Neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase

Journal name:
Nature Medicine
Volume:
18,
Pages:
1407–1412
Year published:
DOI:
doi:10.1038/nm.2885
Received
Accepted
Published online

Chronic low-grade adipose tissue and liver inflammation is a major cause of systemic insulin resistance and is a key component of the low degree of insulin sensitivity that exists in obesity and type 2 diabetes1, 2. Immune cells, such as macrophages, T cells, B cells, mast cells and eosinophils, have all been implicated as having a role in this process3, 4, 5, 6, 7, 8. Neutrophils are typically the first immune cells to respond to inflammation and can exacerbate the chronic inflammatory state by helping to recruit macrophages and by interacting with antigen-presenting cells9, 10, 11. Neutrophils secrete several proteases, one of which is neutrophil elastase, which can promote inflammatory responses in several disease models12. Here we show that treatment of hepatocytes with neutrophil elastase causes cellular insulin resistance and that deletion of neutrophil elastase in high-fat-diet–induced obese (DIO) mice leads to less tissue inflammation that is associated with lower adipose tissue neutrophil and macrophage content. These changes are accompanied by improved glucose tolerance and increased insulin sensitivity. Taken together, we show that neutrophils can be added to the extensive repertoire of immune cells that participate in inflammation-induced metabolic disease.

At a glance

Figures

  1. Neutrophils infiltrate eWAT in mice on HFD, and ablation of neutrophil elastase improves insulin sensitivity in DIO mice.
    Figure 1: Neutrophils infiltrate eWAT in mice on HFD, and ablation of neutrophil elastase improves insulin sensitivity in DIO mice.

    (a) ATNs (Cd11b+Ly6g+F4/80Cd11c cells) as a percentage of SVCs from the eWAT of DIO mice analyzed by FACS. n = 3–4 mice per time point. Error bars, s.e.m. (b) Adipose tissue from C57BL/6J mice on chow or HFD stained for caveolin, Ly6g and Cd11b. The merged Ly6g+Cd11b+ cells (yellow) indicated by white arrowheads are ATNs. Scale bars, 100 μm. (c) FACS analyses showing ATN content (ATNs as a percentage of SVCs) in 4-month-old C57BL/6J mice fed chow and 20-week-old mice fed HFD for 12 weeks. Error bars, s.e.m. *P < 0.05 by two-way analysis of variance (ANOVA) and Bonferroni's post test. (d) Neutrophil elastase mRNA in eWAT of DIO mice normalized to RNA PolII. (e) Neutrophil elastase activity in the eWAT of male C57BL/6J mice fed chow or 60% HFD for 12 weeks. Chow-fed mice (n = 5) and HFD-fed mice (n = 8). (f) GTT in 7-h–fasted C57BL/6J mice fed 60% HFD for 12 weeks that were orally administered vehicle or neutrophil elastase (NE) inhibitor (GW311616A) every day for 2 weeks. Vehicle-treated mice (n = 12) and inhibitor-treated mice (n = 8). (g) GTT in 5-month-old C57BL/6J mice fed normal chow and administered 1 mg kg−1 recombinant mouse neutrophil elastase for 7 d. n = 10 mice per group. (h) Intraperitoneal GTT (IP-GTT) on 7-h–fasted mice fed HFD for 10 weeks, using an intraperitoneal dose of 1 mg kg−1 glucose, in NEKO mice and in WT mice matched on weight or age. n = 8–10 mice per group. (i) ITT using 0.6 U kg−1 insulin injected intraperitoneally in weight-matched WT and NEKO mice fed HFD for 6 weeks. n = 8–10 mice per group. (j) FACS showing ATNs in WT and NEKO mice on HFD for 12 weeks (left). FACS showing ATNs in 12-week–HFD mice treated with vehicle or the neutrophil elastase inhibitor (GW311616A) for 2 weeks (right). *P < 0.05 by two-way ANOVA and Bonferroni's post test. Error bars (cj), s.e.m.

  2. A high degree of insulin sensitivity in NEKO mice.
    Figure 2: A high degree of insulin sensitivity in NEKO mice.

    Hyperinsulinemic-euglycemic clamp studies in WT and NEKO knockout mice fed HFD for 10–11 weeks. (ag) Glucose infusion rate (GIR; a), total glucose disposal rate (GDR; b), insulin-stimulated glucose disposal rate (Is-GDR; c), basal HGP (d), clamp HGP (e), percentage suppression of HGP (f) and FFAs in 6-h–fasted WT and NEKO mice (basal) and in the same mice at the end of the clamp procedure (clamp) (g). (h) Percentage suppression of lipolysis calculated from the data in g. *P < 0.05 by two-way ANOVA and Bonferroni's post test. Error bars (ah), s.e.m. (i) Western blot analysis of total Akt and pAkt in the liver and adipose tissue (eWAT) of WT and NEKO mice either before (–Ins) or after (+Ins; tissue harvested at 3 min for liver and at 7 min for eWAT) acute insulin injection.

  3. Neutrophils infiltrate the liver in HFD-fed mice and cause impaired insulin signaling by degradation of Irs1.
    Figure 3: Neutrophils infiltrate the liver in HFD-fed mice and cause impaired insulin signaling by degradation of Irs1.

    (a) IHC of liver sections obtained from 4-month-old chow-fed WT mice and 20-week-old WT (WT HFD) and NEKO (NEKO HFD) mice fed HFD for 12 weeks. Red indicates lipid content in hepatocytes stained with boron-dipyrromethene (BODIPY), blue indicates nuclei stained with DAPI, and green indicates neutrophils stained with Ly6g (1A8). Scale bars, 50 μm. (b) Neutrophil elastase activity in the livers of male C57BL/6J mice fed chow or an HFD for 12 weeks. Chow-fed mice (n = 5) and HFD-fed mice (n = 8). (c) Western blots for total Irs1 and heat shock protein 90 (Hsp90) as the loading control in WT and NEKO mice fed an HFD for 12 weeks. A densitometry analysis was performed and is represented below the blot. (d) The amount of liver Irs1 quantified by Meso Scale Discovery (MSD) analyses from fasted 4-month-old C57Bl6/J mice on chow diet. Mice were injected with saline or recombinant mouse neutrophil elastase. Tissues were harvested 2 h after neutrophil elastase injection. AU, arbitrary units. (e) Acute insulin response in 4-month-old C57BL/6J mice on chow treated with recombinant mouse neutrophil elastase. A submaximal insulin dose (0.1 U−1 kg) was used, and liver samples were obtained at basal (−Ins) and at the indicated times points (Ins 3′, 3 minutes after insulin injection; Ins 7′, 7 minutes after insulin injection). MSD was used to analyze the total amount of Akt and pAkt. (f) Western blots of protein harvested from primary mouse hepatocytes treated with recombinant mouse neutrophil elastase for 4 h. Irs1/actin refers to the amount of Irs1 protein normalized to the amount of Actin protein. (g) Amount of IRS1 in cryopreserved human hepatocytes treated with purified human neutrophil elastase for 6 h. Shown is a representative graph generated from data from one of at least three independent experiments. IRS1/Actin refers to the amount of Irs1 protein normalized to the amount of Actin protein. (h) Quantification of western blots of primary mouse hepatocytes treated with neutrophil elastase for 4 h and spiked with insulin for 5 min to obtain insulin induction. Shown is a representative graph generated from data from one of at least three independent experiments. (i) Quantification by MSD of total AKT and pAKT in protein harvested from primary human hepatocytes treated with human neutrophil elastase for 6 h and spiked with insulin for 5 min to obtain insulin induction. The graph was generated with data from at least two independent experiments performed in triplicate. *P < 0.05 by two-way ANOVA and Bonferroni's post test. (j) Representative glucose output assay in primary mouse hepatocytes from at least three independent experiments performed in duplicate or triplicate. *Significantly (P < 0.05) higher than basal, #significantly (P < 0.05) higher than insulin and glucagon by Student's t test. C.p.m., counts per million. Error bars (bj), s.e.m.

  4. NEKO mice have low inflammatory tone.
    Figure 4: NEKO mice have low inflammatory tone.

    (a) Quantitative PCR (qPCR) of the indicated genes from IP-macs harvested from WT and Tlr4 knockout mice treated with vehicle, LPS or recombinant mouse neutrophil elastase. *Significantly higher (P < 0.05) than all other treatment groups, #significantly lower (P < 0.05) than LPS-treated WT and significantly higher (P < 0.05) than all other treatment groups by Student's t test. (b,c) qPCR analysis of inflammatory gene expression in liver (b) and adipose tissue (c) of WT and NEKO mice on an HFD. Also shown are western blots of liver IκB and Hsp90 (b, inset) and adipose IκB and Hsp90 (c, inset). (d,e) Quantification of SVCs from eWAT of chow-fed WT, HFD-fed WT and NEKO mice stained for F4/80, Cd11b and Cd11c and analyzed by FACS. Cells that are triple positive for all three markers are referred to as ATM1 (d), and cells that are positive for F4/80 and Cd11b and negative for Cd11c are referred to as ATM2 (e). (f) Serum cytokines measured using the Millipore Luminex assay from WT and NEKO mice on an HFD. *P < 0.05 by Student's t test (c,f). (g) Glucose uptake in eWAT explants harvested from WT and NEKO mice and incubated ex vivo in the absence or presence of insulin followed by measurement of 2-DOG uptake. *P < 0.05 by two-way ANOVA and Bonferroni's post test (d,e and g). Error bars (ag), s.e.m.

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Author information

  1. These authors contributed equally to this work.

    • Saswata Talukdar &
    • Da Young Oh

Affiliations

  1. Department of Medicine, University of California, San Diego, La Jolla, California, USA.

    • Saswata Talukdar,
    • Da Young Oh,
    • Gautam Bandyopadhyay,
    • Jianfeng Xu,
    • Joanne McNelis,
    • Min Lu,
    • Pingping Li,
    • Jachelle Ofrecio,
    • Michael Lin &
    • Jerrold M Olefsky
  2. Pfizer, Cardiovascular, Metabolic and Endocrine Diseases (CVMED)–Diabetes Prevention and Remission, Cambridge, Massachusetts, USA.

    • Dongmei Li,
    • Qingyun Yan,
    • Yimin Zhu &
    • Martin B Brenner
  3. Present address: Pfizer, CVMED–Diabetes Prevention and Remission, Cambridge, Massachusetts, USA.

    • Saswata Talukdar

Contributions

S.T. and D.Y.O. designed and performed the experiments. S.T., D.Y.O. and J.M.O. analyzed and interpreted data and co-wrote the manuscript. All other authors performed experiments and contributed to discussions. This work was supported by grants to J.M.O., as detailed above.

Competing financial interests

The authors declare no competing financial interests.

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