Increased Ifi202b/IFI16 expression stimulates adipogenesis in mice and humans
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Obesity results from a constant and complex interplay between environmental stimuli and predisposing genes. Recently, we identified the IFN-activated gene Ifi202b as the most likely gene responsible for the obesity quantitative trait locus Nob3 (New Zealand Obese [NZO] obesity 3). The aim of this study was to evaluate the effects of Ifi202b on body weight and adipose tissue biology, and to clarify the functional role of its human orthologue IFI16.
The impact of Ifi202b and its human orthologue IFI16 on adipogenesis was investigated by modulating their respective expression in murine 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocytes. Furthermore, transgenic mice overexpressing IFI202b were generated and characterised with respect to metabolic traits. In humans, expression levels of IFI16 in adipose tissue were correlated with several variables of adipocyte function.
In mice, IFI202b overexpression caused obesity (Δ body weight at the age of 30 weeks: 10.2 ± 1.9 g vs wild-type mice) marked by hypertrophic fat mass expansion, increased expression of Zfp423 (encoding the transcription factor zinc finger protein [ZFP] 423) and white-selective genes (Tcf21, Tle3), and decreased expression of thermogenic genes (e.g. Cidea, Ucp1). Compared with their wild-type littermates, Ifi202b transgenic mice displayed lower body temperature, hepatosteatosis and systemic insulin resistance. Suppression of IFI202b/IFI16 in pre-adipocytes impaired adipocyte differentiation and triacylglycerol storage. Humans with high levels of IFI16 exhibited larger adipocytes, an enhanced inflammatory state and impaired insulin-stimulated glucose uptake in white adipose tissue.
Our findings reveal novel functions of Ifi202b and IFI16, demonstrating their role as obesity genes. These genes promote white adipogenesis and fat storage, thereby facilitating the development of obesity-associated insulin resistance.
KeywordsAdipogenesis Beiging IFI16 Ifi202b Obesity Zfp423
Brown adipose tissue
Early B cell factor 2
New Zealand Obese obesity 3 (quantitative trait locus)
New Zealand Obese
Peroxisome proliferator-activated receptor γ
Quantitative trait locus
Stem cell antigen 1
Tübingen Family (study)
Uncoupling protein 1
White adipose tissue
Gonadal white adipose tissue
Subcutaneous white adipose tissue
Zinc finger protein
Obesity, an excessive expansion of adipose tissue, associates with metabolic perturbations that increase the risk for non-alcoholic fatty liver disease, type 2 diabetes and coronary heart disease. The increase in adipocyte size (hypertrophy) and adipocyte number (hyperplasia) both contribute to the development of obesity . Prolonged overnutrition and low levels of physical activity result in adipose tissue dysfunction, characterised by adipocyte hypertrophy, macrophage infiltration, impaired insulin signalling and insulin resistance . In addition, inflammatory cytokine levels rise and, together with excessive amounts of NEFA, promote ectopic fat deposition and lipotoxicity in the liver, muscle and pancreas. These effects ultimately contribute to the development of insulin resistance [3, 4]. Two types of adipose tissue can be distinguished: white adipose tissue (WAT), which stores excess energy as triacylglycerol and brown adipose tissue (BAT), which is specialised in the dissipation of energy through the production of heat . Relatively high quantities of BAT are associated with lower body weight and BAT decreases with age . In addition, a third type of fat cell, referred to as beige or brite (brown in white), has been recognised in certain kinds of WAT depots [7, 8].
Adipocyte commitment and differentiation are complex processes tightly controlled by a transcriptional cascade composed of several transcription factors, among which zinc finger protein (ZFP) 423, CCAAT/enhancer-binding proteins (C/EBPs) and Kruppel-like factors (e.g. KLF15) play an essential role [9, 10]. In 3T3-L1 murine pre-adipocytes, C/EBPβ is induced early in the differentiation process to activate the expression of C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ), the two master regulators of terminal adipocyte differentiation [11, 12].
Previously, to identify obesity and diabetes genes, we performed linkage studies in an intercross of lean C57BL/6J and obese New Zealand Obese (NZO) mice resulting in a major obesity quantitative trait locus (QTL), Nob3 (NZO obesity 3), on chromosome 1 . By positional cloning, we defined Ifi202b (IFN-activated gene 202B) as a putative obesity gene . We further showed that Ifi202b plays a role as a diabetes gene as its overexpression in primary islet cells inhibited proliferation . IFN-inducible protein IFI202b and the human orthologue IFI16 belong to the p200 family of transcriptional modulators of cell proliferation, differentiation, apoptosis and inflammation [16, 17].
The aim of the current work was to provide direct functional evidence for Ifi202b as an obesity gene and to clarify the role of its human orthologue IFI16 in obesity-associated insulin resistance. To study the impact of Ifi202b on adipose tissue biology and to investigate the underlying mechanisms of its obesogenic potency, we overexpressed IFI202b in 3T3-L1 pre-adipocytes and in lean C57BL/6J mice lacking endogenous Ifi202b because of a vast deletion upstream of exon 2 . The effects of the human orthologue IFI16 were analysed in Simpson–Golabi–Behmel syndrome (SGBS) pre-adipocytes, as well as in obese human volunteers.
C57BL/6J mice overexpressing IFI202b, herein referred to as B6-Tg(Ifi202b) mice, were generated by Ozgene (Perth, WA, Australia). c-Myc-tagged Ifi202b cDNA downstream of the human ubiquitin C promoter was integrated into the ROSA26 locus. C57BL/6J wild-type (B6-wt) littermates were used as control animals. NZO mice were taken from our own colony (NZO/HIBomDife, Nuthetal, Germany). For details of housing and diet conditions, see electronic supplementary materials (ESM) Methods.
Characterisation of mice
Blood glucose, plasma variables (insulin, NEFA and triacylglycerol levels), glucose tolerance and body composition were assessed as described previously . Plasma adiponectin and leptin levels were measured using the Mouse Adiponectin/Acrp30 and Mouse/Rat Leptin ELISA (DY1119 and MOB00, respectively; R&D Systems, Minneapolis, MN, USA). Histological analysis of WAT, BAT and liver tissues was carried out using H&E staining (see ESM Methods for further details).
Intraperitoneal ITT and insulin sensitivity
For the i.p. ITT, 12- and 20-week-old non-fasted mice were injected with insulin (0.75 IU/kg body weight; Actrapid Penfill; Novo Nordisk, Mainz, Germany) and blood glucose levels were measured at various time points. To examine hepatic and muscle insulin sensitivity, after a 6 h fast, mice were injected with NaCl (control) or insulin (0.75 IU/kg body weight). Twenty minutes later, mice were killed by cervical dislocation under isoflurane anaesthesia and samples were taken for western blot analysis.
Ex vivo lipolysis assay
Gonadal WAT (WATgon) explants from 8-week-old mice were stimulated with 10 μmol/l isoprenaline (known as isoproterenol in the USA) (Sigma-Aldrich, St Louis, MO, USA) or isoprenaline plus insulin (178 μmol/l) in 160 μl of high-glucose DMEM (PAN-Biotech, Aidenbach, Germany). After 2 h, release of NEFA was detected (NEFA-HR(2); Wako Chemicals, Neuss, Germany).
Cell culture and differentiation
For details of murine 3T3-L1 and human SGBS cell culture and differentiation conditions, see ESM Methods. WT-1 brown pre-adipocytes  tested negative for mycoplasma and cultivated and differentiated as described , with modified dexamethasone concentration (5 μmol/l).
Overexpression of IFI202b and analysis of lipid droplet formation in 3T3-L1 pre-adipocytes
3T3-L1 pre-adipocytes were infected with a c-Myc-Ifi202b encoding adenovirus (ViraQuest, North Liberty, IA, USA). Empty adenovirus was used as a control. Lipid droplet formation in IFI202b-overexpressing 3T3-L1 adipocytes was assessed by fluorescent staining. A detailed protocol for IFI202b overexpression in pre-adipocytes can be found in ESM Methods.
siRNA-mediated knockdown of IFI202B/IFI16 in pre-adipocytes
Expression of IFI202b in murine 3T3-L1 and WT-1 pre-adipocytes, as well as IFI16 in human pre-adipocytes was suppressed via electroporation using specific siRNA (IFI16: J-020004-05-0050; Dharmacon, Lafayette, CO, USA) as described previously .
Isolation and differentiation of primary adipogenic precursor cells
Adipogenic precursor cells were isolated from subcutaneous WAT (WATsc) of 8-week-old mice fed a standard diet (V153x R/M-H; ssniff, Soest, Germany), as described . Stem cell antigen 1 (SCA1)-positive cells [22, 23] were purified by FACS (BD FACSAria III; BD Biosciences, San Jose, CA, USA) and differentiated, as previously described , with minor modifications.
RNA extraction and expression analysis
RNA from cells was isolated and reverse transcribed for quantitative real-time PCR analyses. Expression levels of Ifi202b, Zfp423, Pparg, Cebpa, Adipoq, Glut4 (also known as Slc2a4), Fabp4, Plin1, Hsd11b1, Pref1 (Dlk1), Ucp1, Cidea, Prdm16, Tcf21, Tle3, Ebf2, Ppargc1a, Ppara, Il4, Il6, Il10, Tnfa (Tnf) and Ccl2 were analysed in murine samples. In human samples, IFI16, ZNF423, CEBPA, PPARG, ADIPOQ, FASN, GLUT4 (SLC2A4), PLIN1 and IL6 were analysed. Genes were detected using specific TaqMan Gene Expression Assays and the 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) and gene expression profiling of WATgon of 8-week-old mice was performed by OakLabs (Henningsdorf, Germany). For details see ESM Methods.
Protein extraction and western blotting
Liver, muscle, BAT and WAT tissues from mice were homogenised and analysed by western blotting . A monoclonal anti-IFI202b antibody was raised against the C-terminal peptide 1–21: C-MSNRNLRSSTNSEFSEGQHQ. Primary antibodies against c-Myc (1:500 dilution; Clontech, Saint-Germain-en-Laye, France), uncoupling protein 1 (UCP1; 1:1000; ab10983; Abcam, Cambridge, UK), total Akt (t-Akt; 1:1000; no. 9272; Cell Signaling, Danvers, CO, USA), p-Akt (Ser473; 1:1000; no. 9271; Cell Signaling), HDAC1 (1:25,000; ab7028; Abcam) and GAPDH (1:20,000; AM4300; Thermo Fisher Scientific), and appropriate horseradish peroxidase-labelled secondary antibodies (Dianova, Hamburg, Germany) were applied. Experimental controls were used to validate antibodies.
IFI16 expression in human adipose tissue
Previously, IFI16 expression in adipose tissue was measured in 166 obese individuals . Here, we compared those exhibiting the 10% highest levels of IFI16 with those with the 10% lowest levels of expression. Human phenotyping , IFI16 expression analyses  and basal and insulin-stimulated glucose uptake into isolated adipocytes were measured as previously described . All study protocols were approved by the ethics committee of the University of Leipzig (Leipzig, Germany). All participants gave written informed consent before taking part in the study.
Tagging SNP analysis in humans
From the ongoing Tübingen Family (TÜF) study for type 2 diabetes, healthy adults at increased risk for type 2 diabetes were recruited . In the present study, a subset of 1896 participants with complete leukocyte and CRP measurements and a second subset of 420 subjects with complete IL6 measurements were selected for genotyping. Tagging SNPs covering the common genetic variation were identified and genotyping was conducted, as detailed in ESM Methods. Association analyses were performed by multiple linear regression analysis (least squares method) to adjust for the confounding variables sex, age and BMI. The study adhered to the Declaration of Helsinki, and its protocol was approved by the local ethics boards (Ethics Committees of the Eberhard Karls University Tübingen, Tübingen, Germany and the Karolinska Institute, Stockholm, Sweden). Informed written consent was obtained from all participants.
All data are reported as mean ± SEM. Statistical analysis was performed using Student’s t test for single comparisons and one-way ANOVA with Tukey’s post hoc test for differences between more than two groups. Differences between two groups over time were evaluated using a two-way ANOVA with Bonferroni’s or Sidak’s correction for multiple comparisons (GraphPad Prism version 6.00; GraphPad, La Jolla, CA, USA). For SNP analysis, JMP 11.2.0 (from SAS, Cary, NC, USA) was used for statistical analysis. Significance was accepted at p < 0.05, p < 0.01 and p < 0.001. Samples were randomised, and no data were omitted. The experimenters were not blind to group assignment.
Overexpression of IFI202b increases body weight and fat mass
Plasma variables detected in B6-wt and B6-Tg(Ifi202b) mice at the post-absorptive state
383.90 ± 30.31
(n = 10)
511.78 ± 41.63
(n = 9)
271.20 ± 31.78
(n = 10)
403.70 ± 47.75
(n = 9)
228.80 ± 13.23
(n = 10)
463.89 ± 43.21
(n = 8)
1.81 ± 0.11
(n = 10)
2.28 ± 0.12
(n = 9)
1.21 ± 0.07 (n = 10)
1.57 ± 0.08 (n = 9)
1.27 ± 0.05
(n = 10)
2.08 ± 0.08
(n = 8)
2.91 ± 0.83
(n = 7)
5.77 ± 1.36
(n = 9)
7.83 ± 1.50
(n = 10)
41.83 ± 5.81
(n = 12)
IFI202b expression impairs insulin sensitivity and induces fatty liver
IFI202b expression increases during early stages of adipocyte differentiation
Ifi202b mRNA levels were markedly increased during adipogenesis, reaching a maximum at 24 h after application of the differentiation cocktail and then decreasing over time, with the lowest levels in mature adipocytes (ESM Fig. 3a). As IFI202b is thought to function as a transcriptional modulator, we tested whether its subcellular localisation changes during the differentiation process. Nuclear and cytosolic fractions of confluent 3T3-L1 pre-adipocytes, as well as of cells at different time points after induction of differentiation, were isolated and endogenous IFI202b was detected by western blotting (ESM Fig. 3b). Shortly after induction of adipogenesis (24 h), the total amount of IFI202b increased, as did the quantity of IFI202b that appeared in the nuclear fraction (ESM Fig. 3b). This observation was also confirmed by immunocytochemistry. At 24 h, IFI202b was mostly present in the nucleus of the cells, whereas on day 3 it was mainly located in the cytosol (ESM Fig. 3c).
Overexpression of IFI202b increases Zfp423 mRNA levels in 3T3-L1 cells and in adipose tissue of B6-Tg(Ifi202b) mice
Role of the human orthologue IFI16 in SGBS adipocyte differentiation
Ifi202b enhances the white adipocyte phenotype and inhibits the thermogenic gene program
Elevated Zfp423 expression was also detected in WATgon of 8-week-old B6-Tg(Ifi202b) mice as compared with B6-wt mice (Fig. 6c). Recently, Shao and colleagues described ZFP423 as a critical regulator maintaining white adipocyte identity through the suppression of thermogenic genes, such as Ebf2 and Prdm16 . We screened our transcriptome data for differentially expressed white-selective and thermogenic genes and detected higher mRNA levels of white marker genes (Tcf21 and Tle3, Fig. 6d) and strikingly lower levels of beige-like marker genes (Ebf2, Ppargc1a, Ppara, Cidea and Ucp1, Fig. 6e, f), which also resulted in a stable downregulation of UCP1 (Fig. 6g). UCP1 was detectable in WATgon from B6-wt mice but absent in all samples from Ifi202b transgenic mice, supporting our hypothesis that IfI202b promotes the white adipocyte phenotype. In line with this, overexpression of IfI202b in WT-1 brown adipocytes resulted in a significant decrease of thermogenic genes including Prdm16, Cidea and Ucp1 (ESM Fig. 4c–g). Even though we also detected lower expression levels of Ucp1 mRNA in BAT from B6-Tg(Ifi202b) mice (Fig. 6h), this effect was not translated into protein levels (Fig. 6g).
Thus, the impaired browning capacity of WAT in Ifi202b transgenic mice might be responsible for the significantly lower body temperature of B6-Tg(Ifi202b) mice (Fig. 6i). Taken together, the increased expression of IfI202b induces Zfp423 expression, resulting in a lack of beiging/browning of WAT, finally leading to a lower body temperature and obesity.
Elevated expression of human IFI16 associates with larger adipocytes and increased inflammatory cytokines
Our data provide direct functional evidence for the role of Ifi202b/IFI16 as an obesity gene and shows that it adjusts adipocyte commitment, maintenance of white adipocyte identity, fat cell size and the inflammatory state of adipose tissue. In 3T3-L1 and primary adipocyte progenitor cells, IfI202b induced Zfp423 expression and thereby activated pro-adipogenic genes, enhanced whitening and inhibited the adipocyte thermogenic gene program. The consequent larger lipid droplets and elevated triacylglycerol accumulation are evident in IFI202b-overexpressing mice, which exhibited elevated fat mass, adipocyte hypertrophy and insulin resistance. Data from human studies revealed that individuals expressing high levels of IFI16 in adipose tissue exhibited larger fat cells, elevated CRP plasma concentrations and IL6 mRNA levels and impaired insulin-stimulated glucose uptake in adipose tissues than those with low IFI16 expression.
As IFI202b is mainly expressed in WAT and nearly absent in BAT , we hypothesised that it induces pathways involved in adipocyte whitening. We identified ZFP423 as a downstream target of IfI202b and were able to block the IFI202b-specific effects by suppressing ZFP423. ZFP423 is a vital white adipocyte determination factor with dual roles. First, ZFP423 is a marker of committed pre-adipocytes and regulates Pparg levels, thereby inducing adipocyte differentiation . Second, ZFP423 maintains white adipocyte identity via the suppression of thermogenic gene expression. Inactivation of ZFP423 in mature adipocytes leads to the white-to-beige conversion of WAT cells via induction of the brown fat cell determination factors Prdm16 and Ebf2 . Besides Zfp423, we detected an upregulation of the relevant adipogenic marker genes Cebpa, Pparg, Plin1 and Hsd11b1 in 3T3-L1 pre-adipocytes and in primary precursor cells of B6-Tg(Ifi202b) mice, indicating that IFI202b improves the commitment and differentiation capacity of pre-adipocytes. The fact that thermogenic genes including Ebf2, Cidea and Ucp1 were reduced in WATgon of B6-Tg(Ifi202b) mice indicates that IFI202b mediates the maintenance of white fat cell identity by suppressing thermogenic genes. This provides a possible explanation for the lower body temperature and subsequent development of obesity in B6-Tg(Ifi202b) mice. As seen with IFI202b in 3T3-L1 cells, inhibition of IFI16 in human SGBS pre-adipocytes suppressed the expression of adipogenic genes (ZNF423 in particular).
Often, elevated food intake is a main driver for the development of obesity. Although we found that food intake was not significantly affected by IFI202b overexpression in young mice, it was significantly increased in 25-week-old B6-Tg(Ifi202b) mice as compared with B6-wt mice (ESM Fig. 6). Together with the lack of WAT beiging, this could have contributed to the elevated body weight.
Patil et al recently showed that the adipose tissue-specific overexpression of the transcription factor ID1 (inhibitor of differentiation 1) caused an HFD-induced obesity by diminishing Prdm16. ID1 interacts with early B cell factor 2 (EBF2), thereby suppressing its transcriptional activity and resulting in reduced expression of Prdm16 . PR domain containing 16 (PRDM16) and EBF2 are both required to determine beige/brown adipocyte cell fate . Their genetic manipulation also affects body weight. Transgenic mice overexpressing the transcription factor EBF2 were protected from HFD-induced obesity since EBF2 robustly stimulated beige adipocyte development in the WAT of mice, even under conditions of thermoneutrality . In addition, overexpression of Prdm16 in mice increased their energy expenditure, limited weight gain and improved glucose tolerance on an HFD .
Several genes that are upregulated in response to IFI202b overexpression can participate in elevated fat storage. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which we have previously identified as downstream target of IFI202b , might increase lipid content in adipose tissue by altering the local action of glucocorticoids . Elevated GLUT4 could increase glucose uptake and consequently the fat content as has been shown for the adipocyte-specific GLUT4 transgenic mouse . Perilipin 1 protects triacylglycerol from lipolysis [38, 39] and could thereby contribute to larger adipocytes. In fact, we detected lower lipolysis in response to isoprenaline in Ifi202b transgenic mice.
As hypertrophic adipocytes secrete more leptin and less adiponectin, the leptin-to-adiponectin ratio has been proposed as a useful measure of insulin resistance . Indeed, this ratio was higher in Ifi202b transgenic mice than in B6-wt mice; glucose decrease in ITTs was impaired and insulin levels were not sufficient to suppress isoprenaline-stimulated lipolysis in fat explants of transgenic mice, indicating that IFI202b induces insulin resistance. Obesity associates with dyslipidaemia, which leads to hepatic accumulation of triacylglycerol and to an increased hepatic synthesis of VLDL . IFI202b-overexpressing mice showed enhanced plasma NEFA and triacylglycerol levels, with resulting ectopic hepatic lipid storage. We believe that an elevated action of IFI202b/IFI16 leads to hypertrophic adipocytes, elevated fat storage, induction of inflammatory cytokine expression and finally insulin resistance and fatty liver. Similar to our observations made in B6-Tg(Ifi202b) mice, elevated plasma triacylglycerol levels are often detected in obese individuals and adipose tissue dysfunction appears to be responsible for these abnormalities in lipid metabolism . Pathological expansion of the adipose tissue results in inflammation and increased immune cell and macrophage infiltration, proposed as one mechanism to explain the development of insulin resistance . In fact, humans with high IFI16 mRNA levels displayed higher concentrations of CRP in the plasma and IL6 expression in adipose tissue, effects that are associated with a lower insulin-stimulated glucose uptake.
Thus, IFI proteins could represent possible targets for altering adipogenesis and the development of obesity. Nevertheless, follow-up studies are needed to unravel the specific regulation and putative binding partners of IFI202b and IFI16.
In conclusion, our data show that IFI202b and its human orthologue IFI16 are key players in the development of obesity and insulin resistance: (1) by modulating fat storage in adipose tissue and liver; (2) by inhibiting beiging of WAT; and (3) by inducing inflammatory markers.
We gratefully thank M. Niehaus, C. Gumz, A. Teichmann and S. Ernst of the German Institute of Human Nutrition Potsdam-Rehbrücke (Nuthetal, Germany) for their skilful technical assistance as well as S. Gohlke for sharing her knowledge and skills concerning WT-1 cells with us. We kindly thank U. Smith of the University of Gothenburg for discussions on this project. Furthermore, the authors thank the staff of the Max Rubner Laboratory (Nuthetal, Germany) for animal care and technical support. Some of the data were presented in a talk at the 53rd EASD Annual meeting in 2017.
MS, KS, AG, CB, MR, WJ, CW, HS, AF, HUH, NK, MB and PFP contributed to the acquisition and analysis of data as well as to the critical interpretation of the results. AS, HV, HGJ, TJS, WJ and MS were responsible for the major study concept and critical revision of the manuscript. AS, MS, HV and KS participated in the drafting and writing of the manuscript. AG, CB, MR, WJ, CW, HS, AF, HUH, NK, MB, PFP and TJS were involved in the design of the study. All authors contributed to the interpretation of data, edited the manuscript, and approved its final version for publication. AS is the guarantor of this work.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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