1. Volume 25, Issue 5, Pages 1063-1074.e3 (May 2017)
Muramyl Dipeptide-Based Postbiotics Mitigate Obesity-Induced Insulin Resistance via IRF4 
Joseph F. Cavallari, Morgan D. Fullerton, Brittany M. Duggan, Kevin P. Foley, Emmanuel Denou, Brennan K. Smith, Eric M. Desjardins, Brandyn D. Henriksbo, Kalvin J. Kim, Brian R. Tuinema, Jennifer C. Stearns, David Prescott, Philip Rosenstiel, Brian K. Coombes, Gregory R. Steinberg, Jonathan D. Schertzer 
Cell Metabolism 
Volume 25, Issue 5, Pages e3 (May 2017)
DOI: /j.cmet
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2. Cell Metabolism 2017 25, 1063-1074.e3DOI: (10.1016/j.cmet.2017.03.021)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1 MDP Lowers Diet-Induced Insulin Resistance
(A and B) Experimental design for MDP administration at the inception of HFD (A) and after long-term HFD feeding (B).
(C) Glucose tolerance test (2 g/kg) of standard chow-fed WT mice treated with MDP for 5 weeks. n = 9–10 mice for each group.
(D and E) Glucose (2 g/kg; D) and insulin (1 IU/kg; E) tolerance tests of 60% HFD-fed WT male mice treated with MDP for 5 weeks. n = 8–10 mice for each group.
(F) Glucose tolerance test (1.5 g/kg) of 60% HFD-fed Nod2−/− mice treated with MDP for 5 weeks. n = 7–9 mice for each group.
(G and H) Glucose tolerance test (1 g/kg) of WT mice with obesity that was established using 45% HFD for 10 weeks; then mice were treated for 3 days with saline, 100 μg MDP (G), or 100 μg iE-DAP (γ-D-Glu-meso-diaminopimelic acid; H). n = 8–13 mice for each group.
(I) Peripheral tissue glucose disposal rate and hepatic glucose production during hyperinsulinemic-euglycemic clamps in WT 60% HFD-fed mice treated with MDP for 5 weeks. n = 5 mice for each group.
(J) Glucose tolerance test (1 g/kg) of mice lacking hepatocyte NOD2 (Nod2HKO) with obesity that was established using 45% HFD for 10 weeks, then treated for 3 days with MDP. n = 7 mice for each group.
∗Significantly different from control group determined by t test or one-way ANOVA, where appropriate. For glucose versus time graphs during glucose tolerance tests, asterisk (∗) indicates HFD-fed saline-injected mice versus HFD-fed and MDP-injected mice; ampersand (&) and pound sign (#) indicate that chow-fed values are significantly different from HFD-fed saline and MDP groups, respectively. Values shown are mean ± SEM. See also Figures S1 and S2.
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4. Figure 2
MDP Improves Glucose Tolerance during Obesity Independently of Diet or Microbiome Composition
(A) Glucose (0.75 g/kg) tolerance test of chow-fed 8-week-old ob/ob mice treated with MDP (4 times per week) for 2 weeks. n = 11 mice for each group.
(B) Glucose (2 g/kg) tolerance test of 60% HFD-fed WT male mice treated with MDP (4 times per week) for 2 weeks. n = 5–7 mice for each group.
(C) Principle coordinates analysis (PCoA) performed on Bray-Curtis distances in fecal samples of 60% HFD-fed mice treated with MDP for 3 weeks. Black circles represent samples from saline-treated mice, and red triangles represent samples from MDP-treated mice.
(D) Relative abundance of bacteria resolved to the phylum level in fecal samples of 60% HFD-fed mice treated with MDP for 3 weeks. Each bar represents an individual mouse.
(E) Relative abundance of bacteria resolved to the family level in fecal samples of 60% HFD-fed mice treated with MDP for 3 weeks. Each bar represents an individual mouse.
∗Significantly different from control group determined by t test or one-way ANOVA, where appropriate. Values shown are mean ± SEM. See also Figure S3.
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5. Figure 3
IRF4 Mediates the Anti-inflammatory and Insulin-Sensitizing Effects of MDP
(A and B) Irf4 expression in liver (A) and WAT (B) of 60% HFD-fed WT and Nod2−/− mice treated with MDP for 5 weeks. n = 7–10 mice for each group.
(C) Glucose tolerance (2 g/kg) test of 60% HFD-fed Irf4−/− mice treated with MDP for 5 weeks. n = 6 mice for each group.
(D) NF-κB activity in liver and WAT of 60% HFD-fed WT and Irf4−/− mice treated with MDP for 5 weeks. n = 9–10 for WT mice; n = 6 for Irf4−/− mice.
(E and F) Transcript expression levels of inflammatory (E) and immune cell (F) markers in WAT of WT and Irf4−/− 60% HFD-fed mice treated with MDP for 5 weeks. n = 9–10 for WT mice; n = 6 for Irf4−/− mice.
∗Significantly different from WT control group and #significantly different from Irf4−/− control group determined by t test. Values shown are mean ± SEM. See also Figure S4.
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6. Figure 4 MDP Increases Hepatic Insulin Sensitivity during Endotoxemia
(A) Glucose tolerance test (2 g/kg) of 60% HFD-fed C3H/HeJ mice treated with MDP for 5 weeks. n = 12–13 mice for each group.
(B) Experimental design for MDP administration during endotoxemia in chow-fed mice.
(C–E) Glucose tolerance tests (2 g/kg) of WT (C), Nod2−/− (D), and Nlrp3−/− (E) mice treated with MDP for 3 days and challenged acutely with LPS. n = 8–10 mice for each group.
(F) Peripheral tissue glucose disposal rate and hepatic glucose production during hyperinsulinemic-euglycemic clamps in WT chow-fed mice treated with MDP for 3 days and challenged acutely with LPS. n = 3–4 mice for each group.
(G) Glucose tolerance test (2 g/kg) of mice lacking hepatocyte NOD2 (Nod2HKO) treated with MDP for 3 days and challenged acutely with LPS. n = 5–6 mice for each group.
∗Significantly different from control group determined by t test. Values shown are mean ± SEM. See also Figure S5.
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7. Figure 5
IRF4 Dictates Peptidoglycan-Specific Effects on Insulin Sensitivity during Endotoxemia
(A) Glucose tolerance test (2 g/kg) of Irf4−/− mice treated with MDP for 3 days and challenged acutely with LPS. n = 12–16 mice for each group.
(B) HOMA-IR of WT and Irf4−/− mice treated with MDP for 3 days and acutely challenged with LPS. n = 8–11 for each group.
(C–E) Glucose tolerance tests (2 g/kg) of WT (C), Nod1−/− (D), and Irf4−/− (E) mice treated with FK565 for 3 days and challenged acutely with LPS. n = 9–10 mice for each WT group. n = 8 mice for each Nod1−/− group. n = 12–16 mice for each Irf4−/− group.
∗Significantly different from control group determined by t test. Values shown are mean ± SEM. See also Figure S6.
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8. Figure 6 The Orphan Drug Mifamurtide Is a Potent Insulin Sensitizer
(A and B) Glucose tolerance tests (2 g/kg) of WT mice treated with multiple MDP doses (0, 50, and 100 μg; A) or 0 versus 20 μg MDP (B) for 3 days and challenged acutely with LPS. n = 9–16 mice for each group. Within line graphs, asterisk (∗) indicates that values are significantly different (saline control versus 50 μg MDP). Pound sign (#) indicates that values are significantly different (saline control versus 100 μg MDP). Ampersand (&) indicates that values are significantly different (50 μg MDP versus 100 μg MDP).
(C) Glucose tolerance test (2 g/kg) of WT mice treated with one injection of the NOD2 activator M-TriLYS (100 μg) and challenged acutely with LPS. n = 10 mice for each group.
(D) Glucose tolerance test (2 g/kg) of WT mice treated with a low dose of mifamurtide (50 μg/mouse, which is the molar equivalent to 20 μg MDP) for 3 days and challenged acutely with LPS. n = 9–10 mice for each group.
(E) Glucose tolerance test (2 g/kg) of Nod2−/− mice treated with mifamurtide for 3 days and challenged acutely with LPS. n = 5 mice for each group.
(F–H) Glucose tolerance test (1.5 g/kg; F), body mass (G), and expression of Irf4 (H) in whole WAT versus adipocytes versus stromal vascular fraction of WT 60% HFD-fed mice treated with a low dose of mifamurtide for 5 weeks. n = 10–12 mice for each group.
∗Significantly different from control group determined by t test or one-way ANOVA, where appropriate. For glucose versus time graphs during glucose tolerance tests, asterisk (∗) indicates that HFD-fed saline-injected mice are different from MDP-injected mice; pound sign (#) indicates HFD-fed saline-injected mice are different from mifamurtide-injected mice; ampersand (&) indicates HFD-fed MDP-injected mice are different from mifamurtide-injected mice. Values shown are mean ± SEM.
Cell Metabolism  , e3DOI: ( /j.cmet )
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