Protectin DX alleviates insulin resistance by activating a myokine-liver glucoregulatory axis

Journal name:
Nature Medicine
Volume:
20,
Pages:
664–669
Year published:
DOI:
doi:10.1038/nm.3549
Received
Accepted
Published online

We previously demonstrated that low biosynthesis of ω–3 fatty acid–derived proresolution mediators, termed protectins, is associated with an impaired global resolution capacity, inflammation and insulin resistance in obese high-fat diet–fed mice1. These findings prompted a more direct study of the therapeutic potential of protectins for the treatment of metabolic disorders. Herein we show that protectin DX (PDX) exerts an unanticipated glucoregulatory activity that is distinct from its anti-inflammatory actions. We found that PDX selectively stimulated the release of the prototypic myokine interleukin-6 (IL-6) from skeletal muscle and thereby initiated a myokine-liver signaling axis, which blunted hepatic glucose production via signal transducer and activator of transcription 3 (STAT3)-mediated transcriptional suppression of the gluconeogenic program. These effects of PDX were abrogated in Il6-null mice. PDX also activated AMP-activated protein kinase (AMPK); however, it did so in an IL-6–independent manner. Notably, we demonstrated that administration of PDX to obese diabetic db/db mice raises skeletal muscle IL-6 levels and substantially improves their insulin sensitivity without any impact on adipose tissue inflammation. Our findings thus support the development of PDX-based selective muscle IL-6 secretagogues as a new class of therapy for the treatment of insulin resistance and type 2 diabetes.

At a glance

Figures

  1. PDX prevents lipid-induced insulin resistance.
    Figure 1: PDX prevents lipid-induced insulin resistance.

    (a) Preclamp glycemia (left) and clamp glucose excursion (right) in vehicle-treated, saline-infused (Saline), vehicle-treated, lipid-infused (Lipid) and PDX-treated, lipid-infused (Lipid + PDX) mice. (b) GIR (mg per kg body weight per minute) (left) and mean GIR for the final 60 min of clamp (right) in Saline, Lipid and Lipid + PDX mice. (c) Peripheral insulin action expressed as fold increase in rate of disappearance (Rd) during the clamp (left) and hepatic insulin action expressed as percentage suppression of hepatic glucose production (HGP, right) in Saline, Lipid and Lipid + PDX mice. (d) Immunoblots for phosphorylated Akt (pAkt) Ser473, total Akt and iNOS in gastrocnemius muscle of clamped Saline, Lipid and Lipid + PDX mice. Dashed lines indicate that lanes were run on the same gel but were noncontiguous. Quantification of densitometry analyses is shown to the right of the representative gels. (e) Immunoblots for pAkt Ser473, total Akt, iNOS, pJNK Thr183/Tyr185 and total JNK in liver of clamped Saline, Lipid and Lipid + PDX mice. Dashed lines indicate that lanes were run on the same gel but were noncontiguous. Quantification of densitometry analyses are shown to the right of the representative gels. AU, arbitrary units; ND, not detected. (f) Chemokines and cytokines in plasma from clamped Saline, Lipid and Lipid + PDX mice. Data are mean ± s.e.m., representative of 6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 calculated using analysis of variance (ANOVA).

  2. PDX stimulates skeletal muscle IL-6 expression.
    Figure 2: PDX stimulates skeletal muscle IL-6 expression.

    (a) Skeletal muscle IL-6 protein expression in gastrocnemius from clamped Saline, Lipid and Lipid + PDX mice. Data are mean ± s.e.m., representative of 6 mice per group. Fold increase in IL-6 mRNA expression over vehicle normalized to Gapdh and fold increase in IL-6 protein secretion in medium over vehicle (b) and immunoblots for pAMPK Thr172 and total AMPK (c) in C2C12 myotubes treated with PDX for 2 h compared to ethanol vehicle (Veh) control. Quantification of densitometry analyses for immunoblots is shown below the representative gels. Data are mean ± s.e.m. for three independent experiments. (d) Immunoblots for pSTAT3 Ser727 and total STAT3 in liver from clamped Saline, Lipid and Lipid + PDX mice (n = 6 mice per group). Dashed lines indicate that lanes were run on the same gel but were noncontiguous. Quantification of densitometry analyses for immunoblots is shown below the representative gels. (e) Relative mRNA expression of Ppargc1a, Pck1 and G6pc normalized to Gapdh in liver from clamped Saline, Lipid and Lipid + PDX mice (n = 6 mice per group). (f) Immunoblots for pAMPK Thr172 and total AMPK in gastrocnemius muscle from clamped Saline (n = 6), Lipid (n = 4) and Lipid + PDX (n = 5) mice. Dashed lines indicate that lanes were run on the same gel but were noncontiguous. Quantification of densitometry analyses for immunoblots is shown below the representative gels. Data are mean ± s.e.m. *P < 0.05, **P < 0.01 calculated using ANOVA.

  3. IL-6 is required for the insulin-sensitizing actions of PDX.
    Figure 3: IL-6 is required for the insulin-sensitizing actions of PDX.

    (a) Preclamp glycemia (left) and clamp glucose excursion (right) in WT or Il6-knockout (KO) lipid-infused (L) mice treated with PDX (P) or vehicle (V). (b) GIR (left) and mean GIR for the final 60 min of clamp (right) in WT or Il6-knockout lipid-infused mice treated with PDX or vehicle. (c) Peripheral insulin action expressed as fold increase in Rd during the clamp (left) and hepatic insulin action expressed as percentage suppression of hepatic glucose production (right) in WT or Il6-knockout lipid-infused mice treated with PDX or vehicle. (df) Skeletal muscle IL-6 content (d), immunoblots for pSTAT3 Ser727 and total STAT3 (e) and relative mRNA expression of Ppargc1a, Pck1 and G6pc normalized to Gapdh (f) in liver from clamped WT or Il6-knockout lipid-infused mice treated with PDX or vehicle. Dashed lines indicate that lanes were run on the same gel but were noncontiguous. Quantification of densitometry analyses for immunoblots is shown below the representative gels. (g) Immunoblots for pAMPK Thr172 and total AMPK in gastrocnemius muscle from clamped WT or Il6-knockout lipid-infused mice treated with PDX or vehicle. Dashed lines indicate that lanes were run on the same gel but were noncontiguous. Quantification of densitometry analyses for immunoblots is shown below the representative gels. WT-LV, n = 6 mice; WT-LP, n = 5 mice; KO-LV, n = 5 mice; KO-LP, n = 6 mice. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 calculated using ANOVA.

  4. PDX therapy improves insulin sensitivity in diabetic mice.
    Figure 4: PDX therapy improves insulin sensitivity in diabetic mice.

    (a) Preclamp glycemia (left) and clamp glucose excursion (right) in genetically obese diabetic db/db mice treated with vehicle (Veh) or treated acutely with PDX (PDX). (b) GIR (left) and mean GIR for the final 60 min of clamp (right) in vehicle- and PDX-treated db/db mice. (c) Relative mRNA expression of Ppargc1a, Pck1 and G6pc normalized to Gapdh in liver from clamped vehicle- and PDX-treated db/db mice. (d) Chemokines and cytokines in epididymal white adipose tissue (eWAT) clamped vehicle- and PDX-treated db/db mice. (e) Skeletal muscle (left) and plasma (right) IL-6 content from clamped vehicle- and PDX-treated db/db mice. Data are mean ± s.e.m. Veh, n = 3; PDX n = 4. *P < 0.05, **P < 0.01 calculated using Student's t-test.

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Author information

Affiliations

  1. Department of Medicine, Québec Heart and Lung Institute, Laval University, Québec, Québec, Canada.

    • Phillip J White,
    • Philippe St-Pierre,
    • Alexandre Charbonneau,
    • Patricia L Mitchell,
    • Emmanuelle St-Amand,
    • Bruno Marcotte &
    • André Marette
  2. Institute of Nutrition and Functional Foods, Laval University, Québec, Québec, Canada.

    • Phillip J White,
    • Philippe St-Pierre,
    • Alexandre Charbonneau,
    • Patricia L Mitchell,
    • Emmanuelle St-Amand,
    • Bruno Marcotte &
    • André Marette

Contributions

P.J.W. and A.M. conceived of the study and wrote the manuscript. P.J.W., P.S.-P., A.C., P.L.M. and E.S.-A. performed mouse experiments. P.J.W., P.L.M., B.M. and E.S.-A. performed cell culture experiments. P.J.W., P.L.M., E.S.-A. and B.M. conducted ELISA and multiplex analyses. P.J.W. and P.L.M. carried out western blotting and PCR. All authors analyzed and discussed data and commented on the manuscript.

Competing financial interests

A.M. and P.J.W. have filed a patent application (PCT/CA2014/000047) with the Canadian Patent Office describing a method and use for the stimulation of muscular IL-6 secretion.

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