Diet-induced obesity increases NF-κB signaling in reporter mice
© Springer-Verlag 2009
Received: 6 November 2008
Accepted: 10 March 2009
Published: 26 August 2009
The nuclear factor (NF)-κB is a primary regulator of inflammatory responses and may be linked to pathology associated with obesity. We investigated the progression of NF-κB activity during a 12-week feeding period on a high-fat diet (HFD) or a low-fat diet (LFD) using NF-κB luciferase reporter mice. In vivo imaging of luciferase activity showed that NF-κB activity was higher in the HFD mice compared with LFD-fed mice. Thorax region of HFD females displayed fourfold higher activity compared with LFD females, while no such increase was evident in males. In male HFD mice, abdominal NF-κB activity was increased twofold compared with the LFD males, while females had unchanged NF-κB activity in the abdomen by HFD. HFD males, but not females, exhibited evident glucose intolerance during the study. In conclusion, HFD increased NF-κB activity in both female and male mice. However, HFD differentially increased activity in males and females. The moderate increase in abdomen of male mice may be linked to glucose intolerance.
Obesity is a disease affecting increasing numbers of global populations, in some regions more than 30% of the adult population . Increased adipose tissue mass, especially in the abdominal region, is associated with diseases such as type-2 diabetes and atherosclerosis . It has been demonstrated that increased fat mass is associated with increased macrophage infiltration, increased release of cytokines, adipokines and free-fatty acids from adipocytes and/or activated macrophages, and local insulin resistance [3–6]. Adipose tissues also serve endocrine functions whereby adipokines and free-fatty acids are released into the circulation . This allows transport to the liver and skeletal muscle, often promoting reduced insulin sensitivity in these organs . Obesity and specifically the enlargement of the abdominal adipose depots are thus considered the major risk factors for the development of insulin resistance, a characteristic feature of type-2 diabetes and the metabolic syndrome .
A central mediator of inflammatory and stress responses is the NF-κB family of transcription factors. As a response to foreign pathogens and general stressful insults, NF-κB is activated in most cell types. In addition, NF-κB activity is linked to cancer development through its regulation of apoptosis, cell proliferation, angiogenesis, metastasis and cell survival . Recent evidence also suggests that NF-κB activation is crucial for development of insulin resistance. For example, Arkan et al.  disabled the inflammatory pathway within macrophages by creating myeloid-specific IκB kinase β (IKKβ) knockout mice. These mice were more insulin sensitive and partially protected from high-fat diet (HFD)-induced glucose intolerance and hyperinsulinemia. Moreover, Cai et al.  reported that the activation of NF-κB in transgenic mice expressing constitutively active IKKβ in hepatocytes (LIKK mice) lead to hyperglycemia and insulin resistance. Conversely, hepatocyte-specific deletion of NEMO (IKK-γ), which completely blocks NF-κB activation, protected against insulin resistance in mice-fed HFD . Furthermore, pharmacological manipulations of the NF-κB system by administering salicylates revealed that these transcription factors are central to the obesity-induced proinflammatory state leading to metabolic syndrome, insulin resistance and type-2 diabetes [14, 15].
Although a number of studies have explored the effect of obesity on inflammatory mediators, surprisingly few studies have directly compared activation of NF-κB itself in obese individuals compared with lean controls. The dynamic regulation of NF-κB activity during weight gain is thus unknown, and it is not known whether increased NF-κB signaling is presented before, simultaneously, or after metabolic parameters are affected. More specifically, the time course of inflammation induced in parallel with obesity on an HFD has not been elucidated, including the organs involved, nor the dynamics of inflammation development.
To address these questions, we have utilized transgenic mice harboring a luciferase gene specifically controlled by 3 NF-κB DNA-binding sites [16–18]. Thus, the luciferase activity directly reflects NF-κB transactivation due to the activation of the NF-κB signaling pathways. One group of NF-κB luciferase mice was switched to HFD, whereas another control group was maintained on LFD. NF-κB activity was monitored as luciferase activity by non-invasive in vivo molecular imaging. In this paper, we analyzed for the first time in a non-invasive manner the in vivo NF-κB response to HFD over time.
Materials and methods
NF-κB luciferase reporter mice
Transgenic mice carrying a transgene with three binding sites for NF-κB (5′GGGACTTTCC′3) coupled to the luciferase gene were used in this study. The transgene is flanked by insulator sequences from the chicken [β]-globin gene  to reduce interference from the genome. The resulting transgenic founder, which was the basis of these experiments, was the result of screening of several founders. The original genetic background was a mix of C57BL/6J and CBA. Subsequently, the mice were backcrossed four times with C57BL/6J prior to this experiment. The mice were housed in accordance with the guidelines of the Federation of European Laboratory Animal Science Associations (FELASA).
Composition of diets (% of food weight; w/w)
In vivo imaging of NF-κB activity
Blood samples were taken at t = 0, 5 and 9 weeks after the start of the experiment. The saphena vein was first exposed by shaving the skin and then punctured allowing the blood to be collected from the surface of the skin into EDTA-coated capillaries. Plasma was isolated after centrifugation for 10 min at 6,000g and stored at −70°C. Concentrations of MCP-1, IL-6, TNF-α insulin, leptin, PAI-1 and resistin were determined in the isolated plasma with a multiplexed immunoassay (Mouse Adipokine LINCOplex kit; Millipore, Billerica, MA) according to the manufacturer’s instructions on a Luminex instrument (BioRad, Hercules, CA). Plasma TNF-α levels were below the detection limit of the assay.
Data were analyzed using the SPSS software package (SPSS 15 for Windows, Chicago, IL). Comparisons of repeated measurements between groups were conducted with mixed model analysis using the Toeplitz covariance structure. Student’s t test was used to compare groups at individual time points. The direction and strength of linear relationships between variables were evaluated using Pearson correlation coefficient and two-tailed test of significance.
Elevated whole body NF-κB activity in mice-fed HFD
In the abdominal region, HFD male mice displayed a twofold increase in NF-κB activity compared with LFD male mice (P = 0.002). In female mice, however, no such difference was found in the abdominal region between the two feeding groups (Fig. 3b). Thus, these results indicate that the high-fat feeding increases NF-κB activity differentially in the abdominal and thoracic region in a sex-specific manner.
Thoracic NF-κB activity associated with relative body weight gain in HFD mice
The enhanced body weight was also manifested in two- to threefold (P < 0.01) increased plasma leptin levels in HFD compared with LFD after 5 and 9 weeks of feeding in both sexes (Fig. 5c).
Abdominal NF-κB activity in male HFD mice associated with glucose intolerance
We next investigated whether the elevated NF-κB activity in male mice given HFD correlated with development of insulin resistance, but no correlation was found between abdominal NF-κB activity and glucose intolerance in HFD after 6 (R = −0.222; P = 0.598) or 11 (R = 0.111; P = 0.793) weeks, as inferred from the area under the curve of the IP-GTT. The difference in metabolic status between males and females was further supported by data showing that HFD male mice had twofold higher resistin levels as compared to LFD males (P = 0.005), an effect not paralleled in female mice (Fig. 6c). Taken together, these data indicate that only males fed with HFD developed glucose intolerance (Fig. 6a), and that the individual NF-κB activity does not correlate with the individual degree of glucose intolerance achieved by HFD feeding.
In our present study, we have for the first time followed the in vivo NF-κB activity non-invasively over time in experimental animals fed with HFD and LFD. We observed that mice-fed LFD increased their whole body NF-κB activity 2.3-fold above the baseline values after 12 weeks. At the same time point, mice-fed HFD displayed about 3.5-fold increased whole body NF-κB activity. This shows that basal NF-κB activity increases in a time-dependent manner in mice ingesting a regular laboratory diet containing low amounts of fat, whereas a diet high in fat content adds to this effect.
The HFD-evoked increase in NF-κB activity was not uniform, but was sex-dependently localized to different body regions. Females, but not males, displayed increased NF-κB activity in the thoracic region. Conversely, the abdominal area of males, but not females, showed enhanced NF-κB activity in response to HFD. Plasma levels of inflammatory cytokines were not enhanced by HFD suggesting that these inflammatory processes are localized to individual tissues rather than at the systemic level.
We observed increased NF-κB activity in the thoracic region of HFD-fed female mice, indicative of increased activity in the thymus and/or thoracic lymph nodes (i.e. axillary, brachial lymph nodes). To what extent HFD may influence and even increase NF-κB activity in these lymphoid tissues is not clear from our studies. Previous experiments indicate that mice-fed HFD have larger thymuses than mice fed with a control diet . Moreover, leptin-deficient ob/ob mice have highly involuted thymuses, and leptin can attenuate LPS-induced and starvation-induced thymic involution [21–23]. Thus, one possibility is that the enhanced thoracic NF-κB activity in the female HFD groups may be explained by elevated leptin levels. In the current study, females gained more weight relative to baseline values than males and it was evident that NF-κB activity showed a stronger association with body weight gain, rather than a high-fat intake per se.
It has been suggested that the ingestion of HFD may induce low-grade inflammation in adipose tissue . Especially, the adipose tissue located in the abdominal region has been linked to the development of diseases such as type-2 diabetes and atherosclerosis . We hypothesized, therefore, that the abdominal region would be the major site of HFD-induced NF-κB activity associated with glucose intolerance. We did observe an increase in abdominal NF-κB activity in response to HFD, but the effect was restricted to male mice and was rather modest compared with the thoracic activity in females. We also found that only males developed glucose intolerance and displayed elevated levels of plasma resistin, indicative of the development of diabetes type 2. Altogether, these data suggest a link between increased NF-κB activity and glucose intolerance. However, we could not find any intra-individual correlation between abdominal NF-κB and parameters of glucose intolerance, possibly due to the limited number of animals.
Owing to the scattering of light from the inmost organs, it is difficult to judge the exact origin of the luciferase signal. By visual inspection, it is most likely that the activity arises in visceral fat and/or mesenteric lymph nodes, and not from liver. Abdominal NF-κB activity may be due to immune cells invading the adipose tissue depots or an alteration of the microbial environment of the colon [25, 26]. Alternatively, enhanced NF-κB activity may be due to HFD-induced activation of T cells in the mesenteric lymph nodes surrounded by visceral fat. A recent work demonstrated that HFD induces atrophy of mesenteric lymph nodes, explained by inflammation-induced stimulation of T cells .
Previous studies utilizing electrophoresis mobility shift assays (i.e. NF-κB DNA binding assay) or phosphorylation assays (NF-κB activation) have suggested enhanced NF-κB activation in obese individuals and experimental animals fed with HFD. For example, NF-κB DNA binding is increased about twofold in the liver, hypothalamus and skin of rodents fed with HFD for 6 months compared with animals fed with a control diet [12, 28]. NF-κB was similarly elevated in the liver and skin in common genetic obesity models of genetic hyperphagia (ob/ob mice and fa/fa rats) [12, 29]. Furthermore, peripheral blood mononuclear cells from obese human subjects have been shown to express enhanced nuclear NF-κB DNA binding .
It is important to note that HFD and obesity typically induce activation of NF-κB about twofold, which is much lower that the 10–100-fold activation typical of acute inflammatory reactions. This is consistent with the view of obesity as a chronic low-grade inflammatory condition.
Increased NF-κB activation in aged animals has been observed previously. Helenius  detected increased NF-κB DNA binding in heart, liver, kidney and brain of older mice and rats, as compared to younger animals, whereas Spencer et al. found increased NF-κB activation in splenic macrophages and lymphocytes of aged mice .
Although these previously published studies have assessed NF-κB activation measured as NF-κB DNA binding or phosphorylation of NF-κB components, none of the previous studies have analyzed NF-κB transactivation. The use of reporter constructs, such as the NF-κB luciferase transgene, we have used in the present reporter mice enables direct analysis of NF-κB transactivation. Thus, the luciferase reporter measures the integrated effects of different protein modifications regulating the NF-κB signal transduction pathway leading to DNA binding and transcriptional regulation, as well as the effects of other genetic and epigenetic factors affecting NF-κB signaling.
Reporter mice are particularly useful for analyzing gene regulation over time in a physiological context as opposed to cell cultures. Our in vivo model is also ideally suited to take into account absorption efficiency, transport in blood or other extracellular fluids, and cellular uptake, metabolism and degradation. Furthermore, the present technology also provides the possibility for elucidating the full anatomical expression profile of the regulatory module of interest. The recent description and validation of reporter mice open new horizons for nutrition research and drug discovery because these novel animal models provide a global view of gene expression following acute, repeated or chronic dietary or pharmacological treatment.
In summary, we are the first to report a dynamic assessment of NF-κB activity as a function of high versus low-fat feeding. The results show that NF-κB activity is more elevated in mice-fed HFD. We find that weight gain in HFD mice may be a strong predictor of NF-κB activity in the thoracic region of female mice. Moreover, male mice displayed a modest, but significant increase in abdominal NF-κB activity possibly derived from abdominal adipose tissue depots.
We would like to thank Anne Randi Enget for technical assistance. This work was supported by grants from The Norwegian Cancer Society, Norwegian Research Council and Throne Holst Foundation and the TNO research program VP9 Personalized Health (to RK and TK). The authors gratefully acknowledge financial support from The European Nutrigenomics Organisation (NuGO). The European Nutrigenomics Organisation linking genomics, nutrition, and health research (NuGO, CT-2004-505944) is a Network of Excellence funded by the European Commission’s Research Directorate General under Priority Thematic Area 5 Food Quality and Safety Priority of the Sixth Framework Program for Research and Technological Development.
Conflict of interest statement
H.C. and R.B. are shareholders in the company Cgene with the commercial rights to the NF-κB-luciferase reporter mice. The authors have full control of all primary data and agree to allow the journal to review the data if requested.
- CDC National Health and Nutrition Examination Survey Data, 2001–2004 US Department of Health and Human Services. Internet: http://www.cdc.gov/nchs/nhanes.htm. Accessed 30 April 2008
- Yusuf S, Hawken S, Ounpuu S, Dans T, Avezum A, Lanas F, McQueen M, Budaj A, Pais P, Varigos J, Lisheng L (2004) Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case–control study. Lancet 364:937–952PubMedView ArticleGoogle Scholar
- Xu H, Barnes GT, Yang Q, Tan G, Yang D, Chou CJ, Sole J, Nichols A, Ross JS, Tartaglia LA, Chen H (2003) Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest 112:1821–1830PubMedGoogle Scholar
- Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr (2003) Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 112:1796–1808PubMedGoogle Scholar
- Fantuzzi G (2005) Adipose tissue, adipokines, and inflammation. J Allergy Clin Immunol 115:911–919 (quiz 920)PubMedView ArticleGoogle Scholar
- Clement K, Viguerie N, Poitou C, Carette C, Pelloux V, Curat CA, Sicard A, Rome S, Benis A, Zucker JD, Vidal H, Laville M, Barsh GS, Basdevant A, Stich V, Cancello R, Langin D (2004) Weight loss regulates inflammation-related genes in white adipose tissue of obese subjects. FASEB J 18:1657–1669PubMedView ArticleGoogle Scholar
- Haugen F, Drevon CA (2007) The interplay between nutrients and the adipose tissue. Proc Nutr Soc 66:171–182PubMedView ArticleGoogle Scholar
- Despres JP (2006) Is visceral obesity the cause of the metabolic syndrome? Ann Med 38:52–63PubMedView ArticleGoogle Scholar
- Handschin C, Spiegelman BM (2008) The role of exercise and PGC1alpha in inflammation and chronic disease. Nature 454:463–469PubMedView ArticleGoogle Scholar
- Baud V, Karin M (2009) Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls. Nat Rev Drug Discov 8:33–40PubMedView ArticleGoogle Scholar
- Arkan MC, Hevener AL, Greten FR, Maeda S, Li ZW, Long JM, Wynshaw-Boris A, Poli G, Olefsky J, Karin M (2005) IKK-beta links inflammation to obesity-induced insulin resistance. Nat Med 11:191–198PubMedView ArticleGoogle Scholar
- Cai D, Yuan M, Frantz DF, Melendez PA, Hansen L, Lee J, Shoelson SE (2005) Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kB. Nat Med 11:183–190PubMedView ArticleGoogle Scholar
- Wunderlich FT, Luedde T, Singer S, Schmidt-Supprian M, Baumgartl J, Schirmacher P, Pasparakis M, Bruning JC (2008) Hepatic NF-kB essential modulator deficiency prevents obesity-induced insulin resistance but synergizes with high-fat feeding in tumorigenesis. Proc Natl Acad Sci USA 105:1297–1302PubMedView ArticleGoogle Scholar
- Yin MJ, Yamamoto Y, Gaynor RB (1998) The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta. Nature 396:77–80PubMedView ArticleGoogle Scholar
- Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M, Shoelson SE (2001) Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta. Science 293:1673–1677PubMedView ArticleGoogle Scholar
- Carlsen H, Moskaug JO, Fromm SH, Blomhoff R (2002) In vivo imaging of NF-kappa B activity. J Immunol 168:1441–1446PubMedGoogle Scholar
- Dohlen G, Carlsen H, Blomhoff R, Thaulow E, Saugstad OD (2005) Reoxygenation of hypoxic mice with 100% oxygen induces brain nuclear factor-kappa B. Pediatr Res 58:941–945PubMedView ArticleGoogle Scholar
- Campbell SJ, Anthony DC, Oakley F, Carlsen H, Elsharkawy AM, Blomhoff R, Mann DA (2008) Hepatic nuclear factor kappa B regulates neutrophil recruitment to the injured brain. J Neuropathol Exp Neurol 67:223–230PubMedView ArticleGoogle Scholar
- Bell AC, West AG, Felsenfeld G (1999) The protein CTCF is required for the enhancer blocking activity of vertebrate insulators. Cell 98:387–396PubMedView ArticleGoogle Scholar
- Mito N, Yoshino H, Hosoda T, Sato K (2004) Analysis of the effect of leptin on immune function in vivo using diet-induced obese mice. J Endocrinol 180:167–173PubMedView ArticleGoogle Scholar
- Dixit VD, Yang H, Sun Y, Weeraratna AT, Youm YH, Smith RG, Taub DD (2007) Ghrelin promotes thymopoiesis during aging. J Clin Invest 117:2778–2790PubMedView ArticleGoogle Scholar
- Hick RW, Gruver AL, Ventevogel MS, Haynes BF, Sempowski GD (2006) Leptin selectively augments thymopoiesis in leptin deficiency and lipopolysaccharide-induced thymic atrophy. J Immunol 177:169–176PubMedGoogle Scholar
- Howard JK, Lord GM, Matarese G, Vendetti S, Ghatei MA, Ritter MA, Lechler RI, Bloom SR (1999) Leptin protects mice from starvation-induced lymphoid atrophy and increases thymic cellularity in ob/ob mice. J Clin Invest 104:1051–1059PubMedView ArticleGoogle Scholar
- Clement K, Langin D (2007) Regulation of inflammation-related genes in human adipose tissue. J Intern Med 262:422–430PubMedView ArticleGoogle Scholar
- Cani PD, Bibiloni R, Knauf C, Waget A, Neyrinck AM, Delzenne NM and Burcelin R (2008) Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice. Diabetes 57:1470–1481Google Scholar
- Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D, Neyrinck AM, Fava F, Tuohy KM, Chabo C, Waget A, Delmee E, Cousin B, Sulpice T, Chamontin B, Ferrieres J, Tanti JF, Gibson GR, Casteilla L, Delzenne NM, Alessi MC, Burcelin R (2007) Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes 56:1761–1772PubMedView ArticleGoogle Scholar
- Kim CS, Lee SC, Kim YM, Kim BS, Choi HS, Kawada T, Kwon BS and Yu R (2008) Visceral fat accumulation induced by a high-fat diet causes the atrophy of mesenteric lymph nodes in obese mice obesity (Silver Spring)Google Scholar
- De Souza CT, Araujo EP, Bordin S, Ashimine R, Zollner RL, Boschero AC, Saad MJ, Velloso LA (2005) Consumption of a fat-rich diet activates a proinflammatory response and induces insulin resistance in the hypothalamus. Endocrinology 146:4192–4199PubMedView ArticleGoogle Scholar
- Katiyar SK, Meeran SM (2007) Obesity increases the risk of UV radiation-induced oxidative stress and activation of MAPK and NF-kappaB signaling. Free Radic Biol Med 42:299–310PubMedView ArticleGoogle Scholar
- Ghanim H, Aljada A, Hofmeyer D, Syed T, Mohanty P, Dandona P (2004) Circulating mononuclear cells in the obese are in a proinflammatory state. Circulation 110:1564–1571PubMedView ArticleGoogle Scholar
- Helenius M, Kyrylenko S, Vehvilainen P, Salminen A (2001) Characterization of aging-associated up-regulation of constitutive nuclear factor-kappa B binding activity. Antioxid Redox Signal 3:147–156PubMedView ArticleGoogle Scholar
- Spencer NF, Poynter ME, Im SY, Daynes RA (1997) Constitutive activation of NF-kappa B in an animal model of aging. Int Immunol 9:1581–1588PubMedView ArticleGoogle Scholar