Inflammation Is Necessary for Long-Term but Not Short-Term High-Fat Diet–Induced Insulin Resistance
Article Figures & Tables
Figures
- FIG. 1.
In 3 days of HFD, body weight, fat mass, and adipocyte size are increased significantly. Eight-week-old C57BL6J male mice were fed NCD until they were subjected to 60% HFD. HFD was treated to the mice 0 (NCD), 3 days (3 D), 1 week (1 W), 2 weeks (2 W), 5 weeks (5 W), or 10 weeks (10 W) before death. At the age of 18 weeks, all mice were killed and subjected to several analyses. A: Body weight of the mice at the end of the experiment. n = 8 at each time point. Increase of body weight started to be observed by 3 days of HFD. Body weight of mice with 5-week HFD and 10-week HFD was not significantly different. **P < 0.05. B: Epididymal (Epid.) fat mass. n = 8 at each time point. **P < 0.05; ***P < 0.001. C: Histology analysis of epididymal adipose tissue. The epididymal adipose tissues were subjected to hematoxylin-eosin staining. n = 4 at each time point. D and E: Further qualitative changes of adipocyte size and morphology by short-term HFD were assessed by histology analysis of whole-mount epididymal adipose tissue. D: BODIPY (boron-dipyrromethene) staining of whole-mount adipose tissue from the mice treated with NCD or HFD. Adipocyte size was markedly increased by 3 days of HFD. n = 6. Signal intensity of BODIPY staining was adjusted for optimal measurement of adipocyte size using ImageJ software. E: Distribution of adipocyte size in epididymal adipose tissue from mice treated with NCD or HFD. (A high-quality digital representation of this figure is available in the online issue.)
- FIG. 2.
HFD induces systemic insulin resistance in 3 days. A: C57BL/6J male mice were treated with NCD or HFD and subjected to oral glucose (Glc) tolerance test. n = 8 at each time point. ***P < 0.001 NCD vs. 3-day HFD. B: Blood insulin levels at time 0 and 10 min after glucose injection. Basal insulin level was significantly elevated by 3 days of HFD and then further increase was detected significantly by 5 and 10 weeks of HFD. n = 8. *P < 0.05 NCD vs. 3-day HFD; ***P < 0.001 NCD vs. 3-day HFD; †P < 0.05 3-day HFD vs. 10-week HFD; ††P < 0.01 3-day HFD vs. 10-week HFD. C–H: Hyperinsulinemic-euglycemic clamp was performed in mice treated with HFD for different periods; n = 8 (NCD, and 3-day and 1-week HFD mice) or n = 6 (10-week HFD mice). C: Basal blood glucose level (before clamp). **P < 0.01. D: Basal hepatic glucose production. *P < 0.05; **P < 0.01. E: GIR. **P < 0.01. F: ISGDR. **P < 0.01. G: Insulin-dependent suppression of hepatic glucose. **P < 0.01. H: Suppression of plasma FFA level by insulin. *P < 0.05; **P < 0.01.
- FIG. 3.
Macrophage (MP) infiltration and polarization increase in 3 days of HFD. Mice were treated with NCD (N) or HFD for a series of periods as indicated: 3D, 3 days; 2W, 2 weeks; 10W, 10 weeks. After 10 weeks from when first mice were fed an HFD, mice were killed and epididymal adipose tissues were taken for further analyses. A: Flow cytometry analyses of immune cells in stromal vascular fraction of adipose tissue. #P < 0.05; ##P < 0.01; ###P < 0.001; n = 4 (NCD and 2-week HFD mice) or n = 6 (1-week and 10-week HFD mice). B: Immunohistochemistry analysis of epididymal adipose tissue. The epididymal adipose tissues were stained with antibodies against MAC2 and then subjected to hematoxylin-eosin staining. n = 4. C: Whole-mount immunohistochemistry analysis of epididymal adipose tissue from NCD or 3- or 7-day HFD-treated mice. Samples were stained with DAPI (blue) or antibodies against CD11b (red) and CD11c (green) and visualized by multiphoton confocal microscopy. Scale bar denotes 50 μm. (A high-quality digital representation of this figure is available in the online issue.)
- FIG. 4.
Inflammatory gene expression is induced in 3 days after HFD. C57BL/6J mice were treated with an HFD. Mice were fed an HFD for 0 (0 or N [NCD]), 1 (1), 3 (3), or 7 days (7), or 16 weeks (H). mRNA levels of inflammatory genes from epididymal adipose tissue (WAT), brown adipose tissue (BAT), liver, and skeletal muscle were measured by quantitative real-time RT-PCR analysis. n = 10 at each time point. *P < 0.05; **P < 0.01; ***P < 0.001. iNOS, inducible nitric oxide synthase; SAA3, serum amyloid A3.
- FIG. 5.
Adipocytes become more chemoattractive to macrophages after 3 days of HFD. A: Macrophage migration assays using primary ACM from mice treated with NCD (N) or HFD. ACM were obtained by incubating mouse primary adipocytes in serum-free Dulbecco’s modified Eagle’s medium for 12 h. 3d, 3 days; 1w, 1 week. B: MCP-1 protein expression from the primary adipocytes. C: MCP-1 protein secretion from those adipocytes. *P < 0.05; **P < 0.01.
- FIG. 6.
Rag1 KO mice develop insulin resistance with increased macrophage activation in 1 week of HFD. A: Macrophage infiltration and M1 polarization were increased by short-term HFD. Rag1 KO mice were treated with HFD for 1 week and killed, and epididymal adipose tissues were taken for further analyses. Total macrophages (double positive; F4/80+, CD11b+) and CD11c+ macrophages (triple positive cells; F4/80+, CD11b+, CD11c+) were increased in adipose tissue in 1 week of HFD. *P < 0.05 KO-NCD vs. KO HFD; ***P < 0.001 KO-NCD vs. KO HFD; #P < 0.05 wild-type (WT)-NCD vs. WT HFD; ##P < 0.01 WT-NCD vs. WT HFD. n = 6. B: Epididymal (Epid.) adipose tissue mass in NCD and 1-week HFD-treated Rag1 KO mice. C: Expression of inflammatory genes was promoted in 1-week HFD-fed Rag1 KO mice. mRNA levels of inflammatory genes from epididymal adipose tissue were measured by quantitative real-time RT-PCR analysis. D–F: HFD rapidly induces systemic insulin resistance in Rag1 KO mice. D: Rag1 KO mice (8 weeks old, male) were treated with NCD or HFD for 3 days (3d) (i) or 1 week (1w) (ii) and subjected to oral glucose (Glc) tolerance test. E: Serum insulin levels of Rag1 KO mice treated with NCD or HFD (for 1 week) during oral glucose tolerance. 0 min, before glucose injection; 10 min, 10 min after glucose injection. F: WT or Rag1 KO mice were treated with NCD or HFD for 1 week and subjected to insulin tolerance test with intraperitoneal injection of insulin (IP-ITT). Blood glucose levels were measured at 0, 30, 60, 90, and 120 min after insulin (0.4 unit/kg) injection. G: Glucose-uptake assays using primary adipocytes from mice treated with NCD or HFD for 1 week. iNOS, inducible nitric oxide synthase; SAA3, serum amyloid A3. *P < 0.05; **P < 0.01; ***P < 0.001.
- FIG. 7.
Macrophage depletion using clodronate-liposomes does not affect short-term HFD-dependent adipocyte insulin resistance. Mice were treated with NCD or 60% HFD in the presence or absence of clodronate injection. Mice were given clodronate injection (100 mg/kg i.p.) 3 days before HFD, which was followed by a second and third injection every 3 days (at days 0 and 3). A: Flow cytometry analysis of CD11c+ macrophages in adipose tissue. V, vehicle (empty liposomes); C, clodronate-liposomes. B: Whole-mount immunohistochemistry analysis of epididymal adipose tissue. Veh, vehicle; Clod, clodronate-liposomes. C: Cytokine concentration in the mouse sera injected with vehicle and clodronate. W, week. *P < 0.05. D: Glucose-uptake assays using primary adipocytes from mice treated with NCD or HFD for 1 week with or without clodronate injection. White columns denote relative glucose uptake activity without insulin. Black columns denote relative glucose uptake activity with insulin. Cld, clodronate-liposomes. E: Oral glucose tolerance test (OGTT) of mice treated with HFD for 3 days. #P < 0.05 Veh-NCD vs. Veh-HFD; *P < 0.05 Cld-NCD vs. Cld-HFD; **P < 0.01 Cld-NCD vs. Cld-HFD; ***P < 0.001 Cld-NCD vs. Cld-HFD. n = 6. F: Glucose tolerance test of mice treated with HFD for 7 days. #P < 0.05 Veh-NCD vs. Veh-HFD; ##P < 0.01 Veh-NCD vs. Veh-HFD; ###P < 0.001 Veh-NCD vs. Veh-HFD; *P < 0.05 Cld-NCD vs. Cld-HFD; **P < 0.01 Cld-NCD vs. Cld-HFD. n = 6. G: Homeostasis model assessment of insulin resistance (HOMA-IR). H–J: Hematopoietic cell-specific JNK deficiency does not affect short-term HFD-induced insulin resistance. For the generation of radiation chimera, C57BL/6J mice received a lethal dose of 10 Gy of ionizing radiation, followed by tail vein injection of 107 bone marrow cells either from wild-type (WT) or JNK KO mice. After 6 weeks of bone marrow reconstitution, mice were subjected to 3 or 7 days of HFD, followed by hyperinsulinemic-euglycemic clamp. n = 4 to 5. H: GIR. I: ISGDR. J: Insulin-dependent hepatic glucose suppression. BMT, bone marrow transplantation. (A high-quality digital representation of this figure is available in the online issue.)
- FIG. 8.
Lipid contents of liver and skeletal muscle after 3 days of HFD. Eighteen-week-old C57BL6J male chow-fed mice were started in 60% HFD at day 0 for 3 days before the analyses. A separate group of age-matched chow-fed mice was studied as controls. For the macrophage depletion experiment, two shots of clodronate injection (100 mg/kg i.p.) were given at day −3 and 0. Veh, vehicle; Clod, clodronate-liposomes; TG, triacylglyceride; NS, not significant. A: Lipid contents in liver. B: Lipid contents in skeletal muscle. n = 8. *P < 0.05; **P < 0.01.
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