Intermedin Restores Hyperhomocysteinemia-induced Macrophage Polarization and Improves Insulin Resistance in Mice

Abstract

Hyperhomocysteinemia (HHcy) is a condition characterized by an abnormally high level of homocysteine, an inflammatory factor. This condition has been suggested to promote insulin resistance. To date, the underlying molecular mechanism remains largely unknown, and identifying novel therapeutic targets for HHcy-induced insulin resistance is of high priority. It is well known that intermedin (IMD), a calcitonin family peptide, exerts potent anti-inflammatory effects. In this study, the effects of IMD on HHcy-induced insulin resistance were investigated. Glucose tolerance and insulin tolerance tests were performed on mice treated with IMD by minipump implantation (318 ng/kg/h for 4 weeks) or adipocyte-specific IMD overexpression mice (Adipo-IMD transgenic mice). The expression of genes and proteins related to M1/M2 macrophages and endoplasmic reticulum stress (ERS) was evaluated in adipose tissues or cells. The expression of IMD was identified to be lower in the plasma and adipose tissues of HHcy mice. In both IMD treatment by minipump implantation and Adipo-IMD transgenic mice, IMD reversed HHcy-induced insulin resistance, as revealed by glucose tolerance and insulin tolerance tests. Further mechanistic study revealed that IMD reversed the Hcy-elevated ratio of M1/M2 macrophages by inhibiting AMP-activated protein kinase activity. Adipo-IMD transgenic mice displayed reduced ERS and lower inflammation in adipose tissues with HHcy. Soluble factors from Hcy-treated macrophages induced adipocyte ERS, which was reversed by IMD treatment. These findings revealed that IMD treatment restores the M1/M2 balance, inhibits chronic inflammation in adipose tissues, and improves systemic insulin sensitivity of HHcy mice.

Keywords: AMP-activated kinase (AMPK); adipose tissue; homocysteine; insulin resistance; macrophage.

FIGURE 1.

IMD expression was inhibited in HHcy mice. A, plasma samples were collected from HHcy or age-matched control (Ctl) mice for an IMD ELISA assay (n = 3 for controls; n = 4 for HHcy). B, mRNA expression of IMD was detected in eWAT, livers, and skeletal muscles of HHcy and control mice by real-time PCR (n = 4). C, Western blotting analysis of IMD expression in eWAT. Quantitative analysis is shown in the right panel (n = 3/group). N.D., not detected; *, p < 0.05; **, p < 0.01.

FIGURE 2.

IMD improved HHcy-induced insulin resistance in adipose tissues. A, mRNA expression of IMD in eWAT, livers, and skeletal muscles (SK) of Adipo-IMD-tg and control (Ctl) mice (n = 4). B, protein expression of IMD in eWAT (top panel) and peritoneal macrophages (bottom panel) by Western blotting analysis. C, body weights of controls and Adipo-IMD-tg mice (n = 6, 8/group). D, MRI analysis of fat in Adipo-IMD-tg mice. E, H&E staining of eWAT from control and Adipo-IMD-tg mice (n = 4 for controls; n = 5 for Adipo-IMD-tg mice). F, GTT and ITT were examined in Adipo-IMD-tg mice (n = 4–5/group). G, fasting glucose, fasting serum insulin levels, and HOMA index of controls or Adipo-IMD-tg mice with HHcy (n = 4 and n = 5 mice for each group, respectively). H, phosphorylated AKT (Ser-473) was detected in eWAT. The expression of pAKT was quantified by total AKT, as shown in the right panel (n = 3/group). N.D., not detected; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 3.

Adipo-IMD-tg mice displayed declined M1 but an enhanced M2 phenotype. A and B, eWAT (A) or subcutaneous white adipose tissues (B) were digested, and a stromal vascular fraction was collected for the flow cytometry assay. Gated on 7AAD-F4/80+ cells, CD11c and CD206 expression was analyzed (n = 3–6/group). Ctl, control. C, the mRNA expression of M1 markers (Cd11c, Il-12, and inos) and M2 markers (Cd206, Il-10, and Arg1) was examined in eWAT of Adipo-IMD-tg and control mice (n = 4). *, p < 0.05; **, p < 0.01.

FIGURE 4.

IMD ameliorated the Hcy-induced M1/M2 imbalance in vitro. A–D, peritoneal macrophages were treated with Hcy, IMD, or Hcy + IMD. Quantitative PCR showed the expression of Cd11c and Cd206 (A), Il-12 and Il-10 (B), inos (C), and Arg1 (D) for differently treated macrophages (n = 4). Ctl, control. E, the protein expression of iNOS and Arg 1 was detected by Western blotting assay. Center panel, quantitative data of iNOS expression (n = 2). Bottom panel, quantitative data of Arg1 expression (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

IMD reversed the Hcy-induced M1/M2 imbalance through the AMPK pathway. A, after being treated with Hcy, IMD, or Hcy + IMD, peritoneal macrophage cells were analyzed for the expression of phosphorylated AMPKα. Quantitative data, shown in the right panel, are from three independent experiments. Ctl, control. B, the expression of AMPK was knocked down by siRNA. The knockdown efficiency was verified by real-time PCR. C and D, Arg1 (C) and iNOS (D) expression was detected by Western blotting assay. Quantitative data in the bottom panels are results from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 6.

IMD reversed HHcy-induced ER stress and inflammation. A, eWAT from control (Ctl) or Adipo-IMD-tg mice were collected for pPERK, PERK, p-eIF2α, eIF2α, and ATF6 Western blotting assays. B, quantitative analysis for pPERK, p-eIF2α, and ATF6. C, eWAT from control or Adipo-IMD-tg mice were analyzed for p-JNK, t-JNK, and p-P65 expression. Total protein extract was used for p-JNK and t-JNK detection, and nuclear protein extract was used for p-P65 detection. D, quantitative analysis for p-JNK and p-P65 (n = 4/group). *, p < 0.05; **, p < 0.01.

FIGURE 7.

Macrophages were the mediators of the IMD/Hcy effect on ER stress and inflammation in adipocytes. A and B, peritoneal macrophage cells were treated with Hcy, IMD, or Hcy + IMD (H+I), and the cell culture supernatants were added to the culture of differentiated 3T3-L1 adipocytes. pPERK, PERK, p-eIF2α, eIF2α, and ATF6 (A) and p-JNK (B) were detected in the treated 3T3-L1 adipocytes. Quantitative data in the bottom panels are results from three independent experiments. *, p < 0.05; **, p < 0.01. Ctl, control.

FIGURE 8.

IMD treatment improved Hcy-induced insulin resistance. Osmotic minipumps containing IMD were implanted subcutaneously in HHcy mice. A, GTT and ITT were examined in IMD infusion mice (n = 7 for controls (Ctl); n = 6 for the IMD group). B, the body weights of controls and IMD infusion mice after 4 weeks of Hcy feeding (n = 7 for controls; n = 6 for the IMD group). C, fasted glucose, fasted serum insulin levels, and HOMA index of controls or IMD infusion mice with HHcy (n = 5/group). D, phosphorylated AKT (Ser-473) levels were detected in eWAT. The expression of pAKT was quantified by total AKT, shown in the right panel (n = 2/group). E, IMD restored the Hcy-induced M1/M2 imbalance through activation of the AMPK pathway. M1/M2 imbalance promotes ER stress and chronic inflammation in adipose tissues. IMD reversed Hcy-induced ER stress and inflammation in adipocytes as a result of improvement in macrophage polarization. Through these mechanisms, IMD treatment reversed HHcy-induced insulin resistance in adipose tissues. *, p < 0.05; **, p < 0.01; ***, p < 0.001.