5 cells * Units: micromolar, micromolar/10^5
 cells #### File: Fig_1B_Primary_Mouse_A_SVF.csv **Description:** Mouse
 adipose tissue derived fibroblasts and adipocytes were cultured and media
 release of nucleosides quantified over 48 hours ##### Variables * Cell
 counts: Number of cells in control well for normalization * Condition
 (48hrs): media, adipocytes, or fibroblasts * Measure: concentration, net
 release (subtracting media alone), net release per 10^5 cells *
 micromolar, micromolar/10^5 cells #### File: Fig_1D_hypertrophic.csv
 **Description:** Mouse adipose tissue derived mature and hypertrophic
 adipocytes were cultured and media release of nucleosides quantified over
 48 hours ##### Variables * Cell type: mature vs. hypertrophic adipocytes *
 technical replicates: replicates or fold change * Measure: concentration,
 net release (subtracting media alone), net release per 10^5 cells *
 micromolar, fold change over mature #### File: Fig_2A_Mouse_Chips.csv
 **Description:** Adipose tissue from mice was removed and cultured in PDSM
 flow chambers. Released nucleosides were measured over time in effluent
 media  ##### Variables * Diet: control vs. obese * Run time (hour): hours
 of media collection * Measure: micromolar, area under the curve #### File:
 Fig_2B_Mouse_adipose_IF.csv **Description:**  ##### Variables * Group:
 control vs. obese mice * Excluded: x indicates which samples were
 excluded. Reason for exclusion noted below * Measure: concentration in uM
 #### File: Fig_3A_primary_human_adipocytes_fibroblasts.csv
 **Description:** Human adipose tissue SVF adipocytes and fibroblasts were
 cultured and media release of nucleosides quantified over 48 hours #####
 Variables * Cell counts: Number of cells in control well for normalization
 * Condition (48hrs): media, adipocytes, or fibroblasts * Measure:
 concentration, net release (subtracting media alone), net release per 10^5
 cells * micromolar, micromolar/10^5 cells #### File:
 Fig_3B_Human_adipose_chip.csv **Description:** Human adipose tissue
 explants were minced and put into PDMS flow chamber. Nucleoside release
 was quantified in media effluent over time ##### Variables * Sample * Run
 time: in hours * Nucleoside concentrations: in micromolar #### File:
 Fig_4A_adipose_ENT_expression.csv **Description:** Human adipose tissue
 explants were lysed for RNA ##### Variables * TPCL#: subject ID * BMI:
 body mass index in kg/m^2 * Group: obese vs. nonobese * Site: anatomical
 site of adipose tissue * Measure: RQ * ENT1-4: RQ of expression  ####
 File: Fig_4B_Adipocyte_vs_SVF_ENT1.csv **Description:** Human adipose
 tissue explants were digested and adipocytes separated from SVF  #####
 Variables * Subject: subject ID * BMI: body mass index in kg/m^2 *
 Measure: ENT1 RQ * Adipocytes: ENT1 expression in adipocytes * SVF: ENT1
 expression in SVF #### File: Fig_5A_MS5_3T3_ENTi.csv
 **Description:** Adipocytes and MS5 cells were cultured alone or with ENT
 inhibitor. Nucleoside concentrations were measured over 72 hours #####
 Variables * Cell: 3T3-L1 adipocytes or MS5 cells * Condition: treatment
 with control media, ENTi, or NBMPR * Time: 0, 24, 48, or 72 hours *
 Nucleosides: nucleoside concentration in micromolar #### File:
 Fig_5B_mouse_adipose_IF_ENTi.csv **Description:** Obese mice were treated
 with ENTi or vehicle. Adipose interstitial fluid was collected and
 nucleosides measured ##### Variables * Drug: ENTCi vs. vehicle * Excluded:
 x indicates samples excluded for undetectible levels * Nucleoside
 concentrations in micromolar #### File:
 Fig_6A_mouse_adipose_cytokine_expression.csv **Description:**  #####
 Variables * Diet: Nonobese vs. Obese * Treatment: Vehicle vs. ENTi *
 Cytokine dCT: dCT of respective cytokine * Cytokine dCT ave: dCT of
 control condition of respective cytokine * Cytokine RQ: RQ of respective
 cytokine #### File: Fig_6B_mouse_macrophage_cytokine_expression.csv
 **Description:** Mouse macrophages were treated with control media,
 guanidine, or adipocyte conditioned media ##### Variables * cytokine: IL6,
 TNFa, or IL1b * NT DCt: no treatment DCt * Treatment: control, ACM, or rG
 * ssRNA: present (+) or absent (-) #### File:
 Fig_S1_mouse_SVF_adipocyte_nucleosides.csv **Description:** Mouse adipose
 tissue SVF cells were derived from control and obese mice and
 differentiated into adipocytes. Nucleoside release was then quantified in
 the media ##### Variables * Diet: Nonobese vs. obese * Measure:
 Concentration (micromolar), Net release over media alone (micromolar), or
 net release per 10^5 cells (micromolar/10^5 cells)  * Excluded: x
 indicates sample excluded due to outliers * Cell counts (X10^5): cell
 counts * Nucleoside: nucleoside concentrations in units indicated ####
 File: Fig_S2_mouse_plasma_nucleosides.csv **Description:** Plasma from
 control and obese mice was assayed for nucleoside concentration #####
 Variables * Diet: control vs. obese mice * nucleoside: Nucleoside
 concentration in micromolar #### Fike: Figure_S1.tif A: Nucleoside release
 from cultured adipocytes derived from control (n=3) and obese (HFD; n=5)
 mice. B: Nucleoside levels in plasma from control and obese mice (n=6).
 #### File: Subject1_20x_1_CAV1.tif **Description:** IHC image of adipose
 tissue at 20X magnification from subject 1 stained for caveolin 1 ####
 File: Subject1_20x_1_DAPI.tif **Description:** IHC image of adipose tissue
 at 20X magnification from subject 1 stained for DAPI #### File:
 Subject1_20x_1_Lipidtox.tif **Description:** IHC image of adipose tissue
 at 20X magnification from subject 1 stained for lipid #### File:
 Subject1_20x_1_ENT1.tif **Description:** IHC image of adipose tissue at
 20X magnification from subject 1 stained for ENT1 #### File:
 Subject1_20x_1_Merge.tif **Description:** IHC image of adipose tissue at
 20X magnification from subject 1 all channels merged #### File:
 Subject2_20x_CAV1.tif **Description:** IHC image of adipose tissue at 20X
 magnification from subject 2 stained for caveolin 1 #### File:
 Subject2_20x_DAPI.tif **Description:** IHC image of adipose tissue at 20X
 magnification from subject 2 stained for DAPI #### File:
 Subject2_20x_Lipidtox.tif **Description:** IHC image of adipose tissue at
 20X magnification from subject 2 stained for lipid #### File:
 Subject2_20x_ENT1.tif **Description:** IHC image of adipose tissue at 20X
 magnification from subject 2 stained for ENT1 #### File:
 Subject2_20x_Merge.tif **Description:** IHC image of adipose tissue at 20X
 magnification from subject 2 all channels merged #### File:
 Subject2_10x_CAV1.tif **Description:** IHC image of adipose tissue at 10X
 magnification from subject 2 stained for caveolin 1 #### File:
 Subject2_10x_DAPI.tif **Description:** IHC image of adipose tissue at 10X
 magnification from subject 2 stained for DAPI #### File:
 Subject2_10x_ENT1.tif **Description:** IHC image of adipose tissue at 10X
 magnification from subject 2 stained for ENT1 #### File:
 Subject2_10x_Merge.tif **Description:** IHC image of adipose tissue at 10X
 magnification from subject 2 all channels merged #### File:
 Subject3_20x_CAV1.tif **Description:** IHC image of adipose tissue at 20X
 magnification from subject 3 stained for caveolin 1 #### File:
 Subject3_20x_DAPI.tif **Description:** IHC image of adipose tissue at 20X
 magnification from subject 3 stained for DAPI #### File:
 Subject3_20x_Lipidtox.tif **Description:** IHC image of adipose tissue at
 20X magnification from subject 3 stained for lipid #### File:
 Subject3_20x_ENT1.tif **Description:** IHC image of adipose tissue at 20X
 magnification from subject 3 stained for ENT1 #### File:
 Subject3_20x_Merge.tif **Description:** IHC image of adipose tissue at 20X
 magnification from subject 3 all channels merged #### File:
 Supplementary_Tables.doc **Description:** Supplementary tables from
 manuscript. Note: in table S1, *biospecimen number is a number assigned to
 each specimen that cannot be tracked back to individual patients*, and
 therefore is not considered a direct identifier. Specimens were released
 to laboratories in a de-identified manner. ##### Variables * Diet: control
 vs. obese mice * nucleoside: Nucleoside concentration in micromolar ##
 Human subjects data Subjects undergoing procedures at UCLA signed consent
 to allow any remnant specimens to be de-identified and used in research.
 Identifying data was not attached to these remnants, which were
 distributed in a de-identified manner.