Monocyte chemoattractant protein-1 (MCP-1)/chemokine (C-C motif) ligand (CCL) 2 (CCL2) secreted from white adipose tissue (WAT) in obesity has been reported to contribute to tissue macrophage accumulation and insulin resistance by inducing a chronic inflammatory state. MCP-1 has been shown to be elevated in the fatty liver of lipoatrophic A-ZIP-transgenic (A-ZIP-Tg) mice. Treatment of these mice with the CC chemokine receptor (CCR) 2 antagonist has been shown to ameliorate the hyperglycaemia, hyperinsulinaemia and hepatomegaly, in conjunction with reducing liver inflammation. However, since CCR2 antagonists can block not only MCP-1 but also MCP-2 (CCL8) and MCP-3 (CCL7), it remains unclear whether MCP-1 secreted from the liver could contribute to hyperglycaemia, hyperinsulinaemia and hepatomegaly in conjunction with liver inflammation, as well as to the M1 and M2 states of macrophage polarisation.
To address these issues, we analysed the effects of targeted disruption of MCP-1 in A-ZIP-Tg mice.
MCP-1 deficiency alone or per se resulted in a significant amelioration of insulin resistance in A-ZIP-Tg mice, which was associated with a suppression of extracellular signal-regulated protein kinase (ERK)-1/2 and p38 mitogen-activated protein kinase (p38MAPK) phosphorylation in liver. Although MCP-1 deficiency did not reduce the expression of macrophage markers, it increased the expression of the genes encoding M2 macrophage markers such as Arg1 and Chi3l3, as well as significantly reducing the triacylglycerol content of livers from A-ZIP-Tg mice.
Our data clearly indicated that MCP-1 deficiency improved insulin resistance and hepatic steatosis in A-ZIP-Tg mice and was associated with switching macrophage polarisation and suppressing ERK-1/2 and p38MAPK phosphorylation.
KeywordsA-ZIP-Tg mice Extracellular signal-regulated protein kinase Hepatic steatosis Insulin signalling Lipoatrophic diabetes Macrophage polarisation Monocyte chemoattractant protein-1
p38 Mitogen-activated protein kinase
Adipose tissue macrophages
- A-ZIP-Tg × MCP1−/−
A-ZIP transgenic MCP-1 homozygous knockout
Brown adipose tissue
Chitinase 3-like 3
Chemokine (C-C motif) ligand
Chemokine (C-C motif) receptor
Extracellular signal-regulated protein kinase
Glucose tolerance test
Insulin receptor beta
Monocyte chemoattractant protein
White adipose tissue
Obesity is associated with increased infiltration of macrophages into the adipose tissues. These adipose tissue macrophages (ATMs) are currently considered to be a major cause of obesity-associated chronic low-grade inflammation via the secretion of a wide variety of inflammatory molecules [1, 2], including TNF-α, IL-6  and monocyte chemoattractant protein-1 (MCP-1). These inflammatory molecules may have local effects on white adipose tissue (WAT) physiology as well as potential systemic effects on other organs that culminate in insulin resistance.
Among the inflammatory molecules upregulated in the adipose tissues of obese animals and humans, MCP-1 is a member of the cysteine–cysteine (C-C) chemokine family and promotes the migration of inflammatory cells by chemotaxis and integrin activation . Both Mcp-1 (also known as Ccl2) mRNA expression in WAT and plasma MCP-1 levels have been found to correlate positively with the degree of obesity in the individual . In addition, increased production of MCP-1 in WAT precedes the production of other macrophage markers during the development of obesity . Mice overproducing MCP-1 in adipocytes showed macrophage recruitment in WAT and exhibited insulin resistance in the skeletal muscles (SKM) and liver . Regarding the mechanisms, it was reported that MCP-1 stimulates the phosphorylation of extracellular signal-regulated protein kinase (ERK) through the C-C chemokine receptor (CCR) 2  and activation of ERK-1/2 induces insulin resistance via decreased tyrosine phosphorylation of insulin receptor beta (IR-β)  as well as increased serine phosphorylation of IRS-1 .
Macrophage activation has been operationally defined across two separate polarisation states: M1 and M2. M1, or ‘classically activated’, macrophages are induced by proinflammatory mediators such as lipopolysaccharide, whereas M2, or ‘alternatively activated’, macrophages generate high levels of anti-inflammatory cytokines such as IL-10, arginase-1(Arg1), chitinase 3-like 3 (Chi3l3) and TGF-β . It was previously reported that disruption of MCP-1, or its receptor CCR2, in obese mice resulted in decreased macrophage infiltration in WAT and improved metabolic function [11, 12]. Moreover, ATMs from obese Ccr2-deficient mice produce M2 markers at levels similar to those seen in lean mice . These data suggest that the MCP-1/CCR2 axis contributes to macrophage polarisation. Therefore, the phenotypic switch in ATM polarisation is thought to lead to amelioration of insulin resistance.
In contrast to obesity, lipoatrophic diabetes is caused by a deficiency of WAT and is characterised by severe hepatic steatosis and insulin resistance. Moitra and colleagues have generated lipoatrophic A-ZIP transgenic (A-ZIP-Tg) mice, which are profoundly insulin resistant and hyperlipidaemic [13–15]. In addition, these mice exhibit severe hepatic steatosis and at the same time a chronic state of inflammation as indicated by high systemic levels of inflammatory cytokines such as IL-1β, IL-6, IL-12 and MCP-1 [16–18]. At the very least, MCP-1 is most abundantly expressed in the liver from A-ZIP-Tg mice . Moreover, treatment of the lipoatrophic A-ZIP-Tg mice with a CCR2 antagonist has been shown to ameliorate the hyperglycaemia, hyperinsulinaemia and hepatomegaly, in conjunction with reducing liver inflammation. However, since CCR2 antagonist can block not only MCP-1, but also MCP-2 (chemokine [C-C motif] ligand [CCL] 8) and MCP-3 (also known as CCL7), it remains unclear whether MCP-1 secreted from liver could contribute to hyperglycaemia, hyperinsulinaemia and hepatomegaly in conjunction with liver inflammation, as well as to M1/M2 polarisation.
In this study, we hypothesised that MCP-1 secreted from the liver might also play an important role in the regulation of macrophage polarisation and insulin resistance-causing kinases such as ERK and p38 mitogen-activated protein kinase (p38MAPK) in liver. To address these issues, we analysed the effects of a targeted disruption of MCP-1 in lipoatrophic A-ZIP-Tg mice. We showed for the first time that a targeted disruption of MCP-1 alone in lipoatrophic diabetic A-ZIP-Tg mice resulted in decreased ERK-1/2 and p38MAPK phosphorylation and increased alternative M2 activation of macrophages, with at the same time an amelioration of insulin resistance and hepatic steatosis.
Generation of A-ZIP-Tg×Mcp1 −/− mice
A-ZIP/F-1 (A-ZIP-Tg) mice were generous gifts from C. Vinson of the National Cancer Institute at Frederick, MD, USA. Mcp1 −/− mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and C57BL/6 mice from CLEA Japan (Fujinomiya, Shizuoka, Japan). A-ZIP/F-1 mice were on an FVB/N (FVB) background. MCP-1 homozygous-knockout mice were on a C57BL/6 background, which had been backcrossed to C57BL/6 mice for 10 generations.
To generate A-ZIP-Tg Mcp1 +/− mice, conceptuses that were obtained by in vitro fertilisation of ova from Mcp1 −/− mice and sperm from A-ZIP-Tg mice were implanted into pseudo-pregnant foster mothers as previously described . To generate A-ZIP-Tg×MCP-1 knockout (Mcp1 −/− ) mice and Mcp1 −/− mice, conceptuses that had been obtained by in vitro fertilisation of ova from MCP-1 homozygous-knockout mice and sperm from A-ZIP-Tg×MCP-1 heterozygous-knockout mice were implanted into pseudo-pregnant foster mothers. To generate wild-type (WT) mice and A-ZIP-Tg mice, conceptuses that had been obtained by in vitro fertilisation of ova from C57BL/6 mice and sperm from A-ZIP-Tg×MCP-1 heterozygous-knockout mice were implanted into pseudo-pregnant foster mothers. All experiments in this study were conducted on female mice.
Mice were housed in cages and maintained on a 12-h light/dark cycle. For all experiments, the diet was standard chow (CE-2; CLEA Japan) with the following composition: 25.6% (wt/wt) protein, 3.8% fibre, 6.9% ash, 50.5% carbohydrates, 4% fat and 9.2% water [19–21]. The animal care and use procedures were approved by the Animal Care Committee of the University of Tokyo.
Northern blot analysis
Northern blotting was carried out according to the method described previously [20, 21]. Total RNA was extracted from various tissues with TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Total RNA (15 μg) was loaded onto a 1.3% agarose gel and transferred to a nylon membrane (Hybond N+; GE Healthcare Life Sciences, Hino, Tokyo, Japan). MCP-1 coding sequence cDNA was used as the probe template. The cDNA probe template of MCP-1 was prepared by RT-PCR using specific primers. The forward primer was 5′-CCATGCAGGTCCCTGTC-3′ and the reverse primer was 5′-CTAGTTCACTGTCACAC-3′, as previously described . The corresponding bands were quantified by exposure of BAS2000 to the filters and measurement with BAStation software (Fuji Film, Minato-ku, Tokyo, Japan).
Real-time quantitative PCR
For real-time quantitative PCR analysis, cDNA synthesised from total RNA was analysed. For quantification of gene expression, a set of predesigned primers and probes for each gene (Assays-on-Demand; Applied Biosystems, Carlsbad, CA, USA) were used. These were mouse MCP-1, Mm00441242_m1; mouse MCP-2: Mm01297183_m1; mouse MCP-3, Mm00443113_m1; mouse CCR2, Mm99999051_gH; mouse Emr1, Mm00802530_m1; mouse CD68, Mm00839636_g1; mouse Chi3l3, Mm00657889_mH; mouse TGF-β, Mm03024053_m1; mouse Arg1, Mm01190441_g1; mouse PPARa: Mm00440949_m1; mouse Acyl-coA oxidase: Mm00443579_m1; mouse UCP2: Mm00495907_g1; mouse SREBP1c: Mm00550338_m1 and mouse SCD1: Mm00772290_m1.
The primer sets and the probe for mouse cyclophilin were as follows: the forward primer was 5′-GGTCCTGGCATCTTGTCCAT-3′, the reverse primer was 5′-CAGTCTTGGCAGTGCAGATAAAA-3′; and the probe was 5′-CTGGACCAAACACAAACGGTTCCCA-3′. The relative amount of each transcript was normalised to the amount of mouse cyclophilin mRNA.
Blood sample assays and in vivo glucose homeostasis
Glucose tolerance tests (GTTs) were conducted as previously described with slight modifications [20, 21]. For the GTTs, mice were deprived of food for 6 h and then orally administered with d-glucose (1.5 g per kg body weight). Plasma glucose and plasma triglycerol (TG) levels in the fed state were determined using a glucose B-test and TG E-type test (Wako Pure Chemical Industries, Yodogawa-ku, Osaka, Japan), respectively. Plasma insulin levels were measured with an insulin immunoassay (Shibayagi, Shibukawa, Gunma, Japan). Plasma MCP-1 levels in the fed state were measured using a mouse immunoassay kit (Pierce Biotechnology, Rockford, IL, USA).
Mouse monoclonal anti-phosphotyrosine antibody 4G10 (αPY) was purchased from Merck Millipore (Billerica, MA, USA). Rabbit polyclonal antibody to IR-β was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibodies against ERK-1/2, phospho-ERK-1/2 (Thr202/Tyr204), p38MAPK, phosphor-p38MAPK (Thr180/Tyr182), Akt and phospho-Akt (Ser-473) were purchased from Cell Signaling Technology (Danvers, MA, USA).
Immunoblotting and immunoprecipitation
Immunoblotting and immunoprecipitation were conducted as previously described . In brief, in the fed state, the skeletal muscles from the hind limbs or livers were removed. The samples were homogenised in ice-cold 1% Nonidet P-40-buffer (25 mmol/l Tris-HCl [pH 7.4], 10 mmol/l sodium orthovanadate, 10 mmol/l EGTA and 1 mmol/l phenylmethylsulfonyl fluoride) and centrifuged. For immunoblotting, muscle homogenates containing 5 mg of total protein or liver homogenates containing 15 mg of total protein were incubated with the indicated primary antibodies and horseradish peroxidase-conjugated anti-mouse-IgG secondary antibody and were detected with enhanced chemiluminescence (ECL) reagent (GE Healthcare Life Sciences). For immunoprecipitation, muscle homogenates containing 5 mg of total protein or liver homogenates containing 15 mg of total protein were also incubated with the indicated antibodies followed by addition of protein G-Sepharose. The immunoprecipitates were washed with 1% Nonidet P-40-buffer three times. The immunoprecipitates were subjected to immunoblotting with the indicated primary antibodies and horseradish peroxidase-conjugated anti-mouse-IgG secondary antibody and were detected with ECL reagent.
Histological analyses and TG content in liver
Livers from WT mice, A-ZIP-Tg mice and A-ZIP-Tg×Mcp1 −/− mice were fixed overnight in 10% formalin (vol./vol.). Samples were routinely embedded in paraffin. Approximately 5 μm-thick slices obtained from these liver samples were stained with haematoxylin and eosin. The liver homogenates were extracted and their TG content was determined as previously described .
Results are expressed as means ± SEM. The Student’s t test was performed to compare two groups. Data involving more than two groups were assessed by ANOVA. Values of p < 0.05 were considered statistically significant.
For details of the hyperinsulinaemic–euglycaemic clamp study and antibodies used in immunoprecipitation and immunoblotting, please refer to the ESM Methods.
Plasma MCP-1 concentration and Mcp-1 mRNA expression in fatty liver were increased in lipoatrophic A-ZIP-Tg mice
Many previous studies have reported elevated plasma MCP-1 concentration and Mcp-1 mRNA expression in WAT of obese and diabetic mice [7, 11, 23–25]. As reported , plasma MCP-1 concentrations were significantly elevated in A-ZIP-Tg mice compared with WT mice (Fig. 1a). A previous study showed that, in WT mice, Mcp-1 mRNA was not detected in any tissues , whereas in A-ZIP-Tg mice, as previously reported , Mcp-1 mRNA was most abundantly expressed in the liver among all the tissues we examined, including brown adipose tissue (BAT), except for WAT, because lipoatrophic A-ZIP-Tg mice still have BAT but not WAT, as reported in other studies [12–14, 18] (Fig. 1b). It has been reported that MCP-1 is abundantly expressed in Kupffer cells, as well as in hepatocytes in the liver [26, 27]
MCP-1 deficiency decreased body weight gain and liver weight in A-ZIP-Tg mice
MCP-1 deficiency ameliorated glucose tolerance in lipoatrophic A-ZIP-Tg mice
Livers from A-ZIP-Tg×Mcp1 −/− mice showed increased markers of alternatively activated M2 macrophages
Because a previous report showed that ATMs isolated from obese Ccr2-deficient mice expressed alternatively activated M2-macrophage markers , we hypothesised that livers from A-ZIP-Tg×Mcp1 −/− mice might contain M2-polarised macrophages more abundantly. Interestingly, characteristic M2-macrophage marker genes such as Chi3l3, Arg1 and Tgfb1 were significantly increased in livers from A-ZIP-Tg×Mcp1 −/− mice (Fig. 4f–h). In contrast to liver, characteristic M2-macrophage marker genes such as Chi3l3, Arg1 and Tgfb1 were not significantly changed in SKM and BAT from A-ZIP-Tg×Mcp1 −/− mice (ESM Fig. 7). Thus, MCP-1 deficiency did not reduce macrophage markers but rather induced macrophages to shift to a M2-polarised state in livers from A-ZIP-Tg mice. These data suggest that MCP-1 deficiency enhances M2 polarisation in livers of A-ZIP-Tg mice.
MCP-1 deficiency decreased liver TG content in A-ZIP-Tg mice
MCP-1 deficiency enhanced insulin signalling in livers of A-ZIP-Tg mice
MCP-1/CCL2 secreted from WAT in obesity has been reported to contribute to tissue macrophage accumulation and insulin resistance by inducing a chronic inflammatory state. MCP-1 has been shown to be elevated in the fatty liver of lipoatrophic A-ZIP-Tg mice. Treatment of these mice with CCR2 antagonist has been shown to ameliorate their hyperglycaemia, hyperinsulinaemia and hepatomegaly, in conjunction with a reduction in liver inflammation. However, since CCR2 antagonist can block not only MCP-1, but also MCP-2 and MCP-3, it remains unclear whether MCP-1 secreted from liver could contribute to hyperglycaemia, hyperinsulinaemia and hepatomegaly in conjunction with liver inflammation, as well as to M1/M2 polarisation.
To address these issues, we analysed the effects of targeted disruption of MCP-1 in A-ZIP-Tg mice. In the current study, we showed for the first time that targeted disruption of MCP-1 alone in lipoatrophic diabetic A-ZIP-Tg mice ameliorated insulin resistance and hepatic steatosis, which was associated with decreased ERK-1/2 and p38MAPK phosphorylation (Fig. 6) and alternative M2 activation of macrophages. Although food intake was not significantly different when adjusted by body weight (ESM Table 1), the A-ZIP-Tg mice with MCP-1 deficiency tended to exhibit increased Ppara and exhibited significantly increased Ucp2 expression in the liver (ESM Fig. 8), raising the possibility that the body weight and liver weight reduction (ESM Table 2) were due to increased energy expenditure, and that it could also contribute to the reduction in liver triacylglycerol content. However, it has been technically extremely difficult to prepare enough A-ZIP-Tg×Mcp1 −/− mice to unequivocally prove increased energy expenditure in these mice. We would like to carry out this experiment in a future study.
The amounts of tyrosine phosphorylation of IR-β and serine phosphorylation of Akt were increased in livers from A-ZIP-Tg×Mcp1 −/− mice (Fig. 7). These data suggested that the amelioration of insulin resistance by disruption of MCP-1 in A-ZIP-Tg mice, such as increased tyrosine phosphorylation of IR-β and serine phosphorylation of Akt in liver, seemed to be related to decreased ERK-1/2 and p38MAPK activation , at least in part.
Although the disruption of MCP-1 in A-ZIP-Tg mice improved insulin resistance, the gene expression of macrophage markers such as Emr1 and Cd68 in the liver were not decreased compared with those seen in A-ZIP-Tg mice (Fig. 4d,e). It was recently reported that the induction of M2 markers in resident macrophages in the liver controls hepatic lipid metabolism . In this study, the M2-specific genes Chi3l3 and Arg1 were increased in livers from A-ZIP-Tg×Mcp1 −/− mice compared with A-ZIP-Tg mice (Fig. 4f,g). These data are consistent with the results that ATMs from obese Ccr2-deficient mice express M2 markers at levels similar to those found in lean mice . However, treatment with CCR2 antagonist reduced the macrophage marker gene Cd68 mRNA and Tnf-α mRNA in the livers of A-ZIP-Tg mice . These results might be derived from differences between a chronic and an acute inhibition of CCR2 or between targeted disruption of MCP-1 and simultaneous inhibition of MCP-1/MCP-2/MCP-3 by CCR2 antagonist in the liver of A-ZIP-Tg mice.
Previous studies have shown that CCR2 promoted obesity-induced hepatic steatosis in db/db mice , that a CCR2 antagonist (propagermanium) also reduced liver TG content in db/db mice , and that another CCR2 antagonist (RS 504393) ameliorated hepatomegaly but did not decrease hepatic TG content significantly (p = 0.144) . These results raised the possibility that inhibition of MCP-1 and/or MCP-2 and/or MCP-3 by CCR2 inhibition could be involved in the suppression of hepatic steatosis. In this study, we showed for the first time that targeted disruption of MCP-1 in A-ZIP-Tg mice by itself exhibited decreased liver TG content in the liver (Fig. 5b), clearly indicating that MCP-1 plays an important role in the onset of hepatic steatosis in the liver in A-ZIP-Tg mice.
In conclusion, we showed for the first time that targeted disruption of MCP-1 by itself ameliorated insulin resistance and hepatic steatosis, and at the same time decreased phosphorylation of ERK-1/2 and p38 MAPK (increased tyrosine phosphorylation of IR-β and serine phosphorylation of Akt) and induced alternative M2 activation of macrophages in the fatty liver from lipoatrophic diabetic A-ZIP-Tg mice. These results suggest that MCP-1 derived from tissues other than WAT, such as fatty liver, could also play an important role in the regulation of whole body insulin sensitivity, hepatic steatosis, macrophage polarisation and phosphorylation of ERK-1/2 and p38 MAPK. The current study also suggests that modulating MCP-1 in the liver would be useful therapy for diabetes and fatty liver.
We are grateful to K. Miyata for technical assistance.
This work was supported by Grant-in-aid for Scientific Research (S) (20229008) (to T.K.), Grant-in-aid for Scientific Research on Innovative Areas (Research in a proposed research area) ‘Molecular Basis and Disorders of Control of Appetite and Fat Accumulation’ (to T.Y.), Funding Program for Next Generation World-Leading Researchers (NEXT Program) (to T.Y.), Targeted Proteins Research Program (to T.K.), the Global COE Research Program (to T.K.) and Translational Systems Biology and Medicine Initiative (to T.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
YN researched, analysed and interpreted data and wrote the manuscript. TY wrote the manuscript, researched, analysed and interpreted data and contributed to the conception and design of this study. MI and MO-I researched, analysed and interpreted data and edited the manuscript. MF and MY researched data and edited the manuscript. KU analysed data and edited the manuscript. TK contributed to the conception and design of this study and edited the manuscript. YN, TY and TK are the guarantors for the content of this article. All authors have approved the final version of the manuscript.