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

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.

Intermedin (IMD), 3 also known as adrenomedullin 2 (AM2), is a 53-amino acid peptide belonging to the calcitonin gene-related peptide/calcitonin family (1,2). IMD shares a common family of G-protein-coupled receptors with another calcitonin gene-related peptide/calcitonin family member, adrenomedullin (AM), which is involved in obesity and its related metabolic disorders (3)(4)(5). IMD regulates the central and peripheral circulation and water-electrolyte homeostasis and protects the myocardium from ischemia-reperfusion injury by inhibiting oxidative stress in both the heart and kidney (6 -9). The observation of decreased IMD in the plasma of diabetic rats linked IMD with diabetes (8). Previous studies from our group demonstrated that IMD inhibits scavenger receptors and foam cell formation of macrophages and, consequently, protects mice from atherosclerosis (10,11). Recently published data from our group showed that IMD reduced insulin resistance in high-fat diet-induced obese mice through elevating thermogenesis in brown adipose tissue (12).
Chronic inflammation is considered a pathological issue for aggravating insulin resistance (13,14). However, the effect of IMD on inflammation aggravated insulin resistance remains largely unknown. In a bacterial LPS-induced atypical orchitis rat model, IMD attenuated the expression of pro-inflammatory cytokines TNF␣, IL-6, and IL-1␤ (15). Down-regulation of IMD was observed in atopic dermatitis (16). These results showed that IMD exerts potential anti-inflammation effects. In this study, we explored the effects of IMD on macrophage inflammation and adipose insulin resistance.
Hyperhomocysteinemia (HHcy) indicates a plasma homocysteine (Hcy) level Ͼ10 mol/liter. Evidence showed that Hcy induced the production of several pro-inflammatory cytokines in macrophages in both mice and humans (17)(18)(19)(20). A metaanalysis and Mendelian randomization analysis from 4011 cases and 4303 controls showed a causal association of Hcy level with the development of type 2 diabetes in humans (21). Our previous studies revealed that HHcy induces insulin resistance and adipokine resistin elevation from adipocytes (22). In a mechanism study of HHcy promoting insulin resistance, endoplasmic reticulum stress (ERS), inflammation pathway activation, and macrophage infiltration were found to be induced by HHcy in adipocytes and adipose tissues (23).
Macrophages play an essential role in regulating the inflammation state in adipose tissues. Insulin resistance was promoted by a transition in macrophage polarization from an alternative M2 state to a pro-inflammatory M1 state (13,14,24,25). It has been reported that HHcy promotes inflammatory monocyte differentiation (19). Combined with LPS, Hcy induced the M1 polarization of a macrophage cell line, RAW264.7, in vitro (26). However, whether HHcy regulates the M1/M2 balance in insulin-resistant mice and the effects of IMD in HHcy-induced insulin resistance remain unknown.
Our study aimed to explore the mechanisms of HHcy-enhanced ERS and the consequent insulin resistance in adipocytes and the effects of IMD on these processes, revealing IMD as a novel factor on HHcy-induced insulin resistance in adipose tissues of mice.
Animal Model-All studies followed the guidelines of the Animal Care and Use Committee of Peking University and the Guide for the Care and Use of Laboratory Animals. For the HHcy mouse model, male C57BL/6J mice, 6 weeks old, were fed normal mouse chow with or without 1.8 g/liter DL-Hcy added to the drinking water for 4 weeks, as described previously (20,23). Alzet osmotic minipumps (model 2004) containing 318 ng/kg/h IMD or saline were implanted subcutaneously into male C57BL/6J mice for infusion. After 3 days, the mice were fed Hcy in drinking water for 4 weeks. For adipocyte-specific IMD overexpression (Adipo-IMD-tg) mice, human intermedin gene expression was driven by the Fabp4 promoter in mice with a C57BL/6J background. For the glucose tolerance tests (GTTs), mice were fasted for 12 h and then injected with glucose (3 g/kg body weight) intraperitoneally. For the insulin tolerance test (ITT), mice were fasted for 4 h, and then 2 IU/kg insulin was injected intraperitoneally. Blood glucose levels were evaluated at 0, 15, 30, 60, 90, or 120 min. After 3 days of adaptation, mice were killed. Blood, epididymal fat pads, and subcutaneous fat pads were taken. Body composition was measured in nonanesthetized mice using an EchoMRI TM device (Echo Medical Systems). The homeostasis model assessment (HOMA) index was calculated by using the following formula: [fasting insulin (mIU) ϫ fasting glucose (mmol/liter)]/22.5.
ELISA-Mouse plasma IMD was detected using an ELISA kit from Cloud-Clone Corp. No significant cross-reactivity or interference between IMD and analogues was observed.
siRNA for AMPK-AMPK siRNA was designed and chemically modified by the manufacturer (Ribobio). Sequences corresponding to the siRNA of AMPK were as follows: sense, 5-GCAGAAGAUUCGGAGCCUU dTdT-3; antisense, 3-dTdT CGUCUUCUAAGCCUCGGAA-5. Transfection of mouse peritoneal macrophages with the siRNA (50 nmol/l) in vitro was performed with Oligofectamine (Invitrogen) according to the instructions of the manufacturer.
Flow Cytometry-Adipose tissues were minced and digested with 1 mg/ml type I collagenase for 40 min. After centrifugation, stromal vascular fractions were collected at the bottom of the tubes. Then the stromal vascular fractions were stained with the macrophage marker F4/80 allophycocyanin, the dead cell marker 7-aminoactinomycin D, the M2 marker CD206 FITC, and the M1 marker CD11c phycoerythrin (BD Biosciences) and analyzed on a FACSCalibur flow cytometer (BD Biosciences).
Cell Culture-Mouse peritoneal macrophages were treated with 300 M Hcy, 20 nM IMD, or HcyϩIMD for 24 h in DMEM (Invitrogen) supplemented with 2% FBS. Cells were then used for M1/M2 marker or pAMPK detection. For the co-culture assay, the cells were changed to fresh medium for an additional 24 h. Subsequently, cell culture supernatants were collected. 3T3-L1 cells were cultured in growth medium, high-glucose DMEM, supplemented with 10% FBS. Two days post-confluence, cells were induced to differentiate with a standard mixture consisting of growth medium with 1 mol/liter dexamethasone, 10 g/ml bovine insulin, and 0.25 mmol/liter 3-isobutyl-1-methylxanthine. After 3 days in differentiation medium, cells were treated with growth medium with 10 g/ml bovine insulin for 3 days and then maintained in growth medium alone. Cells were considered mature adipocytes 8 days post-induction of differentiation and then treated with supernatant from cultured macrophages for 24 h.
Statistical Analysis-All data are reported as mean Ϯ S.E. Data were analyzed with GraphPad Prism software. Statistical analysis involved one-way analysis of variance for multiple comparisons, then Tukey-Kramer post-hoc testing, and Student's unpaired t test for comparisons between two groups. p Ͻ 0.05 was considered statistically significant.

IMD Expression Was Decreased in HHcy
Mice-After being fed with 1.8 g/liter Hcy in the drinking water for 4 weeks, HHcy mice displayed insulin resistance (23). To validate whether IMD plays a role in HHcy-induced insulin resistance, we detected the expression level of IMD in HHcy mice. Compared with controls, the plasma IMD levels of HHcy mice were markedly lower (Fig. 1A). IMD was abundant in adipose tissues compared with livers or skeletal muscles (Fig. 1B). Thus, we proposed that adipose tissue is the pivotal organ where IMD participates in glucose metabolism. Consistent with systemic IMD down-regulation, the mRNA and protein levels of IMD were apparently decreased in the adipose tissues of HHcy mice (Fig. 1, B and C).
IMD Reversed HHcy-induced Insulin Resistance in Adipose Tissue-To investigate the role of adipocyte IMD in HHcy-mediated insulin resistance, we generated adipose tissue (Fabp4)specific knockin IMD gene (Adipo-IMD-tg) mice. Adipo-IMD-tg mice showed an accurate transgenic gene band, but wild-type mice did not (data not shown). The mRNA levels of IMD were substantially increased in epididymal white adipose tissues (eWAT) but not in livers or skeletal muscles ( Fig. 2A). It was reported that Fabp4 is expressed in macrophages (27). Therefore, the expression of the IMD transgene in macrophages was also investigated. Adipo-IMD-tg mice exhibited significantly enhanced protein levels of IMD in eWAT, whereas no changes in IMD expression in macrophages were detected (Fig. 2B). The body weights of Adipo-IMD-tg mice were similar to those of controls after feeding with Hcy for 4 weeks (Fig. 2C). The fat-to-weight ratio in Adipo-IMD-tg mice was similar to those of controls (Fig. 2D). No difference was observed in the size of adipocytes in eWAT tissues from Adipo-IMD-tg mice and control mice (Fig. 2E). Consistent with our data published previously (23), HHcy mice had impaired insulin sensitivity, as verified by GTT and ITT (data not shown). However, the GTT and ITT results indicated improved glucose tolerance and insulin tolerance, respectively, in Adipo-IMD-tg mice with HHcy (Fig. 2F). Moreover, fasted serum insulin levels and the HOMA index were significantly reduced in Adipo-IMD-tg mice (Fig.  2G). The insulin sensitivity indicator phosphorylated AKT (Ser-473) was reduced in adipose tissues of HHcy mice but elevated in the Adipo-IMD-tg mouse group (Fig. 2H). These results indicated that IMD overexpression ameliorates systemic and adipose tissue insulin resistance in HHcy mice.
IMD Attenuated Hcy-induced M1/M2 Imbalance-To further explore the mechanisms underlying IMD-ameliorated insulin resistance with HHcy, the balance of pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages was detected in the adipose tissues of Adipo-IMD-tg mice. In a flow cytometry assay gated on F4/80-positive macrophages, CD11c was labeled as M1, whereas CD206 was labeled as an M2 marker (28). In eWAT, an increased proportion of M2 macrophages was observed in Adipo-IMD-tg mice (Fig. 3A). Moreover, a reduced percentage of M1 macrophages was observed in subcutaneous white adipose tissues (Fig. 3B). This imbalance of M1/M2 was further confirmed by examination of cell surface markers (CD11C for M1 and CD206 for M2), cytokines (IL-12 for M1 and IL-10 for M2), and enzymes (iNOS for M1 and Arg1 for M2). Elevated mRNA expression of M2 markers, including Cd206, Il-10, and Arg1, and reduced mRNA expression of inos were observed in the eWAT of Adipo-IMD-tg mice compared with control mice (Fig. 3C). To verify the effect of IMD on the M1/M2 cell balance in vitro, thioglycolate-elicited peritoneal macrophages were administered (300 M Hcy, 20 nM IMD, or 300 M Hcy ϩ 20 nM IMD) for 24 h. The expressions of M1 and M2 macrophage markers were detected. The administration of IMD reversed Hcy-induced M1 macrophage marker expression (i.e. Cd11c and iNOS; Fig. 4, A, C, and E), whereas it recovered Hcy-inhibited M2 macrophage marker expression (i.e. Cd206, Il-10, and Arg1; Fig. 4, A, B, D, and E). These data indicate that IMD improves the Hcy-induced M1/M2 imbalance.
IMD Restored the Hcy-induced M1/M2 Imbalance by Activating the AMPK Pathway-AMP-activated protein kinase (AMPK) is a key regulator of cellular and systemic energy homeostasis. It has recently been reported that the depletion of AMPK in hematopoietic cells inhibits M2 macrophages but promotes M1 macrophage activation (29). The AMPK pathway was further detected in macrophages treated with Hcy and IMD alone or in combination. The phosphorylation of AMPK␣ was inhibited by Hcy treatment but was reversed by IMD in macro- phages (Fig. 5A). To address the effect of AMPK on Hcy-or IMD-mediated macrophage polarization, the activity of AMPK was abolished by siRNA. Around half of the AMPK expression was knocked down by AMPK siRNA (Fig. 5B). Although IMD reversed the Hcy-induced M1/M2 imbalance, knockdown of AMPK diminished these effects. Moreover, the protein expression of ARG1 was markedly reduced, whereas iNOS expression was enhanced by siAMPK transfection (Fig. 5, C and D). These results suggest that IMD reverses the Hcy-induced M1/M2 imbalance through the AMPK pathway.

IMD Reversed HHcy-induced ER Stress and Chronic Inflammation in Adipose
Tissue-Previous studies from our group showed that HHcy enhances ER stress and insulin resistance in adipocytes. The PERK and ATF6 pathways were involved rather than the XBP1 pathway (23). To address the effect of IMD on HHcy-primed ER stress, adipose tissues from Adipo-IMD-tg mice were analyzed regarding the PERK and ATF6 signaling pathways. Compared with controls, Adipo-IMD-tg mice displayed reduced phosphorylated PERK (pPERK) and phosphorylated eIF2␣ (peIF2␣) levels in adipose tissues with HHcy. Activated/cleaved ATF6 was also repressed in adipose tissues of Adipo-IMD-tg mice (Fig. 6, A and B). Our results suggest that, under the HHcy condition, overexpression of IMD attenuates the ER stress pathways in adipose tissues.
Inflammation promotes HHcy-induced insulin resistance in adipocytes and triggers the activation of JNK, which is downstream of ERS in adipocytes (23). The phosphorylation levels of JNK in the adipose tissues of Adipo-IMD-tg mice were substantially reduced compared with those of wild-type mice under HHcy (Fig. 6, C and D). The activation of the NF-B P65 subunit was significantly inhibited in Adipo-IMD-tg mice (Fig. 6, C  and D). IMD ameliorates HHcy-aggravated inflammation in adipose tissue.

IMD Reversed Hcy-induced ER Stress and Inflammation in Adipocytes as a Result of Improvement in Macrophage
Polarization-In adipose tissues, macrophage polarization is a well known inflammation mediator of cross-talk with adipocytes and interferes with adipocyte insulin signaling (13,14,25). Therefore, whether IMD-or Hcy-mediated M1/M2 imbalance interacts with ER stress and inflammation in adipocytes was investigated by co-culture studies of macrophages and adipocytes in vitro. Mouse peritoneal macrophages were treated with Hcy, IMD, or Hcy ϩ IMD. Cell culture supernatants were added to the culture of differentiated 3T3-L1 adipocytes. PERK and ATF6 signaling pathways were detected in the treated adipocytes. Supernatant from Hcy-treated macrophages enhanced the levels of pPERK, peIF2␣, and cleaved ATF6 in adipocytes, whereas the Hcy ϩ IMD group reversed these effects (Fig. 7A). IMD reversed the Hcy-induced activation of JNK (Fig. 7B). Collectively, these data indicate that soluble factors in the macrophage culture supernatants mediated the IMD reversal of Hcy-induced ER stress and chronic inflammation in adipocytes.
IMD Treatment Improved Hcy-induced Insulin Resistance-To verify the therapeutic effect of IMD on Hcy-induced insulin resistance, we implanted osmotic minipumps containing IMD subcutaneously to elevate the IMD level in HHcy mice. GTT and ITT showed improved glucose intolerance and increased insulin sensitivity in IMD-treated mice (Fig. 8A), whereas the body weight remained similar in the two groups (Fig. 8B). The fasted serum insulin levels and HOMA indexes were significantly reduced in the IMD treatment group (Fig. 8C). In adipose tissues, phosphorylated AKT (Ser-473) was elevated by IMD infusion compared with controls with HHcy (Fig. 8D). These results reveal that IMD administration produced striking benefits for the improvement of HHcy-induced insulin resistance.

Discussion
Our previous study reported that HHcy induces ERS and insulin resistance in adipose tissues (23). In this study, adipocyte-specific IMD gene overexpression or the administration of IMD was shown to ameliorate HHcy-induced insulin resistance. Unlike enhancing thermogenesis in brown adipose tissues of high-fat diet-induced obese mice (12), IMD displayed a 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-F4/80ϩ cells, CD11c and CD206 expression was analyzed (n ϭ 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 ϭ 4). *, p Ͻ 0.05; **, p Ͻ 0.01. distinct role in HHcy-induced insulin resistance. Mechanism studies revealed that Hcy enhances the ratio of M1/M2 by repression of AMPK activity and that IMD administration reverses the effect of Hcy on AMPK activity and M1/M2 balance. The restoration of macrophage polarization contributed to the IMD improvement of Hcy-induced ER stress and chronic inflammation in adipocytes (Fig. 8E).
Inflammatory signals such as JNK activation inhibit the phosphorylation of insulin receptor substrate, which results in insulin resistance (15,30). It has been reported that IMD attenuates pro-inflammatory cytokine expression, including TNF␣, IL-6, and IL-1␤, in the testis in a LPS-induced rat orchitis model (31). The reduction of IMD expression in the skin of atopic dermatitis patients compared with healthy controls indicated that lower levels of IMD are a higher susceptibility factor to inflammatory stimuli (16). This study consistently demonstrated that IMD-specific overexpression in adipose tissues reversed HHcyinduced inflammation and insulin resistance. Although some reports have shown that Fabp4 was expressed in macrophages, no significant expression of transgenic IMD was induced in macrophages compared with adipocytes in Adipo-IMD-tg mice. Supporting our findings, a Fabp4-PPAR␥ overexpression line demonstrated that the transgenic PPAR␥ was specifically expressed in adipocytes (32), and different Fabp4-driven Cre mouse models showed that the floxed alleles were recombined in adipocytes but not in macrophages (33).
Increasing infiltration of pro-inflammatory immune cells, particularly macrophages, is a characteristic of obesity-induced insulin resistance (25,34). Insulin resistance is promoted by a transition in macrophage polarization from an anti-inflammatory M2 activation state to an inflammatory M1 activation state (14,(35)(36)(37). Previous reports have indicated that HHcy promotes the differentiation of Ly6C-high inflammatory monocytes in an atherosclerosis mouse model (19). Another recent study demonstrated that Hcy induces M1 polarization and converts M2 to an M1 subtype in vitro (26). In this study, IMD overexpression reversed the AMPK activation is considered to be a potential therapeutic target for insulin resistance. Activated AMPK stimulates glucose uptake in skeletal muscles and fatty acid oxidation in adipose tissues, and it reduces hepatic glucose production (38,39). The inactivation of macrophage AMPK inhibits adipocyte insulin signaling and glucose uptake in a macrophage-adipocyte co-culture system (40). AMPK activation is related to repressed iNOS in primary human umbilical vein endothelial cells, myocytes, macrophages, and adipocytes (41,42). AMPK␣1 Ϫ/Ϫ macrophages did not acquire the phenotype or the functions of M2 macrophages (43). In agreement with these findings, this study demonstrated that Hcy administration repressed the activation of AMPK but that IMD treatment reversed the effect of Hcy on AMPK activity and the M1/M2 balance. Blockage of AMPK enhanced M1 macrophage marker iNOS expression but repressed M2 macrophage marker ARG1 expression. Our data provided a new mechanism for Hcy and IMD in the regulation of the M1/M2 balance. Unlike other members of the calcitonin gene-related peptide/calcitonin family, no unique receptor for IMD has been identified. Intermedin binds nonselectively to both calcitonin gene-related peptide and adrenomedullin (ADM) receptor complexes: CRLR-RAMP1, 2, and 3 (44). IMD couples with the CRLR-RAMP receptor complexes to activate cAMP production (45). cAMP is known to regulate AMPK activity (46). IMD may be signaling, through CRLR-RAMP receptors, to activate AMPK through cAMP in macrophages. Further studies are needed to clarify this issue.
In white adipose tissues, the metabolic functions are regulated by cross-talk between adipocytes and stromal cells, particularly with macrophages (47). Pro-inflammatory M1 macrophages inhibit insulin sensitivity by cytokine production, whereas anti-inflammatory M2 macrophages reverse the effect of M1 macrophages (36). Our data indicated that IMD improved the M1/M2 imbalance induced by Hcy. The distinct soluble factors produced from Hcy and/or IMD treatment led to ERS in adipocytes. Our findings revealed a possible interaction between macrophages and adipocytes in IMD-and/or HHcy-treated mice.
Our study demonstrated that IMD reverses HHcy-induced ERS, inflammation, and insulin resistance. Hcy elevated the 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-eIF2␣, eIF2␣, and ATF6 Western blotting assays. B, quantitative analysis for pPERK, p-eIF2␣, 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 ϭ 4/group). *, p Ͻ 0.05; **, p Ͻ 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 ϩ IMD (HϩI), and the cell culture supernatants were added to the culture of differentiated 3T3-L1 adipocytes. pPERK, PERK, p-eIF2␣, eIF2␣, 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 Ͻ 0.05; **, p Ͻ 0.01. Ctl, control. M1/M2 ratio by inhibiting AMPK activity, and IMD reversed the M1/M2 imbalance. This study revealed the role of IMD administration in the recovery of insulin sensitivity, which shed light on the molecular mechanisms of Hcy and IMD regulation of the M1/M2 balance, and it provided a new therapeutic strategy for insulin resistance.
Author Contributions-Y. P., Yang Li, Y. Lv, L. S., S. Z., Y. W., Yin Li, G. L., M. X., X. W., and C. J. made substantial contributions to the acquisition of data or the analysis and interpretation of data. Y. P., C. J., and X. W. contributed to the design of the experiments and the writing of the manuscript. All authors approved the final version of the paper. X. W. and C. J. are the guarantors of this work and, as such, had full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Osmotic minipumps containing IMD were implanted subcutaneously in HHcy mice. A, GTT and ITT were examined in IMD infusion mice (n ϭ 7 for controls (Ctl); n ϭ 6 for the IMD group). B, the body weights of controls and IMD infusion mice after 4 weeks of Hcy feeding (n ϭ 7 for controls; n ϭ 6 for the IMD group). C, fasted glucose, fasted serum insulin levels, and HOMA index of controls or IMD infusion mice with HHcy (n ϭ 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 ϭ 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 Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001.