IL-7 receptor deletion ameliorates diet-induced obesity and insulin resistance in mice
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Obesity-induced inflammation plays an important role in the development of insulin resistance and type 2 diabetes. Recent studies have demonstrated that adiposity can be improved by ablating certain inflammatory signalling pathways. Although the IL-7 receptor (IL-7R) is mostly known as a key regulator of T lymphocyte development and homeostasis, its role in obesity and metabolic diseases is unknown. Because IL-7 is markedly increased in the serum of obese individuals and IL-7R (also known as IL7R) is overexpressed in white adipose tissue (WAT) in obesity, we studied the metabolic consequences of genetic Il-7r ablation in mice.
Age-matched Il-7r-deficient (Il-7r KO) and wild-type (WT) littermates were fed a standard chow or high-fat diet (HFD) for 14 weeks. Their serum metabolic variables were measured. The expression of genes and proteins related to insulin resistance and inflammation was evaluated in WAT.
We demonstrated that Il-7r KO mice exhibited significantly reduced body weight gain and visceral adiposity compared with WT controls on both chow and HFD. The expression of signalling molecules involved in adipogenesis was reduced in the WAT of Il-7r KO mice. We also found that Il-7r KO mice had significantly enhanced glucose homeostasis and insulin sensitivity. Consistent with an improved metabolic phenotype, proinflammatory cytokine production and macrophage infiltration was attenuated in the WAT of Il-7r KO mice.
The IL-7R plays an important role in the induction of HFD-induced adipogenesis and insulin resistance in mice.
KeywordsAdipogenesis Inflammation Insulin resistance Interleukin-7 receptor Obesity
Extracellular signal-regulated kinase
Inhibitor of κB kinase β
c-Jun amino-terminal kinase
Mitogen-activated protein kinase
Monocyte chemoattractant protein-1
Suppressor of cytokine signalling
Signal transducer and activator of transcription 5
White adipose tissue
The IL-7 ligand receptor system plays an important role in the normal development and homeostatic maintenance of the immune system . Ligand–receptor interaction leads to a cascade of phosphorylation events mediated by signalling molecules, including Janus kinase (JAK)-1 and -3, signal transducer and activator of transcription 5 (STAT5), and phosphatidylinositol 3-kinase (PI3K) [2, 3]. The physiological result of IL-7 receptor (IL-7R) activation is survival, proliferation and maturation in early B lymphocytes and induction of the activation cascade in mature T lymphocytes . IL-7R is a dimeric complex composed of a high affinity α chain and the common γ chain, which is shared with several other cytokine receptors, namely IL-2, IL-4, IL-9, IL-15 and IL-21 . Mutations of the gene encoding IL-7Rα are associated with a milder form of severe combined immunodeficiency disease, which is characterised by a specific lack of T cells [6, 7].
Obesity is now recognised as a state of chronic low-grade systemic inflammation . Adipose tissue, particularly the visceral fat of obese humans and rodents, is affected by this inflammatory state, which is characterised by increased production and secretion of proinflammatory molecules [9, 10]. Many lines of evidence have shown that the activation of proinflammatory pathways within insulin target tissues can impair insulin signalling [11, 12]. TNF-α was among the first mediators defined as a key factor linking inflammation and insulin resistance . Engagement of the TNF receptor with TNF-α induces inhibitory phosphorylation of serine residues of IRS1 and activates the inhibitor of κB kinase β (IKKβ) and c-Jun amino-terminal kinase (JNK) pathways, two major intracellular regulators of insulin resistance [14, 15]. Several studies have reported that IL-6 mediates insulin resistance in the obese state by inducing suppressor of cytokine signalling (SOCS)-1 and -3, which link IRS1 to ubiquitin-mediated degradation [16, 17]. Accumulating evidence reveals that IL-7 induces the production of proinflammatory cytokines, such as TNF-α and IL-6, in monocytes and macrophages [18, 19]. Chronic activation of the IL-7/IL-7R signalling pathway contributes to persistent inflammation associated with inflammatory pathologies, including atherosclerosis, arthritis, gastritis and colitis in rodent models [19, 20, 21, 22, 23]. Recently, IL-7 expression was reported to be upregulated in the adipose tissue of obese individuals , supporting the notion that IL-7 could be a potential mediator of obesity-associated inflammation. The aim of this study was to investigate whether IL-7R plays a role in the induction of both adiposity and insulin resistance. Therefore, we assessed the effects of whole-body deletion of Il-7r (also known as Il7r) on visceral adiposity and glucose homeostasis in mice.
Animal and diets
C57BL/6J wild-type (WT; stock 000664) and Il-7r-deficient (Il-7r KO; B6.129S7-Il7r tm1Imx /J; stock 002295) mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Il-7r KO mice were backcrossed to a C57BL/6J background for six generations. At 5 weeks of age, WT and Il-7r KO mice were separated by sex and placed on either a chow diet (Purina Rodent Chow 5001, Orient Bio, Seongnam, Gyeonggi-do, South Korea) or high-fat diet (HFD) for 14 weeks. The HFD was composed of 200 g of fat/kg (170 g of lard plus 30 g of corn oil) and 1% cholesterol. All mice were housed in standard cages in a temperature controlled (21 ± 2.0°C) room with a 12 h light–dark cycle and were given free access to purified water.
Food intake of the mice was recorded daily and their body weights were measured weekly. At the end of the experimental period, after being fasted for 16 h, the mice were anaesthetised with diethyl ether and their plasma was obtained and stored at −80°C. The visceral and subcutaneous fat pads were dissected out, weighed, snap frozen in liquid nitrogen and then stored at −80°C. All animal experiments were performed in accordance with the Korean Food and Drug Administration guidelines. Protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Yonsei Laboratory Animal Research Center (YLARC) (Permit no. 2011-0061). All mice were maintained in the YLARC’s pathogen-free facility.
Plasma concentrations of triacylglycerol, total cholesterol, NEFA, HDL-cholesterol and glucose were measured using commercial kits (Bio-Clinical System, Gyeonggi-do, Republic of Korea). Plasma insulin levels were analysed using a mouse insulin ELISA kit (Alpco Diagnostics, Windham, NH, USA). The HOMA-IR index was calculated as fasting plasma glucose concentration (mmol/l) multiplied by fasting insulin level (pmol/l) divided by 22.5. Plasma levels of TNF-α, IL-6 and monocyte chemoattractant protein-1 (MCP-1) were measured using an ELISA kit (Millipore, Billerica, MA, USA).
The epididymal fat pads were fixed in neutral buffered formalin, embedded in paraffin and sectioned at 5 μm. The tissue sections were stained with hematoxylin and eosin (H&E).
Oral glucose tolerance test
Two weeks before the end of the experimental period, mice were fasted for 16 h and administered orally with glucose (gavage with 2 g glucose/kg body weight). Blood glucose was measured using a Precision Q.I.D glucometer (Abbott Laboratories, North Chicago, IL, USA).
RNA extraction and semi-quantitative RT-PCR
Total RNA was isolated from the epididymal white adipose tissue (epiWAT) and subcutaneous WAT (subWAT) of each mouse using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and was reverse transcribed using the Superscript II kit (Invitrogen) according to the manufacturer’s recommendations. PCR was performed as follows: 10 min at 94°C; 30–33 cycles at 94°C for 30 s and 55°C for 30 s; 72°C for 1 min and 10 min incubation at 72°C. PCR products were size-fractionated on a 2% agarose gel and stained with ethidium bromide. The band intensities were quantified using Quantity One analysis software 4.6 (Bio-Rad, Hercules, CA, USA). Sequences of all primers are listed in electronic supplementary material (ESM) Table 1.
Western blot analysis
To obtain total protein, the epiWAT samples obtained from each mouse were homogenised at 4°C in an extraction buffer (composition in mmol/l: 100 Tris-HCl, pH 7.4, 5 EDTA, 50 NaCl, 50 sodium pyrophosphate, 50 NaF and 100 orthovanadate plus 1% Triton X-100, 1 mmol/l phenylmethanesulfonyl fluoride, 2 μg/ml aprotinin, 1 μg/ml pepstatin A and 1 μg/ml leupeptin) and centrifuged at 13,000g for 20 min at 4°C. To obtain membrane protein, the epiWAT was homogenised in a buffer containing 20 mmol/l HEPES, pH 7.4, 4 mmol/l EDTA, 250 mmol/l sucrose, two tablets of protein inhibitor, 1 mmol/l sodium orthovanadate and 1% Triton X-100. The homogenates were centrifuged at 2,000g for 1.5 h at 4°C and the supernatant fractions were centrifuged at 150,000g for 1.5 h at 4°C. Total protein or membrane protein (40 μg) was separated on an 8% SDS-PAGE gel and subsequently transferred onto nitrocellulose membranes (Amersham, Little Chalfont, UK). The membranes were blocked with 5% BSA in Tris-buffered saline/Tween buffer (10 mmol/l Tris-HCl, pH 7.5, 150 mmol/l NaCl and 0.05% Tween 20), incubated with primary antibodies (diluted 1:1,000) overnight at 4°C and washed in Tris-buffered saline with 0.05% Tween 20. Antibodies to the following proteins were commercially obtained from the indicated sources: β-actin, STAT5, phospho-STAT5 and SOCS-1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and extracellular signal-regulated kinase (ERK), phospho-ERK (Thr202/Tyr204), IRS1, phospho-IRS1 (Tyr20), protein kinase B (Akt), phospho-Akt (Tyr176) and GLUT4 from Cell Signaling Technology (Danvers, MA, USA). After washing, the corresponding secondary antibodies were applied for 1 h at room temperature in Tris-buffered saline containing 0.05% Tween-20. Signals were detected using a chemiluminescent detection system (Amersham). The protein bands were then quantified using Quantity One analysis software 4.6 (Bio-Rad).
Immunohistochemistry Paraformaldehyde-fixed, paraffin-embedded epiWAT was cut into 4 μm sections. For antigen retrieval, the slides were autoclaved in 10 mmol/l sodium citrate buffer (pH 6.0). Endogenous peroxidase was blocked with 0.345% H2O2 for 30 min and sections were further blocked with 5% BSA in PBS for 30 min. The sections were incubated overnight at 4°C with the primary antibody F4/80 (1:100) (Abcam, Cambridge, MA, USA) and then incubated with biotinylated anti-mouse secondary antibody (1:200) (Dako, Glostrup, Denmark) for 1 h at room temperature, followed by incubation in avidin–biotin complex solution (Vector, Burlingame, CA, USA). Staining was performed with diaminobenzidine (DAB) (Vector) according to the manufacturer’s protocol. The DAB-stained preparations were visualised with a general optical microscope, TE-2000 (Nikon, Tokyo, Japan).
RT-PCR and Western blotting data are shown as the mean ± SEM of three independent experiments (n = 2 or 3 per experiment) for each group, cumulatively including eight mice. An unpaired Student’s t test analysis was used for all data comparisons between the Il-7r KO and WT control mice fed either chow or an HFD. All statistical analyses were performed with SPSS 21.0 software (SPSS, Chicago, IL, USA), and significance was set at *p < 0.05, **p < 0.01 and ***p < 0.001.
HFD increased Il-7r expression in the WAT of mice
Il-7r KO mice exhibited decreased body weight and visceral fat pad weight
Il-7r KO mice showed decreased plasma lipid levels
Il-7r KO mice displayed modulated expression of genes related to adiposity and thermogenesis
Il-7r KO mice were protected from WAT inflammation induced by HFD
The expression of genes relating to M2 markers, mannose receptor 1 (Mrc1), macrophage galactose N-acetyl-galactosamine-specific lectins 1 (Mgl1, also known as Clec10a) and arginase 1 (Arg1), was significantly increased in the epiWAT of Il-7r KO mice vs WT controls on both chow and HFD, whereas the expression of genes related to M1 markers, cluster of differentiation 68 (Cd68) and complement component receptor 4 subunit (Cd11c, also known as Itgax), decreased (Fig. 5e, f). We also observed a substantial decrease in F4/80 immunostaining and fewer crown-like structures in the epiWAT of Il-7r KO vs WT mice (Fig. 5g).
Il-7r KO mice were protected from HFD-induced insulin resistance
We found that tyrosine phosphorylation of both IRS1 and Akt increased in epiWAT of Il-7r KO mice relative to WT controls on both chow and HFD (Fig. 6f, g). The level of GLUT4 protein found in the membrane fraction of the epididymal adipose tissue was markedly increased in HFD-fed Il-7r KO mice relative to WT mice, whereas the amount of total cellular GLUT4 remained the same when comparing genotypes (Fig. 6h). The ratio of GLUT4 in the plasma membrane to the total amount of GLUT4 was significantly increased in Il-7r KO mice when compared with WT controls on the HFD (Fig. 6h).
The expression of IL-7R was thought to be restricted to haematopoietic cells, primarily B and T lymphocytes. Levels of IL-7R are tightly regulated during lymphocyte development, with alterations occurring depending on cell maturity and activation status . Cosenza et al  demonstrated that IL-7R was also expressed in non-haematopoietic cells, including lung, neuronal, breast, renal and colon cell lines. Through cDNA microarray analysis, we previously observed that Il-7r mRNA was expressed in the WAT of mice, with significantly higher levels in HFD-fed mice than in normal diet-fed mice (T. Park, data not published). These observations prompted us to focus on the role of IL-7R in WAT, related to obesity and metabolic complications.
Oestrogens, the primary female sex hormones, are suggested to reduce visceral adiposity and prevent insulin resistance and glucose intolerance in rodents [27, 28]. For this reason, male mice are more susceptible to diet-induced obesity than female mice. This sex difference was also observed in our present study; female mice exhibited less HFD-induced increase in body weight than male mice (25% vs 32%) (ESM Fig. 1). When mice fed an HFD were subjected to glucose tolerance tests, female mice had lower blood glucose levels than male mice at every time point (ESM Fig. 2). Nevertheless, in this study, mice deficient in Il-7r exhibited a similar extent of decrease in body weight and similar insulin resistance when comparing sexes (ESM Figs 1 and 2). Therefore, the therapeutic effect of IL-7R inhibition in obesity and metabolic diseases could be similar in male and female mice.
Our findings demonstrate that a lack of Il-7r protects mice from HFD-induced insulin resistance by improving glucose homeostasis and insulin sensitivity. When a glucose dose is administered based on the total body weight of mice with different fat mass for the evaluation of oral glucose tolerance, the amount of glucose to which the lean tissue, the principal site of glucose disposal, is exposed could be disproportionately low due to decreased fat mass in Il-7r KO mice compared with that in obese WT mice with similar lean mass. Therefore, it is plausible that lean mice could be misdiagnosed as being less glucose intolerant simply because they receive less glucose for the same lean body mass. Nevertheless, in this study, fasting blood glucose and insulin levels were also significantly improved in HFD-fed Il-7r KO mice when compared with their WT counterparts. Mechanistically, IRS-1 phosphorylation on tyrosine residue stimulates GLUT4 translocation and therefore improves insulin sensitivity . In recent studies, SOCS-1 has been found to cause insulin resistance in obesity by inhibiting tyrosine phosphorylation of IRS-1 in muscle cells and adipocytes . It has been reported that IL-7R signalling involves the recruitment of STAT5, leading to expression of its target gene, Socs1, in T lymphocyte lineages . In this study, we found that the level of phosphorylated STAT5 and SOCS-1 was reduced in the epiWAT of Il-7r KO mice vs WT controls (Fig. 1b, c). Furthermore, Il-7r KO mice exhibited increased tyrosine IRS-1 phosphorylation and GLUT4 translocation from the cytoplasm to the membrane compared with WT controls (Fig. 6f, h). Therefore, we suggest that the improvement of insulin sensitivity observed in Il-7r KO mice may be associated with decreased expression of Socs1, the target gene of the IL-7R–STAT5 pathway in visceral adipocytes (Fig. 7). Moreover, it is well-established that proinflammatory cytokines, such as TNF-α and IL-6, from enlarged visceral adipose tissue impair GLUT4 translocation to the plasma membrane . Despite HFD feeding, Il-7r KO mice exhibited decreased fat mass and expression levels of proinflammatory cytokines. Thus, improvement of insulin sensitivity in Il-7r KO mice could also be a secondary event followed by decreased fat mass.
Generally, plasma cytokine levels correlate with the expression of proinflammatory adipokines in WAT . In our data, lower levels of plasma proinflammatory cytokines (TNFα, IL-6 and MCP1) were observed in Il-7r KO mice than in WT controls (Fig. 5a). We also found a marked decrease in the gene expression of proinflammatory cytokines in the WAT of Il-7r KO mice vs WT controls (Fig. 5d). The ERK–MAPK pathway plays a key regulatory role in the production of proinflammatory cytokines (TNFα and IL-6) by activation of transcriptional factors such as nuclear factor κ-light-chain-enhancer of activated B cells in adipocytes . In the present study, Il-7r KO mice exhibited decreased expression of p-ERK and Nf-κb in the epiWAT when compared with WT controls (Figs 4b and 5d). Consequently, we suggest that the IL-7R–ERK signalling pathway in WAT may be a factor in the initiation and maintenance of obesity-induced peripheral inflammation (Fig. 7). At the same time, it is also probable that the reduction in fat mass caused by Il-7r deletion may have contributed to alleviation of WAT inflammation.
Adipose tissue expansion in obesity is associated with infiltration of macrophages that localise to crown-like structures surrounding apoptotic adipocytes . We performed immunohistological staining of adipose tissue for the macrophage marker F4/80 and found markedly fewer macrophages in Il-7r KO mice than in WT mice (Fig. 5g). Obesity also leads to a shift in the activation state of macrophages from the M2 anti-inflammatory state to the M1 proinflammatory state . Activated M1 macrophages produce large amounts of proinflammatory mediators, such as TNFα and IL-6, which act on adipocytes to induce an insulin-resistant state . In the present study, expression of M1 marker genes (Cd68 and Cd11c) was dramatically decreased in the WAT of Il-7r KO mice vs WT controls (Fig. 5f), suggesting reduced infiltration of M1 macrophages. Besides the lack of the IL-7R–ERK signalling pathway, the reduction in M1 macrophages is a reasonable mechanism for the decreased WAT inflammation and improved insulin sensitivity observed in Il-7r KO mice.
In conclusion, the present study demonstrates that Il-7r deficiency significantly decreases body and visceral fat pad weights and improves adipose tissue inflammation and insulin sensitivity in mice. These observations implicate IL-7R as a mediator of lipid accumulation in WAT and metabolically triggered inflammation. Further study needs to be done in a conditional knockout mouse model and primary pre-adipocytes to confirm the role of IL-7R in adipogenesis and insulin resistance in WAT.
This research was supported by the SRC program (Center for Food and Nutritional Genomics: Grant no. 2015R1A5A6001906) of the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology and by a grant of Ministry of Health and Welfare, Republic of Korea (HI13D23470200).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
ML and TP designed the study. ML, SJS, MSC, RY and TP participated in acquisition, analysis and interpretation of data. ML and TP drafted the manuscript and ML, SJS, MSC, RY, and TP revised it critically for important intellectual content and approved the final version to be published. TP is the guarantor of this work.
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