The Genetic Ablation of TNF-α Attenuates Wnt-Signaling and Adiposity in High Fat Diet-Induced Obese Mice

A chronic low-grade inflammation is considered as a consequence of obesity, and linked with multiple complications. However, it is under-investigated how inflammatory cytokines mediate adipogenesis. This study investigated the role of Tumor Necrosis Factor α (TNF-α) on adipogenes is over high-fat diet feeding. Three groups of wild type or TNF-α-/mice with the same C57BL/6 genetic background were utilized in this study: wild type fed with a low-fat diet (WT-LFD), wild type fed with a high-fat diet (WT-HFD), and TNF-α-/fed with a HFD (TNF-HFD). After 16-wk feeding, inflammatory cytokine, Wnt pathway and adipogenesis-related genes were analyzed. HFD feeding increased body weight in both WTHFD and TNF-HFD groups, but genetic ablation of TNF-α attenuated HFD-induced obesity. In visceral adipose tissues, HFD elevated Wnt/β-catenin signaling, indicated by decreased phospho-GSK3β and active β-catenin, two key components within the Wnt pathway, and dysregulated adipogenesis, indicated by reduced PPARγ/CEBPα expressions. Whereas, the deletion of TNF-α suppressed Wnt-signaling, and restored expressions of adipogenes is-related genes, which were otherwise decreased in the HFD-induced obese animals. These findings demonstrated a critical role of TNF-α in the regulation of Wnt-signaling and adiposity in mice over a HFD feeding, indicating HFD-induced adipocyte dysfunction could be mitigated by targeting TNF-α and Wnt-signaling.


Introduction
It is well-established that obesity, with inflammation as one of the critical mechanisms, linked to a range of chronic diseases such as type 2 diabetes mellitus (T2DM), nonalcoholic fatty liver disease (NAFLD), cardiovascular disease (CVD), and cancer [1]. Obesity is well-accepted as a chronic inflammatory condition as a consequence of hypertrophic adipocyte expansion [2]. Whereas, how inflammatory cytokines reversely mediate adipogenesis is less investigated. To understand this mechanism and prevent chronic over nutrition-induced adipocyte dysfunction represents a promising approach to diminish the epidemic of obesity and its associated medical complications in our society [3,4].
hibiting PPARγ expression [7,8]. Among many transcriptional factors, PPARγ is one of the major transcriptional factors to orchestrate the adipogenesis. PPARγ regulates lipid and glucose metabolism, and PPARγ target genes critically involve in lipid uptake and adipogenesis [9]. PPARγ agonists have been used in the treatment of hyperlipidemia and hyperglycemia [10], and it has been implicated in the pathology of numerous diseases including cardiovascular diseases and cancer [11]. Therefore, it is important to understand the regulation of PPARγ expression in obesity, thereby providing feasible av-luminescence assay using the QuickPlex SQ 120 (Meso Scale Diagnostics, Rockville, MD). Assays were performed according to the manufacturer's instructions. Briefly, the protein samples including inflammatory cytokines were isolated from visceral fat tissues with lysis buffer prepared according to the protocol for the electrochemiluminescence assay (Meso Scale Discovery, Rockville, MD). Then 25 µl of calibrator standards or samples were added to each well of a 96-well plate, on the bottom of which antibodies for 3 cytokines (IFN-γ, IL-1β, and IL-6) were coated. After washing for 3 times, 50 µl of the detection antibody solution was added to each well. A four-parameter logistic fit curve was generated for each analyte using the standards and the levels of inflammatory cytokines in samples were calculated accordingly. Cytokines are express as ng of cytokine per mg protein. All standards and samples were measured in duplicate.

Real-time PCR for gene expression
Total RNA was extracted with the Trizol reagent (Invitrogen, Carlsbad, CA). The concentration as well as purity of RNA samples was determined by NanoDrop 2000 (Thermo Scientific, Waltham, MA). The first-strand cDNAs were synthesized from 0.5 µg total RNAs using QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA). Real-time PCR was performed on the ViiA 7 Real-Time PCR System (Applied Biosystems, Carlsbad, CA) using the SYBR green PCR reagent kit (Invitrogen, Carlsbad, CA) with the following thermal cycling condi-enues to properly manage adipogenesis and related complications.
Tumor necrosis factor-alpha (TNF-α), which is one of pro-inflammatory cytokines found to be increased in obesity, has been considered an important mediator that causes metabolic dysregulations in the body via adipose tissue dysfunction [12]. Previous animal studies with TNF-α knockout (TNF-α -/-) mice showed that TNF-α deficiency attenuated insulin resistance in peripheral tissues and prevented excessive hepatic lipid accumulation in diet-induced obese mice [13][14][15][16]. However, the cellular mechanism(s), by which TNF-α medicates adipose dysfunction has not been clearly defined. This study aimed to investigate the role of TNF-α in the regulation of Wnt/β-catenin signaling in adipogenesis in mice over HFD feeding. We hypothesized that obesity-promoted TNF-α activates Wnt/β-catenin signaling in adipose tissue, and that its constant activation inhibits the production of PPARγ, thereby leading to further adipocyte dysfunction.

Animals and experimental diets
TNF-α -/mice were purchased from Jackson laboratory (Bar Harbor, ME, USA). It was originally created on 129S/6 background, but has been backcrossed to C57BL/6 for >10 generations. Mice were housed under conditions of controlled temperature (22 ˚C -25 ˚C) and illumination (12:12 hour light-dark cycle, lights on between 7:00 and 19:00) with food and water provided ad libitum. The animal use protocol was approved by the Institutional Animal Care and Use Committee of the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University and the Institutional Animal Care and Use Committee of the University of Massachusetts, Amherst.
All the C57BL/6 wild type and TNF-α -/mice were initially fed a standard rodent chow diet (Teklad#8604, Envigo, Madison, WI). At 8 weeks of age, animals were divided into 3 groups (26 animals (13 males and 13 females) /group): wild type fed with a low-fat diet (WT-LFD, 10 % of total calorie from fat), wild type fed with a high-fat diet (WT-HFD, 60 % of total calorie from fat), and TNF-α -/fed with the same highfat diet (TNF-HFD).A high fat diet, which is commonly used to induce obesity, was purchased from Research Diets (HFD: D12492 vs. LFD: D12450B, New Brunswick, NJ). The dietary compositions were shown in Table 1. As significant gender differences in body weight gain and metabolic parameters exist with male mice more sensitive to high fat induced-induced obesity [17,18], in addition to the body weight and body composition, only male animals were used for molecular analysis in this study to avoid the confounding from gender. At the end of the experiment, mice were euthanized by CO 2 asphyxiation followed by cervical dislocation. Exsanguination was performed by cardiac puncture and blood was collected. The abdomen was opened and epididymis fats were collected into foil packets and frozen in liquid N 2 for subsequent RNA and protein assays.

Inflammatory cytokine assays
The inflammatory cytokines were measured by a chemi- At the beginning of the experiment (8 wks of age), there were no differences across the 3 groups in terms of body weight. As expected, after 16 weeks of high fat diet feeding, the body weight of both wild type mice (WT-HFD) and TNF-α -/-(TNF-HFD) significantly increased when compared with that of wild type mice fed a low fat diet (WT-LFD). It is noteworthy that the genetic deletion of TNF-α reduced the sensitivity of high fat diet-induced obesity. Only after 7 weeks, the body weight of the wild type animals fed a high fat diet (WT-HFD) increased to a degree that was significantly higher than the body weight of those animals fed a low fat diet (WT-LFD), whereas a statistical difference was not observed for the TNFα -/animals (TNF-HFD) until 13 weeks of a high fat diet feeding (p < 0.05). The body weight of the WT-HFD group were significantly higher than that of the TNF-HFD group after feeding for the high fat diet for 9 weeks (p < 0.05). By the end of the experiment, the body weight gains were 53.7% for the WT-LFD group, 115.1% for the WT-HFD group, and 88.9% for the TNF-HFD group. Compared to the WT-LFD group, the body weights were 38.5% higher for the WT-HFD group, but only 15.5% for the TNF-HFD group (p < 0.05) ( Figure 1a).

Inflammatory Cytokines
After observing the differentiated body weight change among groups, we measured 3 inflammatory cytokines in the visceral adipose tissue (IFNγ, IL-1β and IL-6). When compared to the WT-LFD group, the high fat diet (WT-HFD) significantly increased protein levels of IFNγ by 120.3% (p < 0.01), IL-1β by 29.5% (p < 0.05) and IL-6 by 100.6% (p < 0.001). However, the genetic ablation of TNF-α diminished the these elevations and the levels of IFNγ and IL-6 in the TNF-HFD group were significantly lower than in the WT-HFD groups (p < 0.05) with a numerical decrease of IL-1β (p = 0.07) (Figure 2).

The Wnt-signaling pathway
To investigate the Wnt-signaling pathway, we examined tions: 95 ˚C for 10 minutes, followed by 40 cycles of 95 ˚C for 15 seconds and 60 ˚C for 60 seconds. The cycle threshold (Ct) values were defined as the fractional cycle number at which the fluorescence passes an arbitrarily set threshold. The copy number of each transcript was calculated with respect to the GAPDH copy number. Amplification of specific transcripts was confirmed by analyzing melting curve profiles at the end of each PCR. Primer sequences and relative expressions of each genes examined are listed in Table 2.

Western blot analyses
The protein sample was isolated from adipose tissue using RIPA lysis buffer (Thermo Scientific, Waltham, MA) with protease inhibitor cocktail (Santa Cruz Biotechnology, Dallas, TX). The total protein concentrations were determined using a commercially available Pierce TM BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). Western blotting was performed to determine the protein levels of phospho-GSK3β and active β-catenin as well as adipogenesis-related genes Pparγ and Cebpα. Briefly, 40 µg of protein from each treatment was separated by 10% SDS-PAGE and transferred onto a PVDF membrane. After the nonspecific binding was blocked with 10% instant nonfat dry milk, membranes were then probed with specific primary antibodies overnight at 4 ˚C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, CA). Anti-PPARγ, CEBPα, Phospho-GSK3β (Ser9) and GAPDH antibody was purchased from Cell Signaling Technology (Boston, MA, USA). Anti-Active-β-Catenin was purchased from EMD Millipore Corporation (Billerica, MA, USA). The chemiluminescence detection was achieved using ECL Plus Substrate (Amersham Biosciences, Waltham, MA), and band intensities were analyzed using Image J Version 1.49 (https://imagej.nih.gov). The ratios of the density of the proteins were calculated after normalizing to corresponding loading controls.

Statistical analysis
Data are expressed as means ± SEM, and statistical analysis was performed using SAS (Version 9.4, SAS Institute, Cary, NC). One-way ANOVA was applied to test the differences among the 3 groups. The expression of each gene was normalized to the housekeeping gene Gapdh (∆Ct = Ct    Open Access | Page 116 | resents a low-grade inflammatory condition [14,[19][20][21], and the increased inflammatory cytokines are generally considered as a consequence of obesity. This study, on the contrast, demonstrated that genetic ablation of TNF-α attenuated HFD-induced obesity, potentially via attenuating Wnt/β-catenin signaling and restoring the adipogenic PPARγ/CEBPα expression, suggesting that TNF-α plays a causal role in adipocyte dysfunction during adipocyte hypertrophic expansion.

Body weight and body composition
Since the initial discovery of escalated expression of TNF-α in adipose tissue by Hotamisligil and colleagues in 1993 [14], it is well-accepted that the adipose tissue is an active endocrine organ, which releases >50 different cytokines and adipokines [19,20]. In addition to the origins of inflammatory cytokine from adipocytes, adipocyte hypertrophy leads to the infiltration of immune cells, especially macrophages, and these immune cells significantly contribute to the production of pro-inflammatory cytokines [22]. Whereas, it is less understood how inflammatory cytokines influence adiposity, though a few pro-inflammatory cytokines have been shown possessing important metabolic effects, including effect on lipid metabolism [23,24].
Studies pertaining the role of TNF-α on adipocyte dysfunction and obesity are limited and results are highly inconsistent. The study performed by Uysal, et al. in 1997 [16] showed that the body weights were similar between the wild type and TNF-α -/animals regardless feeding with a LF or HF diet throughout the 16-week study, but a significantly decreased body weight for the TNF-α -/model fed a HF diet were observed in a study from others [13]. On the contrast, two other studies in 2012 [15] and 2016 [25] showed that TNF-α -/mice were even prone to gain weight on HF diet when the protein levels of phosphorylated GSK3β and active β-catenin as well as the transcriptional levels of Wnt-signaling downstream target genes, C-Myc and Cyclin D1. As shown in figure 3a and figure 3b, when compared to WT-LFD, high fat diet (WT-HFD) significantly increased the protein levels for both phospho-GSK3β at the Ser 9 site (the inactive form) and active β-catenin (p < 0.01), whereas the genetic deletion of TNF-α (TNF-HFD) significantly diminished these effects (p < 0.01). After observing the mitigation of phosphorylated GSK3β and active β-catenin by TNF-α, we further examined the transcriptional expression of Wnt-signaling downstream genes. As shown in ure 3c,When compared to the WT-HFD group, the deletion of TNF-α (TNF-HFD group) reduced the expression of C-Myc and Cyclin D1 (p < 0.01) which were otherwise up regulated by high fat diet (p < 0.001, WT-LFD vs. WT-HFD).

Adipogenesis
To investigate the influence of TNF-α on adipocyte dysfunction in high fat-induced obesity, we analyzed the expression, at both transcriptional and protein level, of PPARγ and CEBPα that are two critical transcriptional factors involved in adipogenesis. We found that, after a 16-wk high fat diet feeding, the transcriptional expression of Pparγ and Cebpα were significantly suppressed (p < 0.001) by high fat diet (WT-HFD vs WT-LFD), whereas the genetic deletion of TNF-α attenuated this effect (TNF-HFD vs WT-HFD, p < 0.05) (Figure 4). The analysis of the protein levels of these two adipogenesis-related genes recapitulated the transcriptional data ( Figure 4).

Figure 5:
PPARγ and Wnt/β-catenin signaling in the regulation of adipogenesis. At the beginning of high-fat diet feeding, our body may respond via stimulating the progression from preadipocyte to matured adipocyte, resulting in hyperplasia characterized by increased number of matured adipocytes. These adipocytes may secret adipogentic factors, leading to increased level of PPARγ and the proliferation of adipocytes. Whereas, with the continuous high-fat diet feeding, the over-uptake of lipids lead the adipocyte to the hypertrohpic stage, which results to inflammation in the adipose tissue. The increased inflammatory cytokines may activate Wntsignaling, which further inhibits PPARγ expression and leads to the dysregulation of adipogenesis. Values are means ± SEMs, n = 4 animals/ group. * represents a p < 0.05, ** represents a p < 0.01, *** represents a p < 0.001. compared wild type animals. The study of Uysal, et al. [16] also showed that the genetic absence of TNF-α receptors did not attenuate body weight in genetically-induced obese animals (ob/ob) when compared to those ob/ob mice without the deletion of TNF-α receptors. However, another study [26] showed that the ablation of TNF-α receptor protected against diet-induced obesity. These inconsistent results require further investigation to understand the cellular action of TNF-α on adipogenesis.
Diet-induced adiposity includes two stages: hyperplasia characterized by increased number of adipocytes, and hypertrophy featured with enlarged adipocytes. For a short-term over nutrition, our body may respond via stimulating the progression from pre-adipocyte to matured adipocyte, resulting in increased number of adipocytes called adipose hyperplasia. It is reported that, as early as 2 wks of HF diet feeding, an increased expression of PPARγ was observed in the liver tissue [27]. Whereas in chronic over nutrition, the over-uptake of lipids lead the adipocyte to the hypertrophic stage, which results in inflammation in the adipose tissue. Therefore, inflammatory cytokines may play regulatory roles on adipogenesis primarily in the latter hypertrophic stage when inflammation occurs ( Figure 5). In our current study, animals were fed with a HFD (60 kcal% fat) for a long period (16 wks), and had reached the hypertrophic phase as indicated by increased inflammation. Our data clearly demonstrated that the genetic ablation of TNF-α attenuated Wnt/β-catenin signaling, restored the expression of PPARγ, and thereby suppressed HFD-induced obesity over a long-term HFD feeding. Our results may offer an explanation for the previous inconsistent results. TNF-α plays a regulatory role on adipogenesis primarily at the later hypertrophic stage when the inflammation occurs in the adipose tissue. In those previous studies, due to the either low fat content in the diet or the duration of feeding, animals in some of those studies might not reach the hypertrophic stage, and thereby resulted in inconsistent results.
Adipogenesis can be divided into two stages: Mesenchymal stem cells (MSCs) "commitment" to pre-adipocytes and pre-adipocytes differentiation into mature adipocytes. Initially, canonical Wnt-signaling controls the balance of MSC differentiating into myoblasts, osteoblasts or pre-adipocytes [28], and its activation suppresses commitment to the adipocytic lineage, probably suppressing the initiation of adiposity. This is manifested by the fact that genetic deletion of Wnt antagonist Sfrp1 leads to reduction of fat mass concomitant with increased bone mass [29]. On the contrast, our study demonstrated an impact of the canonical Wnt-signaling at the later stage of adipogenesis: The impairment of the proper progression from pre-adipocytes into matured adipocytes. Our results unraveled an important mechanism that TNF-α suppresses adipogenesis via enhancing the canonical Wnt-signaling, which consequently suppressed the adipogenic PPARγ/CEBPα expression, and promoted hypertrophic adipocyte expansion after 16-wk HFD feeding ( Figure 5).

Conclusion
In summary, a chronic low-grade inflammation is generally considered as a consequence of obesity. On the contrast,