LRG1 is an adipokine that promotes insulin sensitivity and suppresses inflammation

  1. Chan Hee J Choi
  2. William Barr
  3. Samir Zaman
  4. Corey Model
  5. Annsea Park
  6. Mascha Koenen
  7. Zeran Lin
  8. Sarah K Szwed
  9. Francois Marchildon
  10. Audrey Crane
  11. Thomas S Carroll
  12. Henrik Molina
  13. Paul Cohen  Is a corresponding author
  1. Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program, United States
  2. Laboratory of Molecular Metabolism, Rockefeller University, United States
  3. Department of Immunobiology, Yale University, United States
  4. Bioinformatics Resouce Center, Rockefeller University, United States
  5. Proteomics Resource Center, Rockefeller University, United States
8 figures, 1 table and 4 additional files

Figures

Figure 1 with 1 supplement
Characterization of primary adipocyte secretome using BONCAT.

(A) Chemical structures of L-methionine (Met) and L-azidohomoalanine (AHA). (B) Schematic diagram of MS-based secretome analysis from primary Visc, SubQ, and Brown adipocytes using BONCAT. (C) …

Figure 1—figure supplement 1
Validation of primary adipocyte differentiation and overview of MS results.

(A) Phase-contrast microscope images of mature Visc, SubQ, and Brown adipocytes on day 6 of differentiation. Scale bars indicate 500 μm. (B) Relative mRNA levels of mature adipocyte genes. (C) …

Profiling in vivo nascent serum proteome using BONCAT.

(A) Schematic of AHA IP injections. (B) Body weights of mice injected with PBS (n=2) or AHA (n=3). (C) In-gel fluorescence analysis of TAMRA-conjugated serum proteins from PBS-pulsed (lanes 1 and 2) …

Figure 3 with 2 supplements
LRG1 is secreted by mature adipocytes and increased in obesity.

(A) Search strategy for identification of novel adipokines with a potential role in whole-body metabolism. (B) Relative Lrg1 mRNA levels across a panel of tissues from 8-week-old male B6 mice. Visc, …

Figure 3—figure supplement 1
Validation of tissue fractionation and quantification of LRG1 in adipocyte CM.

(A) Relative mRNA levels of mature adipocyte markers, Fabp4, Pparg2, and Adipoq and SVF markers, Adgre1 and Pecam1, in indicated tissues fractionated into mature adipocytes and SVF. Data are …

Figure 3—figure supplement 2
LRG1 gain or loss of function does not affect adipogenesis.

(A) Depletion of bovine LRG1 in FBS using Cyt c-conjugated agarose beads. Arrow indicates LRG1. (B) Oil red O stain of Lrg1+/+ or Lrg1-/- primary adipocytes treated with indicated concentrations of …

Figure 4 with 2 supplements
LRG1 overexpression improves glucose homeostasis in C57BL/6 J DIO mice.

(A) Schematic of Ad vector-mediated acute LRG1 overexpression in B6 DIO male mice. (B) Plasma western blot of LRG1 5 days after Ad injection. (C) Quantification of (B). *p<0.05 from two-sided …

Figure 4—figure supplement 1
Metabolic characterization of AAV-transduced B6 mice.

(A) Relative Lrg1 mRNA levels across AAV-transduced B6 males at 21 weeks of age (13 weeks on HFD). *p<0.05, **p<0.01, ***p<0.001 from two-sided Welch’s t-test. (B) Quantification of band intensities …

Figure 4—figure supplement 2
LRG1 loss of function in C57BL/6 J DIO mice elevates fasting blood glucose.

(A) Schematic diagram of murine Lrg1 exon structure. Red arrowhead indicates the region targeted by CRISPR/Cas9 gRNA. (B) Sanger sequencing chromatogram of PCR products amplifying a portion of Lrg1

Figure 5 with 1 supplement
LRG1 overexpression in C57BLKS/J-Leprdb/db mice delays onset of diabetic phenotype and promotes WAT expansion.

(A) Schematic of AAV8-mediated LRG1 overexpression in db/db males. (B) Plasma western blot of LRG1 2 weeks after AAV injection. (C) Quantification of (B). ****p<0.0001 from two-sided Welch’s t-test. …

Figure 5—figure supplement 1
Metabolic characterization of db/db vs. m/m mice and AAV-transduced db/db mice.

(A) Body weights of db/db and littermate m/m male mice at 7 and 10 weeks of age. ***p<0.001, ****p<0.0001 from two-sided Welch’s t-test. n=6 per group. (B) 6 hr fasting blood glucose levels of db/db

Figure 6 with 1 supplement
LRG1 suppresses obesity-associated systemic inflammation.

(A) Representative images from H&E-stained eWAT sections from B6 DIO mice transduced with AAV-eGFP or AAV-LRG1-FL under 10 x (left) and 20 x (right) objectives. Scale bars indicate 100 µm. (B) …

Figure 6—figure supplement 1
Analysis of inflammatory phenotypes with LRG1 gain of function.

(A) Representative images from H&E-stained iWAT sections from B6 DIO mice transduced with AAV-eGFP or AAV-LRG1-FL. Scale bars are indicated. (B) Quantification of CLS from (A). Data are presented as …

Figure 7 with 1 supplement
LRG1 binds extracellular cytochrome c and blocks its pro-inflammatory effect on macrophages.

(A) α-FLAG co-IP of C-terminally FLAG-tagged LRG1 and Cyt c from the serum of AAV-transduced db/db mice at 7 weeks of age. (B) Western blot of Cyt c in the serum of db/db or littermate m/m mice at 7 …

Figure 7—figure supplement 1
Analysis of LRG1-Cyt c interaction.

(A) Quantification of Figure 7B. *p<0.05 from Tukey post hoc test following one-way ANOVA. n=4–5 biological replicates per group. (B) Quantification of Figure 7C. **p<0.01 from Šídák post hoc test …

Author response image 1

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Mus musculus)Lrg1NCBINM_029796.2
Strain, strain background (Mus musculus)C57BL/6 JThe Jackson LaboratoryCat #000664, RRID:IMSR_JAX:000664
Strain, strain background (Mus musculus)BKS.Cg-Dock7m +/+Leprdb/JThe Jackson LaboratoryCat# 000642, RRID:IMSR_JAX:000642
Strain, strain background (Mus musculus)LRG1-KO in C57BL/6 J backgroundThis StudyProduced by the CRISPR and Genome Editing Center (Rockefeller University)
Strain, strain background (Escherichia coli)NEB StableNEBCat# C3040Competent cells
Strain, strain background (Escherichia coli)BJ5183-AD-1 cellsAgilentCat# 200157Electroporation competent, recombination proficient cells carrying the pAdEasy-1 plasmid
Strain, strain background (Escherichia coli)XL-10 Gold ultracompetent cellsAgilentCat# 200314Competent cells
Strain, strain background (Adenovirus)Ad-eGFPThis study; protocol adapted from Luo et al., 2007Produced in Paul Cohen lab (Rockefeller University)
Strain, strain background (Adenovirus)Ad-LRG1-FLThis study; protocol adapted from Luo et al., 2007Produced in Paul Cohen lab (Rockefeller University)
Strain, strain background (AAV8)AAV8-eGFPThis studyProduced by Penn Vector Core (University of Pennsylvania).
Strain, strain background (AAV8)AAV8-LRG1-FLThis studyProduced by Penn Vector Core (University of Pennsylvania).
Cell line (Homo-sapiens)HEK293AInvitrogenCat# R70507
AntibodyAnti-LRG1 (rabbit polyclonal)Sigma-AldrichCat# HPA001888, RRID:AB_1079276Western blot (1:5000)
AntibodyAnti-CFD/Adipsin (sheep polyclonal)R and D SystemsCat# AF5430, RRID:AB_1655868Western blot (1:1000)
AntibodyAnti-Cytochrome c (rabbit monoclonal)Cell Signaling TechnologyCat# 11940, RRID:AB_2637071Western blot (1:1000)
AntibodyAnti-FLAG M2 Magnetic Beads (mouse monoclonal)Sigma-AldrichCat# M8823, RRID:AB_2637089Immunoprecipitation
AntibodyAnti-Cleaved Caspase-3 (Asp175) (rabbit polyclonal)Cell Signaling TechnologyCat# 9661, RRID:AB_2341188WB (1:1000)
AntibodyAnti-Caspase-3 (rabbit polyclonal)Cell Signaling TechnologyCat# 9662, RRID:AB_331439WB (1:1000)
AntibodyAnti-IRS-1 (rabbit polyclonal)Cell Signaling TechnologyCat# 2382, RRID:AB_330333WB (1:500)
AntibodyAnti-β-actin (rabbit polyclonal)GeneTexCat# GTX109639, RRID:AB_1949572WB (1:20000)
AntibodyAnti-vinculin (rabbit polyclonal)Cell Signaling TechnologyCat# 4650, RRID:AB_10559207WB (1:1000)
Peptide, recombinant proteinRecombinant Mouse M-CSFBiolegendCat# 576406
Peptide, recombinant proteinRecombinant human LRG1R and D SystemsCat# 7890-LR
Peptide, recombinant proteinRecombinant human TNFαR and D SystemsCat# 210-TA-020/CF
Peptide, recombinant proteinCytochrome c from equine heartSigma-AldrichCat# C2506
Commercial assay or kitClick-iT Protein Enrichment KitInvitrogenCat# C10416
Sequence-based reagentqPCR primersThis paperSupplementary file 3
Sequence-based reagentpAdTrack-CMVAddgeneRRID:Addgene_16405
Sequence-based reagentpENN.AAV.CB7.CI.eGFP.WPRE.rBGAddgeneRRID:Addgene_105542

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