Hyperhomocysteinemia promotes insulin resistance by inducing endoplasmic reticulum stress in adipose tissue

Abstract

Type 2 diabetes is a chronic inflammatory metabolic disease, the key point being insulin resistance. Endoplasmic reticulum (ER) stress plays a critical role in the pathogenesis of type 2 diabetes. Previously, we found that hyperhomocysteinemia (HHcy) induced insulin resistance in adipose tissue. Here, we hypothesized that HHcy induces ER stress, which in turn promotes insulin resistance. In the present study, the direct effect of Hcy on adipose ER stress was investigated by the use of primary rat adipocytes in vitro and mice with HHcy in vivo. The mechanism and the effect of G protein-coupled receptor 120 (GPR120) were also investigated. We found that phosphorylation or expression of variant ER stress markers was elevated in adipose tissue of HHcy mice. HHcy activated c-Jun N-terminal kinase (JNK), the downstream signal of ER stress in adipose tissue, and activated JNK participated in insulin resistance by inhibiting Akt activation. Furthermore, JNK activated c-Jun and p65, which in turn triggered the transcription of proinflammatory cytokines. Both in vivo and in vitro assays revealed that Hcy-promoted macrophage infiltration aggravated ER stress in adipose tissue. Chemical chaperones PBA and TUDCA could reverse Hcy-induced inflammation and restore insulin-stimulated glucose uptake and Akt activation. Activation of GPR120 reversed Hcy-induced JNK activation and prevented inflammation but not ER stress. Therefore, HHcy inhibited insulin sensitivity in adipose tissue by inducing ER stress, activating JNK to promote proinflammatory cytokine production and facilitating macrophage infiltration. These findings reveal a new mechanism of HHcy in the pathogenesis of insulin resistance.

FIGURE 1.

HHcy promoted insulin resistance in vivo. HHcy animal models were developed by feeding C57BL/6J mice with supplement of Hcy (1.8 g/liter) in drinking water for 4 weeks. A, blood Hcy concentrations; B, body weight; C, ratio of epididymal fat pad and body weight; D, food intake in normal and HHcy mice. E, oral glucose tolerance test. Mice were fed 3 g/kg body weight glucose by gavage. Blood glucose concentrations were detected at 0, 30, 60, 90, and 120 min. F, insulin tolerance test. 1 IU/kg insulin was injected intraperitoneally into each mouse. Blood glucose concentrations were detected at 0, 30, 60, 90, and 120 min. Data are means ± S.E. (error bars) (n = 8). *, p < 0.05 versus normal control mice at the same state.

FIGURE 2.

HHcy induced adipose ER stress in vivo and in vitro. Shown are the protein level of Bip and phosphorylation (p-) of PERK, eIF2α (A), and ATF6 (H) in adipose tissue of normal and HHcy mice. Right-hand panels show the quantification of protein level. Relative protein levels were normalized to levels for normal mice. Data are means ± S.E. (n = 8). *, p < 0.05 versus control mice. B, C, D, and I, time course effect of Hcy (500 μm); E, F, G, and J, dose-response effect of Hcy on phosphorylation of PERK, eIF2α, ATF6, and Bip in primary cultured adipocytes. Bottom panels show quantification of protein level. Relative protein levels were normalized to that for non-Hcy-treated cells. Data are means ± S.E. (error bars) from four separate experiments. *, p < 0.05 versus Hcy-untreated cells.

FIGURE 3.

HHcy induced adipose inflammation in vivo. A, immunohistochemical staining of Mac-3-positive macrophages in epididymal adipose tissue in HHcy mice (right) and normal control mice (left). B, RT-PCR analysis of mRNA levels of MCP-1, TNF-α, IL-6, and PAI-1 in adipose tissue of HHcy and control mice. C, plasma MCP-1 and TNF-α levels in HHcy and control mice. D, phosphorylation (p-) of JNK, p65, and c-Jun in adipose tissue of normal and HHcy mice. Right, quantification of phosphorylation. Relative protein levels were normalized to levels for control mice. Data are means ± S.E. (error bars) (n = 8). *, p < 0.05 versus control mice.

FIGURE 4.

Inhibition of ER stress reversed Hcy-induced adipose inflammation. A and B, attenuation of Hcy (500 μm)-induced MCP-1 and TNFα expression by PBA (5 mm), TUDCA (200 μm), and SP600125 (20 μm) in primary rat adipocytes. Relative mRNA levels were normalized to levels for untreated cells. Data are means ± S.E. (error bars) from four separate experiments. C, attenuation of Hcy (500 μm)-stimulated JNK phosphorylation (p-) and IκB degradation by PBA, TUDCA, and SP60125 in primary rat adipocytes. The bottom panels show quantification of protein level. Relative protein levels were normalized to that for non-treated cells. *, p < 0.01 versus untreated cells; #, p < 0.01 versus Hcy treatment alone.

FIGURE 5.

Macrophage infiltration aggravated Hcy-induced adipose ER stress. Effect of Hcy on phosphorylation (p-) of PERK and eIF2α in primary cultured adipocytes co-cultured with or without peritoneal macrophages. The right-hand panels show quantification of phosphorylation. Relative protein levels were normalized to that for non-Hcy-treated adipocytes. Data are means ± S.E. (error bars) from four separate experiments. *, p < 0.05 versus Hcy-untreated cells; #, p < 0.01 versus adipocytes only.

FIGURE 6.

Inhibiting ER stress or inflammation reversed Hcy-induced insulin resistance. A and C, reversal of Hcy-impaired insulin-stimulated Akt phosphorylation (p-) by PBA (5 mm), TUDCA (200 μm), and SP600125 (20 μm) in primary rat adipocytes. The bottom panels show quantification of phosphorylation. Relative protein levels were normalized to that for non-insulin- and non-Hcy-treated cells. B and D, reversal of Hcy-impaired insulin-stimulated glucose uptake by PBA, TUDCA, and SP60125 in primary rat adipocytes. Data are means ± S.E. (error bars) from four separate experiments. *, p < 0.01 versus insulin treatment alone; #, p < 0.01 versus Hcy treatment alone.

FIGURE 7.

Activation of GPR120 reversed Hcy-induced insulin resistance by antagonizing Hcy-induced inflammation but not ER stress. A, reversal of Hcy-impaired insulin-stimulated Akt phosphorylation (p-) by GW9508 in primary rat adipocytes. The bottom panel shows quantification of phosphorylation. Relative protein levels were normalized to that for non-insulin- and non-Hcy-treated cells. B, reversal of Hcy-impaired, insulin-stimulated glucose uptake by GW9508 in primary rat adipocytes. Data are means ± S.E. (error bars) from four separate experiments. *, p < 0.01 versus insulin treatment alone; #, p < 0.01 versus Hcy treatment alone. C, attenuation of Hcy-stimulated JNK phosphorylation and IκB degradation by GW9508 in primary rat adipocytes. The bottom panels show quantification of protein level. Relative protein levels were normalized to that for non-treated cells. D and E, attenuation of Hcy-induced MCP-1 and TNFα expression by GW9508 in primary rat adipocytes. Relative mRNA levels were normalized to levels for untreated cells. Data are means ± S.E. from four separate experiments. *, p < 0.01 versus untreated cells; #, p < 0.01 versus Hcy treatment alone. F, modified Boyden chamber assay of migration of peritoneal macrophages co-cultured with adipocytes under Hcy with or without GW9508. Right, results of macrophage migration. *, p < 0.01 versus macrophages only; #, p < 0.01 versus Hcy-untreated cells; &, p < 0.01 versus Hcy treatment alone. G, effect of PBA and TUDCA on Hcy-stimulated JNK phosphorylation in primary rat adipocytes. H, effect of SP600125 on Hcy-stimulated eIF2α phosphorylation in primary rat adipocytes. I, effect of TUDCA and GW9508 on Hcy-stimulated JNK and eIF2α phosphorylation in primary rat adipocytes. The bottom panels show quantification of phosphorylation. Relative protein levels were normalized to that for non-treated cells. *, p < 0.01 versus untreated cells; #, p < 0.01 versus Hcy treatment alone.