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Endocrinology, doi:10.1210/en.2007-0978
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Endocrinology Vol. 149, No. 6 2943-2951
Copyright © 2008 by The Endocrine Society

Blockade of Tumor Necrosis Factor (TNF) Receptor Type 1-Mediated TNF-{alpha} Signaling Protected Wistar Rats from Diet-Induced Obesity and Insulin Resistance

Huifang Liang1, Bingjiao Yin1, Hailong Zhang, Shu Zhang, Qingling Zeng, Jing Wang, Xiaodan Jiang, Li Yuan, Cong-Yi Wang and Zhuoya Li

Department of Immunology (H.L., B.Y., H.Z., S.Z., Q.Z., J.W., X.J., L.Y., Z.L.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, People’s Republic of China; and Center for Biotechnology and Genomic Medicine (C.-Y.W.), Medical College of Georgia, Augusta, Georgia 30912

Address all correspondence and requests for reprints to: Zhuoya Li, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan, Hubei 430030, People’s Republic of China. E-mail: zhuoyali{at}mails.tjmu.edu.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TNF-{alpha} plays an important role in the pathogenesis of obesity and insulin resistance in which the effect of TNF-{alpha} signaling via TNF receptor type 1 (TNFR1) largely remains controversial. To delineate the role of TNFR1-mediated TNF-{alpha} signaling in the pathogenesis of this disorder, a TNFR1 blocking peptide-Fc fusion protein (TNFR1BP-Fc) was used for the present study. Wistar rats were fed a high-fat/high-sucrose (HFS) diet for 16 wk until obesity and insulin resistance developed. In comparison with increased body weight and fat weight, enlarged adipocytes, and hypertriglyceridemia in the obese state, the subsequent 4-wk treatment with TNFR1BP-Fc resulted in significant weight loss characterized by decreased fat pad weight and adipocyte size and reduced plasma triglycerides. Furthermore, obesity-induced insulin resistance, including hyperinsulinemia, elevated C-peptide, higher degree of hyperglycemia after glucose challenge, and less hypoglycemic response to insulin, was markedly improved, and the compensatory hyperplasia and hypertrophy of pancreatic islets were reduced. Interestingly, treatment with TNFR1BP-Fc markedly suppressed systemic TNF-{alpha} release and its local expression in pancreatic islets and muscle and adipose tissues. In addition, blockage of TNFR1-mediated TNF-{alpha} signaling in obese rats significantly enhanced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in the muscle and fat tissues. Our results strongly suggest a pivotal role for TNFR1-mediated TNF-{alpha} signaling in the pathogenesis of obesity and insulin resistance. Thus, TNFR1BP-Fc may be a good candidate for the treatment of this disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OBESITY HAS NOW become one of the most prevalent and serious health problems in both developed and developing countries. Because obesity is able to engender insulin resistance, it has been recognized to be associated with type 2 diabetes for decades. Insulin resistance resulting from obesity is also linked to a wide array of other pathophysiological sequelae including hypertension, hyperlipidemia, atherosclerosis, and polycystic ovarian disease (1). It has been estimated that around 30% of obese and 11% of overweight adults in the United States suffer from type 2 diabetes (2). Despite decades of extensive studies, the molecular mechanisms through which obesity leads to insulin resistance and type 2 diabetes still remain elusive (3).

TNF-{alpha} has been widely recognized as an important mediator for insulin resistance by impairing insulin signaling (4, 5, 6). Adipocyte-derived TNF-{alpha} has been shown to play a key role in the control of adipocyte function (7, 8). Studies in both animal models and obese human subjects suggest that elevated production of TNF-{alpha} by adipose tissues is associated with decreased insulin sensitivity (9, 10). The altered TNF-{alpha} expression in adipose tissues is also positively correlated with the degree of obesity and the level of hyperinsulinemia, which is often taken as an indirect evidence for insulin resistance (9, 11). Therefore, weight loss in obese patients is associated with reduced TNF-{alpha} production and ameliorated insulin resistance (12, 13). On the contrary, administration of TNF-{alpha} into animals or humans has been reported to result in reduced insulin sensitivity (14, 15, 16). On the other hand, significantly increased peripheral insulin sensitivity was observed in obese animals by neutralizing TNF-{alpha} (9). Furthermore, TNF-{alpha}-deficient mice were protected from obesity-induced insulin resistance and hyperlipidemia (17).

Two receptors, TNF receptor type 1 (TNFR1) and TNFR2, have been identified for TNF-{alpha}. These two receptors do not share homology in the cytoplasmic domains but exhibit a low degree of similarity in the ligand-binding region located in the extracellular domains, which suggests that they are capable of inducing distinct cellular responses. Recent studies have demonstrated the role for each receptor in the pathogenesis of obesity and insulin resistance (5, 18, 19, 20, 21, 22). However, these results were somehow controversial. Studies from Hotamisligil’s group (19, 22) suggest that TNFR1 is the predominant receptor for mediating insulin resistance associated with obesity and TNF-{alpha}-induced lipolysis. It is also involved in the inhibition of insulin-stimulated glucose transport and insulin receptor autophosphorylation. In contrast, Schreyer and colleagues found that mice lacking TNFR1 (p55–/–) were not protected from insulin resistance induced by a high-fat diet (18).

The present study sought to delineate the role of TNFR1-mediated TNF-{alpha} signaling in the pathogenesis of obesity and insulin resistance. Wistar rats were fed a high-fat/high-sucrose (HFS) diet to induce obesity and insulin resistance. A TNFR1 blocking peptide, originated from screening a phage display library, fused with a human IgG1 Fc fragment (TNFR1BP-Fc) was used to competitively block the interaction of TNF-{alpha} and TNFR1. It has been shown that this reverses insulin resistance in rodents.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines
A murine fibroblast L929 cell line (as target cells for TNF-{alpha}) and a Chinese hamster ovary K1 (CHO-K1) cell line (for stable transfection) were maintained within the laboratory. These cells were grown in DMEM (Life Technologies, Inc., Rockville, MD) supplemented with 10% fetal bovine serum (Life Technologies), 100 IU/ml penicillin, and 100 mg/ml streptomycin at 37 C in 5% CO2.

Construction of the pIg3C-TNFR1BP plasmid
A TNFR1 blocking peptide originated from screening a phage 12-mer peptide library (New England Biolabs, Ipswich, MA) using soluble TNFR1 as bait (23). DNA sequence encoding the TNFR1 blocking peptide was directly synthesized by the Sangon Biological Engineering Technology and Service Co. (Shanghai, China). The two complementary oligonucleotide chains were mixed at a 1:1 ratio and then subjected to forming double-stranded DNA by denaturing at 95 C and subsequent annealing at room temperature. The resulting DNA fragment contains compatible BamHI and KpnI cutting sites at the 5'- and 3'-end, respectively. This fragment was then cloned into a linearized pIg/3C vector, which was kindly provided by Dr. Boquan Jin, Department of Immunology, the Fourth Military Medical University of China. This plasmid contains an upstream signal peptide sequence necessary for the secretion of chimeras and a downstream human IgG1-Fc sequence (24, 25).

Production of human IgG1 Fc fused TNFR1 blocking peptide (TNFR1BP-Fc)
Four micrograms of pIg/3C-TNFR1BP-Fc plasmid or pIg/3C plasmid were cotransfected along with a pcDNA3.1 vector that contains a neo gene into CHO-K1 cells using Lipofectamine 2000 as instructed (Invitrogen, Carlsbad, CA). After 24 h of transfection, the cells were selected with 800 µg/ml G418 (GIBCO, Grand Island, NY) for 8 d. Single clones were obtained by limited dilution (0.3 cells/100 µl·well in 96-well plates) and maintained in the presence of G418.

TNFR1BP-Fc or IgG1Fc (as a control) was purified from the culture supernatants by chromatography using protein A-Sepharose CL-4B beads (Amersham Biosciences, Uppsala, Sweden) (26). The targeted proteins were eluted with 0.1 M glycine-HCl buffer (pH 3.5) and subsequently neutralized with 1.0 M Tris-HCl buffer (pH 8.0). The unpurified and purified proteins were analyzed on 10% SDS-PAGE and stained with Coomassie blue R250. The purified proteins were further passed through 0.45-µm filters (Millipore, Boston, MA) for sterilization.

The concentration for each purified protein was determined by ELISA. An antibody against human IgG was used as a capture antibody to coat the plate. After incubation at 37 C for 1 h with samples or human IgG standard, a horseradish peroxidase-conjugated goat antihuman IgG antibody (1:10,000 dilution) was used to detect the bound proteins. The OD was read under a microplate autoreader (Titertek Multiskan Scanner; Labsystems, Finland) at 492 nm.

Measurement of TNF-{alpha} concentration
Serum samples were collected from the retroorbital sinus under anesthesia by injection of sodium pentobarbital. Tissue extracts were prepared using the method described by Molina et al. (27). Briefly, 0.5 g tissue was homogenized using a polytron homogenizer in 2 ml buffer containing 0.9% NaCl, 10 mM Na2HPO4, 1 mM phenylmethylsulfonyl fluoride, 0.01 mg/ml each of pepstatin, aprotinin, and leupeptin, 0.5% Triton X-100, and 0.05% sodium azide (pH 7.2). The tissue samples were subjected to one freeze/thaw cycle and then sonicated briefly and centrifuged at 12,000 x g for 1 h. The concentration of TNF-{alpha} in the tissue supernatants or in the serum samples was determined by an ELISA kit (eBioscience, San Diego, CA) as instructed.

Indirect immunofluorescence
A total of 1 x 105 L929 cells/ml were grown on coverslips in a six-well culture plate overnight and then treated with 160 ng/ml TNFR1BP-Fc or IgG1Fc for 2 h. Untreated cells served as a control. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 30 min and then blocked with 0.5% BSA/PBS. The fixed cells were then incubated at room temperature with a rabbit polyclonal antibody against TNFR1 or a monoclonal antibody specific to TNFR2 at a dilution of 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA). After washes, the cells were incubated with a corresponding fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Zhongshan Co., Beijing, China) for 30 min and then observed under a fluorescence microscope (Olympus, Tokyo, Japan).

TNF-{alpha} bioassay
L929 cells (2 x 104 cells per well) were seeded in 96-well plates and cultured overnight at 37 C with 5% CO2. The cells were subsequently incubated with different concentrations of TNFR1BP-Fc or IgG1Fc in the presence of 100 U/ml soluble TNF (sTNF)-{alpha} (Peprotech, London, UK) and 1 µg/ml actinomycin D for 24 h. Cell viability was determined by staining for 4 h with 30 mM glucose-PBS containing 0.5 mg/ml of 3-(4,5)-dimethylthiahiazo-(z-yl)-3,5-diphenytetrazoliumromide (Sigma Chemical Co., St. Louis, MO), followed by lysis of the cells with 0.1 ml dimethylsulfoxide. Photometric measurement was carried out at 570 nm on a microplate-autoreader (Titertek Multiskan Scanner, Flow). TNF-{alpha} cytotoxicity was calculated as follows: cell death rate = (1 – ODsample/ODcontrol) x 100%.

Treatment of animals
Male Wistar rats were obtained from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology. Rats were housed individually on a 12-h light, 12-h dark cycle and were cared for in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The study was approved by the Animal Care and Use Committee of Huazhong University of Science and Technology.

For estimation of the serum half-life of TNFR1BP-Fc, five male Wistar rats (400–450 g) received a single iv dose of 1 mg/kg TNFR1BP-Fc. Serum samples were collected at 0.25, 0.5, 1, 3, 6, and 12 h after dosing and at 1, 2, 3, 4, 5, 7, 10, 13, 16, 19, and 22 d after administration and detected for the concentrations of TNFR1BP-Fc protein by ELISA. A capture antibody specific for human H and L chains (Abcam, Cambridge, MA) was used for capture of the fusion proteins from diluted serum samples. A horseradish peroxidase-conjugated antibody specific for human IgG1 Fc fragment (Calbiochem, La Jolla, CA) was used as detecting antibody. The assay was reproduced in triplicate. Serum concentration-time data were analyzed using a model-independent approach based on the statistical moment theory. Pharmacokinetic parameters including the area under the curve (AUC0-{infty}), clearance (CL), volume of distribution (Vss), and elimination half-life (t1/2β) were calculated.

For the study of TNFR1BP-Fc action on obesity and insulin resistance, 4-wk-old male Wistar rats (110–130 g) were used. The animals were fed with HFS diet [20% wt/wt lard (33% of energy), 24.5% wt/wt sucrose (20% of energy)] for 20 wk to induce obesity and insulin resistance. After being fed with HFS for 16 wk, the animals were injected via tail vein with either TNFR1BP-Fc (1 mg/kg in 0.1 ml normal saline solution, NS) or same amount of IgG1Fc or 0.1 ml NS, twice a week. The treatment lasted 4 wk. Eight rats were included in each study group. Rats fed a normal diet (ND) were used as a control. Total body weight, fasting blood glucose, and serum insulin were measured monthly. At the end of experiments, the animals were anesthetized by sodium pentobarbital, and blood samples were collected from the retroorbital sinus. Liver, kidney, pancreas, quadriceps, and epididymal fat pad from each animal were isolated and weighed. Histochemical analysis and Western blot were also performed on those collected tissues.

Metabolic parameters
Glucose and insulin tolerance assays were performed on conscious rats after a 24-h fast. Glucose (3.0 g/kg) or porcine insulin (1 IU/kg) (Sigma) was injected into the peritoneal cavity. Blood was collected by nicking the tail-tip. Glucose levels were measured before injection and after injection at different time points. Blood glucose was measured with a One-Touch Profile glucose meter (Lifescan, Inc., Milpitas, CA). Fasting serum insulin and C-peptide antigen were determined by a double antibody-coated tube RIA kit (Chemclin, Inc., Beijing, China). Insulin resistance was determined by the homoeostasis model assessment index for insulin resistance (HOMA-IR) using the formula: HOMA-IR = fasting insulin (µIU/ml) x fasting glucose (mmol/liter)/22.5. Plasma triglyceride and total cholesterol levels were evaluated using commercial kits as instructed (Sigma and Wako Pure Chemical Industries Ltd., Richmond, VA, respectively).

Immunohistochemistry
The whole pancreas was excised, weighed, and cut into pieces. The tissues were first fixed with 4% paraformaldehyde (in 0.1 mol/liter phosphate buffer) for 24 h and then dehydrated and embedded in paraffin. Some of the tissue sections were subjected to hematoxylin/eosin staining, whereas the rest of sections were used for immunohistochemical analysis of TNF-{alpha} or insulin expression with the avidin-biotin complex method. Briefly, the sections were incubated with primary polyclonal antibody specific to rat TNF-{alpha} (Beijing Zhongshan Biotechnology Co., Beijing, China) or monoclonal antibody to rat insulin (Wuhan Boster Co., Wuhan, China) at 1:200 dilution for 1 h at room temperature, followed by a biotin-conjugated secondary antibody (1:200 dilution; Beijing Zhongshan Biotechnology) for 1 h. After incubation with the peroxidase-labeled streptavidin, the slides were developed in a solution containing diaminobenzidine.

For morphometry measurements, pancreatic tissue area and insulin-positive cell area were determined by computer-assisted measurements using a Nikon microscope (Nikon, Japan) equipped with a color video camera coupled to a computer. Islet size and β-cell mass were analyzed using Image software (HPIAS-1000, China). The number of islets (defined as insulin-positive aggregates at least 25 µm in diameter) was scored. Mean islet size was calculated as the ratio of the total insulin cell area to the total islet number on the sections. The percentage of β-cell fraction was measured as the ratio of the insulin-positive cell area to the total tissue area on the entire section. The β-cell mass was obtained by multiplying the β-cell fraction by the weight of the pancreas (28). For TNF-{alpha} expression, total areas of islets containing TNF-{alpha}-positive cells were circled for image analysis and selected by OD value. The relative level of TNF-{alpha} expression in islets was calculated as the ratio of integral OD value to the integral area (24).

Immunoprecipitation and Western blotting
After an overnight fast, the animals were ip injected with 15 U porcine insulin (Sigma) or an equal volume of PBS solution (29). The animals were killed after 30 min of injection. Quadriceps, liver, and adipose tissues were then isolated and homogenized using a polytron homogenizer at 4 C as described earlier. After centrifugation at 12,000 x g for 15 min at 4 C, the supernatants were collected, and the protein concentrations were determined by the Bradford protein assay reagent (Bio-Rad Laboratories, Richmond, CA). Then, 1 mg of the above prepared proteins was subjected to immunoprecipitation using a polyclonal antibody against insulin receptor substrate 1 (IRS-1, 1 µg/ml) (Santa Cruz Biotechnology). The immune complexes were enriched by incubation with 50 µl protein A-Sepharose CL-4B beads (Amersham). After washing with RIPA buffer [0.15 M NaCl/10 mM phosphate buffer (pH 7.0), 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS], the precipitates were eluted out by boiling the beads with sample buffer for 5 min and were then subjected to Western blot analysis. The membranes were probed with a monoclonal antibody against phosphotyrosine p-Tyr (PY99) (Santa Cruz Biotechnology), or a polyclonal antibody specific to IRS-1, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Beijing Zhongshan Biotechnology). The immunoreactive bands were visualized by enhanced chemiluminescence (Amersham). The relative intensity of each band was determined by laser densitometry.

Statistical analysis
All of the data were analyzed by one-way ANOVA test. Post hoc analysis was performed using Fisher’s exact least significant difference test. Data are presented as mean ± SD. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNFRIBP-Fc efficiently blocks TNFR1-mediated TNF-{alpha} signaling
Previously, we have shown that TNFR1 binding peptide is potent in inhibiting TNF-{alpha}-induced rat peritoneal macrophage activation (30). We also demonstrated that this blocking peptide protected rats from joint damage induced by adjuvant injection (31). In the current study, this blocking peptide was linked with IgG1Fc fragment. We examined the capacity of TNFR1BP-Fc to block TNFR1-mediated TNF-{alpha} signaling. The purity of recombinant TNFR1BP-Fc and IgG1Fc was confirmed by SDS-PAGE, and their molecule masses were about 37 and 36 kDa, respectively (Fig. 1AGo). L929 cells were treated with either 160 ng/ml TNFR1BP-Fc or same amount of IgG1Fc (as a control) before incubation with a TNFR1 antibody or a TNFR2 antibody. It was found that TNFR1BP-Fc exhibited a high capacity to block the interaction between TNFR1 and its specific antibody. As shown in Fig. 1BGo, TNFR1BP-Fc significantly decreased the fluorescence intensity as compared with IgG1Fc or antibody alone (positive control). However, it did not cross-react with TNFR2, because there was no difference observed between TNFR1BP-Fc treatment and the controls (Fig. 1CGo). We then checked the effect of TNFR1BP-Fc on TNF-{alpha} bioactivity. Consistent with the above observations, TNFR1BP-Fc suppressed TNF-{alpha}-induced cell death in a dose-dependent manner (Fig. 1DGo). When 160 ng/ml TNFR1BP-Fc was added into the culture, TNF-{alpha}-induced (100 U/ml) cytotoxicity was completely blocked, suggesting that TNFR1BP-Fc is potent in blocking TNFR1-mediated TNF-{alpha} signaling.


Figure 1
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  		FIG. 1. TNFR1BP-Fc competitively binds to TNFR1 on L929 cells and inhibits TNF-{alpha} cytotoxicity. A, SDS-PAGE analysis of purified recombinant TNFR1BP-Fc and IgGFc. TNFR1BP-Fc and IgGFc were purified by affinity chromatography on Sepharose-protein-A beads and analyzed by SDS-PAGE. B and C, TNFR1BP-Fc competitively binds to TNFR1 but not to TNFR2 on L929 cells. L929 cells (1 x 105/ml) were treated with 160 ng/ml TNFR1BP-Fc fusion protein or Fc protein or medium alone at 37 C for 2 h and stained with a TNFR1 polyclonal antibody or a TNFR2 monoclonal antibody (at 1:500), followed by a corresponding FITC-labeled secondary antibody (1:200). Cells incubated with FITC-labeled secondary antibody alone served as a negative control. D, Blockade of TNF-{alpha}-induced cytotoxicity by TNFR1BP-Fc. L929 cells (2 x 104/100 µl) were incubated with indicated concentrations of TNFR1BP-Fc or IgGFc in the presence of 100 U/ml sTNF-{alpha} and 1 µg/ml actinomycin D for 24 h. Cell viability was measured by 3-(4,5)-dimethylthiahiazo-(z-yl)-3,5-diphenytetrazoliumromide assay. E, The serum half-life of TNFR1BP-Fc. The fusion protein (1 mg/kg) was given iv, and its serum levels at the time shown after injection were detected by ELISA. Each time point represents the average serum concentration for five animals. Data represent the mean ± SD of three independent experiments. Comparison between groups was carried out by ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. IgGFc.

 
To determine the treatment regimen of TNFR1BP-Fc, we ascertained the circulating half-life of the fusion protein after a single injection. The serum levels detected by ELISA indicate that TNFR1BP-Fc has a circulating half-life of about 4 d (Fig. 1EGo), suggesting that serum levels of TNFR1BP-Fc will likely be achieved with twice-weekly injection.

Establishment of an obesity and insulin resistance rat model with HFS diet
We fed 4-wk-old male Wistar rats with HFS diet for 16 wk to establish the model. After being fed with HFS diet for 4 wk, the rats began to gain more weight, and finally they weighed 18% heavier than control rats (Fig. 2AGo, P < 0.05). Interestingly, these rats still maintained normal blood glucose levels (Fig. 2BGo). However, their plasma insulin levels were significantly increased (Fig. 2CGo), suggesting a decreased insulin sensitivity. In line with these results, their HOMA-IR was significantly augmented after 4 wk of HFS and continued to increase in the following weeks (Fig. 2DGo). All of these observations indicate that obesity and insulin resistance had developed in these rats fed a HFS diet.


Figure 2
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  		FIG. 2. Obesity and insulin resistance induced by HFS diet. Four-week-old male Wistar rats were fed HFS diet (n = 24) for 16 wk to induce obesity and insulin resistance. Rats fed ND (n = 8) served as controls. Body weight (A), fasting plasma glucose (B), fasting plasma insulin (C), and HOMA-IR (D) values were analyzed monthly during the period of feeding HFS. Data represent the mean ± SD. Comparison between groups was carried out by ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. ND.

 
TNFR1BP-Fc treatment reverses obesity induced by HFS diet
We next examined the effect of TNFR1BP-Fc on obesity induced by HFS. Namely, 500 µg TNFR1BP-Fc or the same amount of IgG1Fc was administered iv into the rats at 17 wk after feeding with HFS when obesity-linked insulin resistance had developed. The rats were treated twice a week for 4 wk. As expected, the treatment with TNFR1BP-Fc significantly reduced obesity (by 26%, P < 0.05), whereas the treatment with IgG1Fc showed no effect on obesity (Fig. 3AGo). Consistently, the weight for epididymal fat pads was also markedly decreased in the rats treated with TNFR1BP-Fc as compared with that of the rats treated with IgG1Fc or NS (Fig. 3BGo, P < 0.001). Histological analysis of adipose tissues revealed that the sizes of adipocytes became larger in HFS-fed rats than those in ND-fed rats. The treatment with TNFR1BP-Fc, but not with IgG1Fc, diminished their large diameter (Fig. 3CGo). Importantly, there was no significant difference in the weight of other tissues such as liver, kidney, and pancreas examined (data not shown), which suggests that the reduced body weight gain by TNFR1BP-Fc is attributable to the decrease of fat tissue weight. Of note, daily food intake has shown no significant difference between the experimental and control animals, indicating that the decreased body weight was not due to reduced food consumption.


Figure 3
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  		FIG. 3. Blockade of TNFR1-mediated TNF-{alpha} signaling attenuates the development of obesity. HFS-fed rats were given iv 500 µg TNFR1BP-FC, IgGFc, or NS twice a week for 4 wk and then subjected to the following analysis: A, body weight gain; B, the weight of epididymal fat pads; C, the size of adipocytes; and D, the plasma triglyceride. Values are expressed as mean ± SD. Comparisons among groups were done by ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. ND; {dagger}, P < 0.05; {dagger}{dagger}, P < 0.01; {dagger}{dagger}{dagger}, P < 0.001 vs. HFS.

 
Furthermore, the lipid (triglyceride) content in the plasma of HFS-fed rats was much higher than that of ND-fed rats. TNFR1BP-Fc treatment drastically lowered the hypertriglyceridemia (P < 0.001), whereas IgG1Fc treatment had no effect (Fig. 3DGo). Moreover, the total cholesterol levels remained unchanged in the plasma among the four groups (data not shown).

TNFR1BP-Fc treatment increases peripheral insulin sensitivity
Because TNF-{alpha} plays a pivotal role in obesity-induced insulin resistance, we next analyzed the effect of TNFR1BP-Fc on glucose homeostasis and peripheral insulin sensitivity in the above animals. To our surprise, all animals remained euglycemic throughout the study (Fig. 4AGo). In contrast, fasting insulin levels (Fig. 4BGo) and C-peptide levels (Fig. 4CGo) in HFS-fed rats were increased by about 2-fold (P < 0.01). The rise in serum insulin concentrations in the presence of euglycemia indicates that there is a compensatory response to the development of obesity-induced insulin resistance. Therefore, the HOMA-IR was markedly elevated (P < 0.001, Fig. 4DGo). Consistently, a compensatory hypertrophy of pancreatic islets (Fig. 4EGo) with markedly increased insulin-containing cells (Fig. 4GGo) was observed in HFS-fed rats. The mean area of islets (Fig. 4FGo) was 3-fold larger (P < 0.001), and the β-cell mass (Fig. 4HGo) was 2-fold heavier (P < 0.01) in HFS-fed rats than those in ND-fed rats. However, TNFR1BP-Fc treatment significantly suppressed HSF-induced higher levels of insulin and C-peptide by nearly 40% (Fig. 4Go, B and C, P < 0.05), reduced hyperplasia and hypertrophy of pancreatic islets (P < 0.01), and blocked the augmentation of HOMA-IR (Fig. 4DGo, P < 0.01), suggesting that TNFRBP-Fc treatment protected animals from obesity-induced insulin resistance.


Figure 4
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  		FIG. 4. TNFR1BP-Fc treatment enhances peripheral insulin sensitivity. A–D, The obese animals were treated with either TNFR1BP-c or IgGFc for 4 wk. Fasting blood glucose (A), fasting insulin (B), C-peptide (C), and HOMA-IR (D) were determined; E and G, representatives of histological sections of pancreas were stained with hematoxylin and eosin (E) or a rat insulin antibody (G); F and H, mean islet size (F) and the β-cell mass (H) were analyzed by image software from three sections per rat, five rats from each group. Data represent the mean ± SD. Comparisons among groups were done by ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. ND; {dagger}, P < 0.05; {dagger}{dagger}, P < 0.01; {dagger}{dagger}{dagger}, P < 0.001 vs. HFS.

 
To further confirm the above observations, glucose and insulin tolerance assays were performed. Our ip glucose tolerance tests revealed a higher degree of hyperglycemia in the obese HFS group after 15–120 min than that in the ND group. A more significant drop-off in blood glucose levels was observed in TNFR1BP-Fc-treated animals as compared with that of IgG1Fc or NS-treated animals after glucose challenge from 30–120 min (Fig. 5AGo, P < 0.05). Similarly, the hypoglycemic response to a given dose of insulin was less in HFS-fed rats at 15–120 min than that in ND-fed rats (Fig. 5BGo). In contrast to the inefficacy of IgG1Fc, TNFR1BP-Fc treatment caused a much stronger hypoglycemic response to insulin (P < 0.05). Taken together, these results demonstrate that TNFR1BP-Fc treatment increased peripheral insulin sensitivity in HFS-induced obese animals.


Figure 5
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  		FIG. 5. TNFR1PB-Fc treatment enhances the capability of obese rats to glucose and insulin tolerance. The rats after the onset of obesity and insulin resistance were treated with TNFR1BP-Fc for 4 wk before the assays. Glucose (A) and insulin (B) tolerance tests were performed as described in Materials and Methods. Data represent the mean ± SD.

 
TNFR1BP-Fc treatment attenuates TNF-{alpha} production
We next sought to explore the molecular mechanisms by which TNFR1BP-Fc protects animals from HFS-induced obesity and insulin resistance. We first checked the effect of TNFRBP-Fc on the circulating TNF-{alpha} with ELISA. As shown in Fig. 6AGo, the serum levels of TNF-{alpha} were 2.4-fold higher in HFS-fed rats than those in ND-fed ones. Treatment with TNFR1BP-Fc restored circulating TNF-{alpha} levels to those of ND-fed animals (P < 0.01). Similarly, TNF-{alpha} production in HFS-fed rats was significantly decreased in the supernatants of epididymal fat pad and muscle extracts after treatment with TNFRBP-Fc as compared with that of treatment with IgG1Fc or NS (Fig. 6Go, B and C). In situ immunostaining of pancreatic tissue sections was then performed for detection of TNF-{alpha} expression. TNF-{alpha} (brown punctiform particles) was found to be predominantly distributed in islets of the pancreas. Furthermore, TNF-{alpha} expression in the pancreatic islets was significantly increased in HSF-fed rats (P < 0.001). This elevated TNF-{alpha} expression, however, was markedly blocked by TNFRBP-Fc treatment (P < 0.01, Fig. 6Go, D and E).


Figure 6
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  		FIG. 6. TNFR1BP-Fc treatment inhibits TNF-{alpha} production. The production of TNF-{alpha} was assayed in obese rats after 4 wk of treatment with TNFR1BP-Fc or IgGFc. A–C, TNF-{alpha} concentration in serum (A) and in supernatant of epididymal fat pads (B) and muscle extracts (C) were determined by ELISA; D, tissue sections from pancreas were stained with the avidin-biotin complex method using a rat TNF-{alpha} polyclonal antibody; E, intensity of TNF-{alpha} expression in islets was analyzed by image software from three sections per rat, five rats from each group. Values are expressed as mean ± SD. *, P < 0.05, **, P < 0.01, ***, P < 0.001 vs. ND; {dagger}, P < 0.05, {dagger}{dagger}, P < 0.01, {dagger}{dagger}{dagger}, P < 0.001 vs. HFS.

 
TNFR1BP-Fc treatment enhances IRS tyrosine phosphorylation
It has been demonstrated that TNF-{alpha} promotes insulin resistance by disrupting the insulin receptor-mediated signaling (6). Because tyrosine phosphorylation of IRS-1 is the initial step to trigger the downstream insulin signaling, we therefore examined the impact of TNFRBP-Fc treatment on IRS-1 tyrosine phosphorylation. As shown in Fig. 7AGo, tyrosine-phosphorylated IRS-1 was undetectable in all of the tissues examined without insulin stimulation (the first four lanes of each panel). However, after 30 min of insulin stimulation, high levels of tyrosine-phosphorylated IRS-1 were detected in the muscle, fat, and liver tissues derived from ND-fed rats (the last lane of each panel). This induction was down-regulated in the fat (by 80%) and muscle (by 68%) tissues from HFS-fed rats (Fig. 7Go, A and B). Nevertheless, TNFRBP-Fc treatment significantly promoted the tyrosine phosphorylation of IRS-1 in the muscle and fat tissues. On average, the treatment restored the levels of tyrosine-phosphorylated IRS-1 to 92.7% in the muscle and 84.7% in the fat tissues (Fig. 7BGo). In the liver, however, phosphorylated tyrosine of IRS-1 remained unchanged among the three groups fed with HFS diet.


Figure 7
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  		FIG. 7. Blockade of TNFR1 augments IRS-1 tyrosine phosphorylation. Epididymal fat pads, quadriceps, and livers were collected after 30 min of PBS or insulin administration, and 1 mg extract protein was immunoprecipitated by 1 µg/ml polyclonal antibody against IRS-1. Tyrosine phosphorylation levels were examined by immunoblotting with a monoclonal antibody against phosphotyrosine. The same membrane was reprobed with an anti-IRS-1 antibody as a control for equal protein loading. A, Representative Western blot results for each tissue examined; B, relative level of insulin-induced tyrosine-phosphorylated IRS-1 was determined by densitometric analysis and normalized by the total IRS-1 protein. Data represent the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. ND; {dagger}, P < 0.05; {dagger}{dagger}, P < 0.01; {dagger}{dagger}{dagger}, P < 0.001 vs. HFS.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been well demonstrated that TNF-{alpha} plays a pivotal role in the initiation and progression of obesity and its related insulin resistance. Therefore, TNF-{alpha} neutralizing antibodies or sTNFR has been used to block TNF-{alpha} bioactivity clinically as a therapeutic approach for obesity and insulin resistance (32, 33). Unfortunately, both approaches did not show expected effects. One of the possible reasons could be the unknown actions of reverse signaling via transmembrane TNF-{alpha} activated by both reagents. Although a number of studies suggested that TNFR1 is the major mediator responsible for the TNF-{alpha} pathophysiological action in insulin resistance (5, 19, 22), contradictory results have also been reported (18). To address this conflictive issue and explore the feasibility of TNFR1 as a therapeutic target for TNF-{alpha}-mediated obesity and insulin resistance, we used a TNFR1 binding peptide that has been previously confirmed by us to effectively inhibit TNF-{alpha}-induced activation of rat peritoneal macrophages in vitro (30) and alleviate inflammatory damage of adjuvant arthritis in rats (31). In the present study, this blocking peptide was linked to the IgG1 Fc fragment above the hinge region (TNFR1BP-Fc), by which the blocking peptides form a dimer to compete with TNF-{alpha} for receptor binding and to suppress TNF-{alpha} bioactivity including transmembrane TNF-{alpha} reverse signaling.

We treated animals with TNFR1BP-Fc fusion protein after obesity-linked insulin resistance had been developed, which is closer to reality because patients come to the doctor when the disease is present. After being fed with HFS diet for 16 wk, these rats developed obesity characterized by a 50% increase in body weight gain and a 60% increase in fattiness. Their obesity-linked insulin resistance was manifested as hyperinsulinemia to maintain euglycemia and less utilization of glucose in the presence of insulin (Figs. 3Go and 5Go). This model actually resembles the reality of human patients with clinical onset of obesity and insulin resistance.

TNFR1BP-Fc was used to treat animals for 4 wk after the onset of obesity and insulin resistance. It was found that blockade of TNFR1 signaling protected Wistar rats from HFS-induced obesity and adiposity. Although the expression of TNF-{alpha} was reported to be in strong positive correlation with the degree of obesity, the absence of TNF-{alpha} did not have a significant effect on the development of dietary obesity in TNF-{alpha}–/– mice (17). In contrast, our results indicate that blockade of TNFR1 signaling reduced obesity. The treatment of TNFR1BP-Fc resulted in body weight loss, which was mainly due to a decrease in adiposity and adipocyte size (Fig 3Go). These results suggest that TNF-{alpha} may have a direct or indirect effect on the increase of adipose tissue mass. However, TNF-{alpha} has been shown to suppress expression of the enzymes involved in lipogenesis and cause cachexia (34, 35, 36). It appears to be associated with the hormonal milieu of the organism and the relative concentrations of this cytokine. However, the production of TNF-{alpha} in animals with obesity and insulin resistance was far lower than those that can induce a variety of other symptoms, including cachexia (9). Furthermore, TNF-{alpha} potentially augments in vivo adipogenesis, because it may function as a growth factor for preadipocytes (37). Here, we have shown that blockade of TNFR1 signaling prevented hypertriglyceridemia in obese rats. This hyperlipidemia is thought to be the result of decreased lipoprotein lipase activity and increased hepatic lipogenesis (38).

TNF-{alpha} may be one of the key molecules that link obesity with insulin resistance. As shown here, the concentrations of systemic and local TNF-{alpha} were significantly elevated in obese animals and were highly correlated with insulin resistance because these animals needed three times higher insulin to maintain euglycemia. Furthermore, blockade of TNFR1 signaling significantly inhibited TNF-{alpha} expression, which was also correlated with enhanced peripheral insulin sensitivity such as decreased fasting hyperinsulinemia, reduced C-peptide levels and HOMA-IR values, diminished hyperplasia and hypertrophy of pancreatic islets (Fig. 4Go), and an improved hypoglycemic response to insulin (Fig. 5Go). Consistent with our results, TNF-{alpha} has been reported to be overexpressed in the adipose tissue of rodents and humans with obesity and insulin resistance (9, 11), and animals with TNF-{alpha} deficiency had a significantly improved insulin sensitivity in dietary or hypothalamic obesity (17, 39). In addition, a synthetic compound that blocks TNF-{alpha} production has been effective in increasing insulin sensitivity in obese diabetic yellow KK (KKAy) mice (40).

Of note, TNFRBP-Fc treatment only partially reversed insulin resistance (i.e. a 2- to 3-fold increase in insulin-stimulated glucose utilization). This limited effect of TNFRBP-Fc on insulin resistance could be due to the following reasons: 1) the length of treatment and the given dose of TNFRBP-Fc may be not optimal (because nearly all nucleated cells constitutively express TNFR1, the amount of TNFRBP-Fc necessary to sufficiently block TNFR1 could be higher than what we administered) and (2) the existence of other molecules in addition to TNF-{alpha} in the pathogenesis of obesity and insulin resistance. It has been shown that free fatty acid, resistin, and adiponectin also play a key role in obesity-induced insulin resistance both in vitro and in vivo (41, 42, 43).

Although the mechanism for TNF-{alpha} action in obesity-linked insulin resistance is not fully understood, it is well known that TNF-{alpha} can induce IRS-1 serine phosphorylation, thereby interfering in the interaction between insulin receptor and IRS-1, reducing IRS-1 tyrosine phosphorylation and disrupting downstream insulin signaling. In line with this assumption, our data indicate that TNFRBP-Fc treatment enhanced IRS-1 tyrosine phosphorylation, which could be associated with inhibited TNFR1 signaling and reduced TNF-{alpha} production. However, the improved IRS-1 tyrosine phosphorylation can be detected only in the muscle and fat tissues but not in the liver (Fig. 7Go). This observation somehow is consistent with those previously reported data in rodents (44, 45). Moreover, these data also suggest that additional molecular mechanisms may be implicated in the pathogenesis of hepatic insulin resistance. TNF-{alpha} may modulate insulin signaling in the liver either at sites downstream from the insulin receptor or indirectly via alterations in metabolic parameters, e.g. gluconeogenic substrates or glucagon.

In summary, we provided strong evidence demonstrating the importance of TNFR1-mediated TNF-{alpha} signaling in the pathogenesis of obesity and insulin resistance. Blockade of TNFR1 by TNFRBP-Fc not only prevented the development of obesity and enhanced peripheral insulin sensitivity but also attenuated the production of TNF-{alpha}. Unlike the effect of conventional TNF-{alpha} neutralizing antibodies or sTNFR, TNFR1BP-Fc not only blocks TNFR1 signaling but also inhibits the transmembrane TNF-{alpha}-mediated reverse signaling, because it disrupts the interaction of TNFR and transmembrane TNF-{alpha}. Further studies will address the role of transmembrane TNF-{alpha} in obesity-linked insulin resistance, which might give us deeper insights to select anti-TNF-{alpha} compounds that could be useful as therapeutic agents.


   Acknowledgments
 
We thank Drs. Diethard Gemsa (Institute of Immunology, Philipps University, Marburg, Germany) and Richard McIndoe (Center for Biotechnology and Genomic Medicine, Medical College of Georgia, Augusta, GA) for their edition of the manuscript.


   Footnotes
 
This work was supported by the National Natural Science Foundation of China (30471586) and the Hi-tech Research and Development Program of China from the Ministry of Science and Technology of the People’s Republic of China (2004AA215162).

Disclosure Summary: The authors have nothing to disclose.

First Published Online March 13, 2008

1 H.L. and B.Y. contributed equally to this work. Back

Abbreviations: FITC, Fluorescein isothiocyanate; HFS, high-fat/high-sucrose; HOMA-IR, homoeostasis model assessment index for insulin resistance; IRS-1, insulin receptor substrate 1; ND, normal diet; NS, normal saline solution; sTNF, soluble TNF; TNFR1, TNF receptor type 1; TNFR1BP-Fc, TNFR1 blocking peptide-Fc fusion protein.

Received July 17, 2007.

Accepted for publication March 6, 2008.


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 Introduction
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 Results
 Discussion
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