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Science 339 (6116): 218-222

Copyright © 2013 by the American Association for the Advancement of Science

JNK Expression by Macrophages Promotes Obesity-Induced Insulin Resistance and Inflammation

Myoung Sook Han1,2, Dae Young Jung2, Caroline Morel1,2, Saquib A. Lakhani3,*, Jason K. Kim2,4, Richard A. Flavell3, and Roger J. Davis1,2,{dagger}

1 Howard Hughes Medical Institute, Worcester, MA 01605, USA.
2 Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
3 Howard Hughes Medical Institute and Department of Immunobiology, Yale University School of Medicine, New Haven, CN 06520, USA.
4 Department of Medicine, Division of Endocrinology, Metabolism and Diabetes, University of Massachusetts Medical School, Worcester, MA 01605, USA.


Figure 1
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  		Fig. 1. Macrophage JNK promotes the establishment of obesity-induced insulin resistance. (A) Insulin tolerance tests (ITT) were performed on chow diet and HFD-fed {Phi}WT and {Phi}KO mice (4 weeks) by means of intraperitoneal injection of insulin (0.75 U/kg) and measurement of blood glucose concentration (mean ± SEM; n = 7 ~ 10 mice). (B) Glucose tolerance tests (GTT) were performed by means of intraperitoneal injection of glucose (1 g/kg) and measurement of blood glucose concentration (mean ± SEM; n = 7 ~ 10 mice). (C to E) The blood concentration of glucose and insulin in overnight-fasted mice and the blood glucose concentration in fed mice were measured (mean ± SEM; n = 10 mice). (F to J) Insulin sensitivity was measured by using a hyperinsulinemic-euglycemic clamp in conscious mice. The steady-state glucose infusion rate (GIR), hepatic insulin action (HIA), clamp hepatic glucose production (HGP), whole-body glucose turnover, and whole-body glycogen plus lipid synthesis are presented (mean ± SEM; n = 8 ~ 10 mice). (K) Chow-fed (ND) and HFD-fed mice (4 weeks) were fasted overnight and then treated by means of intraperitoneal injection with 1 U/kg of insulin (15 min). Multiplexed enzyme-linked immunosorbent assay was used to detect Akt and activated (pSer473) Akt in the liver, epididymal adipose tissue (WAT), and gastrocnemius muscle (mean ± SEM; n = 3 ~ 5 mice). Representative tissue samples were also examined by means of immunoblot analysis by probing with antibodies to phospho-Akt, Akt, and αTubulin (Tub.). Data [(A) to (E)] are representative of three experiments. Data were pooled from [(F) to (J)] eight to ten and (K) three to five experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as determined with [(A) and (B)] Student’s t test or [(C) to (K)] analysis of variance (ANOVA), with Bonferroni’s posttest correction for multiple comparisons.

 

Figure 2
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  		Fig. 2. Macrophage JNK promotes pancreatic islet dysfunction. (A and B) The morphology of pancreatic islets was examined by using chow-fed (ND) and HFD-fed mice (4 weeks) fasted overnight. Sections were stained with antibodies to insulin, glucagon, or F4/80. DNA was stained with 4',6-diamidino-2-phenylindole (blue). Scale bar, 75 μm. No significant differences between {Phi}WT and {Phi}KO islet infiltration by F4/80+ macrophages were detected (fig. S19). (C) The islet area per section is presented (mean ± SEM; n = 7 ~ 10 mice). (D) The number of β cells per islet that stained with an antibody to the proliferation marker proliferating cell nuclear antigen (PCNA) is presented (mean ± SEM; n = 5 ~ 7 mice). (E) Glucose-induced insulin release measurements were performed by means of intraperitoneal injection of glucose (2 g/kg) and measurement of blood insulin concentration (mean ± SEM; n = 8 ~ 10 mice). (F) Glucose-induced insulin secretion in vitro. Isolated islets were incubated (1 hour) with low glucose (3.3 mM) or high glucose (16.7 mM). Insulin secretion was measured (mean ± SEM; n = 5 mice). Data [(A) and (B)] are representative of [(A) and (B)] five to ten, (E) three, and (F) two experiments. Data [(C) and (D)] were pooled from five to ten experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as determined with (E) Student’s t test or [(C), (D), and (F)] ANOVA, with Bonferroni’s posttest correction for multiple comparisons.

 

Figure 3
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  		Fig. 3. JNK promotes M1 polarization of adipose tissue macrophages. (A to C) The stromal vascular fraction (SVF) of epididymal adipose tissue was isolated from chow-fed and HFD-fed (4 weeks) mice and examined by means of flow cytometry to detect the total number of F4/80+ ATMs, the number of F4/80+CD11c+CD206– (M1 ATMs), and the number of F4/80+CD11c–CD206+ (M2 ATMs) (mean ± SEM; n = 5 mice). (D) Total RNA was isolated from epididymal adipose tissue from chow-fed and HFD-fed {Phi}WT and {Phi}KO mice. The relative expression of mRNA associated with M1-polarized macrophages and M2-polarized macrophages was measured by means of quantitative reverse transcription polymerase chain reaction (RT-PCR) assays (mean ± SEM; n = 8 ~ 10 mice). Data [(A) to (C)] are representative of three experiments. Data (D) were pooled from eight to ten experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as determined with ANOVA, with Bonferroni’s posttest correction for multiple comparisons.

 

Figure 4
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  		Fig. 4. JNK promotes M1 polarization of macrophages in vitro. (A) BMDMs from {Phi}WT and {Phi}KO mice were incubated without or with 100 ng/ml of IFN-{gamma} (36 hours). F4/80+ cells were stained with an antibody to the M1 marker CD11c and examined by means of flow cytometry (mean relative fluorescence intensity ± SEM; n = 3 independent experiments). (B) Total RNA was isolated from BMDMs incubated (8 hours) with 100 ng/ml of IFN-{gamma}. The relative expression of the indicated M1 marker genes was measured by means of quantitative RT-PCR assays (mean ± SEM; n = 5 mice). (C) Chow-fed {Phi}WT and {Phi}KO mice were fasted overnight. Blood was collected from the mice 2.5 hours after intraperitoneal injection of LPS (20 mg/kg) or solvent (Control). The blood concentration of CCL2, CCL5, IL1β, IL6, and TNFα was measured (mean ± SEM; n = 10 mice). Data are representative of (A) three and (C) two experiments. Data (B) were pooled from five experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as determined with ANOVA, with Bonferroni’s posttest correction for multiple comparisons.

 

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