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Endocrinology Vol. 148, No. 12 5991-5997
Copyright © 2007 by The Endocrine Society

Infliximab Restores Glucose Homeostasis in an Animal Model of Diet-Induced Obesity and Diabetes

Eliana P. Araújo, Cláudio T. De Souza, Mirian Ueno, Dennys E. Cintra, Manoel B. Bertolo, José B. Carvalheira, Mário J. Saad and Lício A. Velloso

Department of Internal Medicine, State University of Campinas, Sao Paulo 13083-970, Brazil

Address all correspondence and requests for reprints to: Dr. Lício A. Velloso, Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Sao Paulo 13083-970, Brazil. E-mail: lavelloso{at}fcm.unicamp.br.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TNF-{alpha} plays an important role in obesity-linked insulin resistance and diabetes mellitus by activating at least two serine kinases capable of promoting negative regulation of key elements of the insulin signaling pathway. Pharmacological inhibition of TNF-{alpha} is currently in use for the treatment of rheumatoid and psoriatic arthritis, and some case reports have shown clinical improvement of diabetes in patients treated with the TNF-{alpha} blocking monoclonal antibody infliximab. The objective of this study was to evaluate the effect of infliximab on glucose homeostasis and insulin signal transduction in an animal model of diabetes. Diabetes was induced in Swiss mice by a fat-rich diet. Glucose and insulin homeostasis were evaluated by glucose and insulin tolerance tests and by the hyperinsulinemic-euglycemic clamp. Signal transduction was evaluated by immunoprecipitation and immunoblotting assays. Short-term treatment with infliximab rapidly reduced blood glucose and insulin levels and glucose and insulin areas under the curve during a glucose tolerance test. Furthermore, infliximab increased the glucose decay constant during an insulin tolerance test and promoted a significant increase in glucose infusion rate during a hyperinsulinemic-euglycemic clamp. In addition, the clinical outcomes were accompanied by improved insulin signal transduction in muscle, liver, and hypothalamus, as determined by the evaluation of insulin-induced insulin receptor, insulin receptor substrate-1, and receptor substrate-2 tyrosine phosphorylation and Akt and forkhead box protein O1 serine phosphorylation. Thus, pharmacological inhibition of TNF-{alpha} may be an attractive approach to treat severely insulin-resistant patients with type 2 diabetes mellitus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TYPE 2 DIABETES MELLITUS affects more than 190 million people worldwide, and its prevalence is expected to increase rapidly to reach 330 million by the year 2025 (www.idf.org). Obesity is the most common clinical condition associated with diabetes (1, 2), and proinflammatory factors produced by the enlarged adipose tissue are known to play an important role in the association between obesity and diabetes (3, 4). Several studies have shown that adipose tissue-produced TNF-{alpha} is capable of inducing insulin resistance by activating intracellular serine kinases that hamper insulin signal transduction by targeting key proteins of the insulin signaling machinery (5, 6, 7, 8). The importance of TNF-{alpha} as an inducer of insulin resistance has been thoroughly investigated and approaches leading to both the activation and the inhibition of its signaling have been employed to unravel the mechanisms involved in this phenomenon (6, 7, 9, 10, 11).

In clinical practice, the inhibition of TNF-{alpha} activity has proven to have beneficial effects in diseases such as rheumatoid arthritis, Crohn’s disease, and psoriatic arthritis (12, 13). The anti-TNF-{alpha} monoclonal antibody infliximab is one of the available drugs approved for human use and is currently employed by thousands of patients worldwide (12). Some groups have recently reported an improvement in glycemic control in patients presenting type 2 diabetes and rheumatic or psoriatic arthritis simultaneously (14, 15). In addition, one study has demonstrated a relapse in diabetes after the interruption of infliximab use in a patient with diabetes and psoriatic arthritis (16).

To determine the mechanisms involved in infliximab-induced blood glucose control, we evaluated key molecular steps of the insulin signaling machinery in skeletal muscle, liver, adipose tissue, and hypothalamus of a mouse model of obesity-associated diabetes. Our results show that the impaired signal transduction through the insulin receptor (IR)/insulin receptor substrate-1 (IRS1)/IRS2/Akt/forkhead box protein O1 (FOXO1) pathway is completely restored in muscle, liver, and hypothalamus of infliximab-treated mice. This phenomenon is accompanied by a reduction in IRS1 phosphorylation at serine residue 307 and by reduced activation of c-Jun N-terminal kinase (JNK).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies, chemicals, and buffers
Reagents for SDS-PAGE and immunoblotting were from Bio-Rad (Richmond, CA). HEPES, phenylmethylsulfonyl fluoride, aprotinin, dithiothreitol, Triton X-100, Tween 20, glycerol, and BSA (fraction V) were from Sigma Chemical Co. (St. Louis, MO). [125I]Protein A and nitrocellulose paper (BA85, 0.2 µm) were from Amersham (Aylesbury, UK). Amobarbital and human recombinant insulin (Humulin R) were from Lilly (Indianapolis, IN). The anti-TNF-{alpha} monoclonal antibody infliximab was from Centocor (Horsham, PA). Anti-IR (sc-711, rabbit polyclonal), anti-iIRS1 (sc-560, rabbit polyclonal), anti-IRS2 (sc-8299, rabbit polyclonal), anti-Akt (sc-1618, goat polyclonal), anti-phosphotyrosine (sc-508, mouse monoclonal), anti-phospho-[Ser473] Akt (sc-7985-R, rabbit polyclonal), anti-phospho-[Thr183] JNK (sc-6254, mouse monoclonal), anti-FOXO1 (sc-11350, rabbit polyclonal), and anti-phospho-[Ser256] FOXO1 (sc-22158-R, rabbit polyclonal) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-[Ser307] IRS1 (no. 2381, rabbit polyclonal) was from Cell Signaling Technology (Danvers, MA).

Mouse model and treatment protocols
Male 6-wk-old Swiss (Sw/Uni) inbred strain mice, originally imported from the Jackson Laboratory and currently bred at the State University of Campinas Breeding Center were used in the study. The investigation followed the University guidelines for the use of animals in experimental studies and conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). The animals were maintained on an artificial 12-h light, 12-h dark cycle and housed in individual cages. Mice were treated either with a regular rodent chow or with a fat-rich diet (Table 1Go). At 6 and 14 wk of age (0 and 8 wk after introduction of diets), mice were evaluated for biochemical and hormonal parameters (Table 2Go). After setting 8 wk of fat-rich-diet feeding as the time when all mice have developed diabetes, animals from the fat-rich-diet group were randomly divided and treated either with saline (100 µl/dose, ip, twice a day) or with infliximab (10 µg in 100 µl saline per dose, ip, twice a day). The infliximab dose was adjusted for mice metabolic rates, compared with human metabolic rates (in clinical practice, the human dose is 5.0–10.0 mg/kg every 8 wk). Because the half-life of Ig is about 2–3 wk, we believe that treating experimental animals on a daily basis leads to stable anti-TNF-{alpha} Ig levels at about 7–10 d treatment. All experiments were conducted at 0 or 14 d after starting infliximab use.


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  		TABLE 1. Macronutrient composition of the diets

 

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  		TABLE 2. Diet-induced obesity and diabetes mellitus in Sw/Uni mice: clinical, biochemical, and hormonal parameters

 
Determination of glucose, leptin, and insulin
Serum glucose was determined by the glucose oxidase method, as previously described (17). Serum insulin was determined by RIA, as described (18). Serum leptin was determined using an ELISA kit from Linco/Millipore (Billerica, MA) according to the recommendations of the manufacturer.

Intraperitoneal glucose tolerance test
After 6 h fasting, mice were anesthetized by an ip injection of sodium amobarbital (15 mg/kg body weight), and the experiments were initiated after the loss of corneal and pedal reflexes. After collection of an unchallenged sample (time 0), a solution of 20% glucose (2.0 g/kg body weight) was administered into the peritoneal cavity. Blood samples were collected from the tail at 30, 60, 90, and 120 min for determination of glucose and insulin concentrations.

Insulin tolerance test
Insulin (1.5 U/kg) was administered by ip injection, and blood samples were collected at 0, 5, 10, 15, 20, 25, and 30 min for serum glucose determination. The rate constant for glucose disappearance during an insulin tolerance test (KITT) was calculated using the formula 0.693/t1/2. The glucose t1/2 was calculated from the slope of the least-square analysis of the plasma glucose concentrations during the linear decay phase (19).

Hyperinsulinemic-euglycemic clamp
After 6 h fasting, mice were anesthetized by an ip injection of sodium amobarbital (15 mg/kg body weight), and the experiments were initiated after the loss of corneal and pedal reflexes. Catheters were inserted into the left jugular vein (for infusion) and femoral artery (for blood sampling). A 120-min hyperinsulinemic-euglycemic clamp procedure was conducted as previously described, with minor modifications (20). In obese control mice (OC) and infliximab-treated mice, a bolus injection of insulin (200 mU/kg) was required for reaching stable euglycemic levels. In lean control mice (LC), no bolus insulin was required. During the clamp, insulin was continuously infused at a rate of 5.0 mU/kg body weight·min to raise the plasma insulin concentration to approximately 4.8–5.4 ng/ml. Glucose (5%) was infused at variable rates to maintain plasma glucose at 110 ± 10 mg/dl. All infusions were performed using Harvard infusion pumps (Harvard Apparatus, Holliston, MA). At the end of the clamp procedure, animals were killed by a mix of ketamine and diazepam iv injection.

Cytokine determination
Serum levels of TNF-{alpha}, IL-1ß, and IL-6 were determined in samples obtained at the beginning and at the end of the experimental periods using ELISA kits from Pierce Biotechnology (Rockford, IL), according to the instructions of the manufacturer.

Immunoprecipitation and immunoblotting
Immunoprecipitation and immunoblotting assays were performed as previously described, with minor modifications (21, 22). The abdominal cavities of anesthetized mice were opened, and the animals received an injection of insulin (100 µl, 10–6 mol/liter) or saline (100 µl) through the cava vein. After different intervals (described in Results), fragments (3.0 x 3.0 x 3.0 mm) of white adipose tissue, gastrocnemius muscle, liver, and hypothalamus were excised and immediately homogenized in solubilization buffer at 4 C [1% Triton X-100, 100 mmol/liter Tris-HCl (pH 7.4), 100 mmol/liter sodium pyrophosphate, 100 mmol/liter sodium fluoride, 10 mmol/liter EDTA, 10 mmol/liter sodium orthovanadate, 2.0 mmol/liter phenylmethylsulfonyl fluoride, and 0.1 mg aprotinin/ml] with a Polytron PTA 20S generator (model PT 10/35; Brinkmann Instruments, Westbury, NY). The protein concentration of the supernatants was determined by the Bradford dye binding method. Aliquots of the resulting supernatants containing 2.0 mg total protein were used for immunoprecipitation with antibodies against IR, IRS1, or IRS2 at 4 C overnight, followed by SDS-PAGE, transfer to nitrocellulose membranes, and blotting with anti-phosphotyrosine antibodies. In direct immunoblot experiments, 0.2 mg protein extracts obtained from each tissue were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-IR, anti-IRS1, anti-IRS2, anti-Akt, anti-phospho-[Ser473] Akt, anti-FOXO1, anti-phospho-[Ser256] FOXO1, anti-phospho [Ser307] IRS1, and anti-phospho-[Thr183] JNK antibodies. Specific bands were labeled with [125I]protein A, and visualization was performed by exposure of the membranes to RX-films.

Statistical analysis
Specific protein bands present in the blots were quantified by digital densitometry (ScionCorp, Inc., Frederick, MD). Mean values ± SEM obtained from densitometric scans and the values for blood insulin, leptin, cytokines, and glucose, body weight, and food intake were compared using the Mann-Whitney U test. A P value of <0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Metabolic characteristics of the experimental animals
Table 2Go shows that after 8 wk, the consumption of a fat-rich diet led to a significant increase in body mass, blood glucose, insulin, and leptin levels. These effects were accompanied by increased glucose and insulin areas under the curve during a glucose tolerance test and to insulin resistance, as determined by the constant of glucose decay during an insulin tolerance test. In addition, obese mice had higher blood levels of TNF-{alpha}, IL-6, and IL-1ß than control mice. Based on these results, we decided to start treating mice with infliximab at this time.

Infliximab modulates blood TNF-{alpha} and leptin levels
As expected, the treatment with infliximab caused a reduction of TNF-{alpha} concentration in blood (Table 3Go). This was not accompanied by significant modification of IL-1ß and IL-6, although a tendency for reduction could be noticed. In addition, the blood concentration of leptin was significantly reduced by infliximab (Table 3Go).


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  		TABLE 3. Effect of infliximab treatment on serum hormone and cytokine levels in diet-induced obese Sw/Uni mice

 
Infliximab restores glucose homeostasis
Fourteen days of treatment with infliximab resulted in no significant changes in mean daily food intake (Fig. 1AGo) and in body mass (Fig. 1BGo). However, the treatment produced a significant reduction in fasting glucose levels (Fig. 1CGo) that was accompanied by an increased glucose decay constant during the insulin tolerance test (Fig. 1DGo) and by a reduced glucose area under the curve during the glucose tolerance test (Fig. 1EGo). The insulin area under the curve during the glucose tolerance test was not affected by infliximab treatment (Fig. 1FGo). In addition, infliximab treatment caused a significant increase in glucose infusion rate during the hyperinsulinemic-euglycemic clamp (Fig. 2Go).


Figure 1
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  		FIG. 1. Metabolic parameters. Mean 24-h food intake (A), body mass (B), and blood glucose levels (C) were determined before (D0) and 14 d (D14) after the beginning of treatment in obese Sw/Uni mice treated with saline (OC in A and C; {blacksquare} in B) or with infliximab (I in A and C; bullet in B). KITT during an insulin tolerance test (D); and, glucose (E) and insulin (F) areas under curves during a glucose tolerance test were determined in lean Sw/Uni (LC), obese Sw/Uni treated with saline (OC) or obese Sw/Uni treated with infliximab (I) for 14 d. In all experiments, values are means ± SEM; n = 5. *, P < 0.05 vs. OC (D14) in C and vs. LC in D–F; {dagger}, P < 0.05 vs. OC in D and E.

 

Figure 2
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  		FIG. 2. Hyperinsulinemic-euglycemic clamp. Steady-state glucose infusion rates obtained from 120-min infusion of 5% unlabeled glucose during a hyperinsulinemic-euglycemic clamp procedure. Analysis was performed in lean Sw/Uni (LC), obese Sw/Uni treated with saline (OC), or obese Sw/Uni treated with infliximab (I) for 14 d. In all experiments, values are means ± SEM; n = 4. *, P < 0.05 vs. LC; {dagger}, P < 0.05 vs. OC.

 
Infliximab improves insulin signal transduction in skeletal muscle, liver, and hypothalamus
High-fat feeding is known to produce severe impairment of insulin signaling in several strains of mice and rats (10, 23, 24). In this study, we evaluated insulin signaling through the IR/IRS1/IRS2/Akt/FOXO1 pathway in skeletal muscle, liver, adipose tissue, and hypothalamus of Sw/Uni mice fed on regular and fat-rich chow. Impairment in all steps of insulin signaling was detected in skeletal muscle, liver, and hypothalamus but not in adipose tissue of fat-rich diet-treated mice (Fig. 3Go). To determine the effect of TNF-{alpha} inhibition on insulin signal transduction, Sw/Uni mice fed on a fat-rich diet were treated with infliximab for 14 d and evaluated for insulin signaling in the tissues, as above. As shown in Fig. 4Go, significant improvement in insulin signal transduction through all elements of the IR/IRS1/IRS2/Akt/FOXO1 pathway was detected in skeletal muscle, liver, and hypothalamus of mice treated with infliximab. No significant change was observed in any of the steps of the insulin signaling cascade in adipose tissue.


Figure 3
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  		FIG. 3. Insulin signal transduction in lean and control obese mice. Twelve-week-old LC or OC Sw/Uni mice were anesthetized and acutely treated through the cava vein with saline (100 µl) (–) or insulin (100 µl, 10–6 mol/liter) (+). After 5 (IR, IRS1, and IRS2), 10 (Akt), or 20 (FOXO1) minutes, fragments were obtained from skeletal muscle (A), liver (B), adipose tissue (C), and hypothalamus (D) and used in immunoprecipitation and immunoblotting experiments. In immunoprecipitation (IP) experiments, samples containing 2.0 mg total protein extracts were incubated with respective antibodies (as stated in the right margin), and immunocomplexes recovered with protein A Sepharose were separated, under denaturing conditions, by SDS-PAGE. Nitrocellulose transfers were blotted (IB) with respective antibodies. In direct immunoblotting (IB) experiments, samples containing 0.2 mg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with respective antibodies. The bar graphs represent means ± SEM of densitometric values obtained for the bands of LC and OC treated with insulin (+). In all experiments, n = 5. *, P < 0.05 vs. respective LC.

 

Figure 4
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  		FIG. 4. Insulin signal transduction in infliximab-treated mice. Obese Sw/Uni mice treated for 14 d with saline (OC) or infliximab (I) were anesthetized and acutely treated through the cava vein with saline (100 µl) (–) or insulin (100 µl, 10–6 mol/liter) (+). After 5 (IR, IRS1, and IRS2), 10 (Akt), or 20 (FOXO1) minutes, fragments were obtained from skeletal muscle (A), liver (B), adipose tissue (C), and hypothalamus (D) and used in immunoprecipitation and immunoblotting experiments. In immunoprecipitation (IP) experiments, samples containing 2.0 mg total protein extracts were incubated with respective antibodies (as stated in the right margin), and immunocomplexes recovered with protein A Sepharose were separated, under denaturing conditions, by SDS-PAGE. Nitrocellulose transfers were blotted (IB) with respective antibodies. In direct immunoblotting (IB) experiments, samples containing 0.2 mg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with respective antibodies. The bar graphs represent means ± SEM of densitometric values obtained for the bands of OC and I treated with insulin (+). In all experiments, n = 5. *, P < 0.05 vs. respective OC.

 
Infliximab inhibits JNK activation and reduces IRS1 serine phosphorylation
Because TNF-{alpha} inhibits insulin action by activating serine kinases known to promote serine phosphorylation and molecular inhibition of at least one important substrate of the insulin signaling cascade, IRS1, we decided to evaluate the effect of infliximab treatment on threonine phosphorylation/activation of JNK and serine phosphorylation of IRS1. Figure 5Go shows that infliximab significantly reduced the threonine phosphorylation of JNK in skeletal muscle, liver, and hypothalamus of Sw/Uni mice fed on a fat-rich diet. This effect was accompanied by a reduction in serine phosphorylation of IRS1 in the same tissues. No significant modification of JNK threonine phosphorylation and IRS1 serine phosphorylation was detected in the adipose tissue of infliximab-treated mice.


Figure 5
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  		FIG. 5. JNK activation and IRS1 serine phosphorylation. Obese Sw/Uni mice, treated for 14 d with saline (OC) or infliximab (I) were anesthetized, and fragments from skeletal muscle, liver, adipose tissue, and hypothalamus were obtained for preparation of protein extracts used in immunoblotting experiments. Samples containing 0.2 mg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-phospho-[Thr183] JNK (A) or anti-phospho-[Ser307] IRS1 (B) antibodies. The bar graphs represent means ± SEM of densitometric values obtained for the bands of OC and I. In all experiments, n = 5. *, P < 0.05 vs. OC.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studies performed in the last 15 yr have provided strong evidence to support a mechanistic role of inflammation in the pathogenesis of insulin resistance and type 2 diabetes mellitus (3, 5, 6, 10, 11). Cytokines produced by the adipose tissue of obese subjects can activate systemic proinflammatory signaling (7, 8, 25). When acting in insulin-sensitive tissues, these proinflammatory cytokines disrupt the insulin signaling machinery by activating serine kinases that target proteins such as the IR, IRS1, and IRS2, producing molecular resistance to insulin action (7, 26, 27). In addition, more recently, the effect of locally produced cytokines to inhibit insulin action was shown in the hypothalamus of experimental animals fed on a high-fat diet (10, 28) and in the liver of humans and rodents with nonalcoholic steatohepatitis, a condition known to be associated with hepatic insulin resistance (29, 30).

Several proinflammatory cytokines and proteins are thought to play a pathogenetic role in obesity-associated insulin resistance (3, 5, 6, 10). However, due to its properties of powerfully controlling gene transcription of other cytokines and acting, by itself, as an effector of inflammation, TNF-{alpha} is regarded as one of the central determinants of this phenomenon (7, 8).

The mechanisms involved in TNF-{alpha}-induced insulin resistance have been thoroughly studied using approaches such as the inhibition of TNF-{alpha} or TNF-{alpha} receptors and genetic/pharmacological blockade of key elements of the TNF-{alpha} signaling pathway (3, 5, 6, 7, 8, 10). All these approaches led, invariably, to the improvement of insulin action in the various cellular and organismal systems tested.

Because inhibition of TNF-{alpha}, using monoclonal antibodies, is currently used for the treatment a number of inflammatory diseases, and considering that recent studies have reported the improvement of glycemic control in patients on infliximab (14, 15, 16), we decided to evaluate the mechanisms involved in infliximab-induced improvement of glucose homeostasis in an animal model of diet-induced obesity and diabetes.

Swiss mice are related to the diabetes-prone AKR mice (31) and on a high-fat diet become rapidly obese and diabetic (22). These phenomena are accompanied by increased blood levels of proinflammatory cytokines, including TNF-{alpha}, as shown here and elsewhere (32), and by reduced blood levels of adiponectin (32). Thus, high-fat diet-fed Swiss mice are regarded as a good experimental model for diabetes and the metabolic syndrome (22, 32).

Initially, we evaluated the effect of infliximab treatment upon cytokine/adipokine levels in blood and upon several parameters related to glucose homeostasis in obese Sw/Uni mice. After 2 wk treatment, blood TNF-{alpha} and leptin were significantly reduced, whereas the levels of IL-1ß and IL-6 displayed only a tendency of reduction. In other studies, infliximab treatment produced variable results concerning blood cytokine concentration, and this may be explained by differences in species/strain and protocol specificities such as the dose used and the duration of treatment (33, 34, 35, 36, 37). Regarding the metabolic parameters, infliximab produced a significant fall in fasting blood glucose levels and glucose area under the curve values during a glucose tolerance test. Because these outcomes were accompanied by a significant increase of KITT, with no change in insulin area under the curve during a glucose tolerance test, and by improved glucose consumption during the hyperinsulinemic-euglycemic clamp, we believe that most of the effect of infliximab was due to the improved peripheral action of insulin. In addition, although food intake and body mass did not decrease significantly, the reduction of blood leptin levels suggests that a beneficial effect on the anorexigenic action of this adipokine was achieved. This is further supported by the improved insulin signal transduction in hypothalamus, because both hormones act synergistically in this region of the brain (38).

To determine the effect of infliximab treatment upon insulin action, we evaluated insulin signal transduction through the IR/IRS1/IRS2/Akt/FOXO1 pathway in skeletal muscle, liver, adipose tissue, and hypothalamus of obese mice treated or not with infliximab. In preliminary experiments, we observed that all but adipose tissue presented features of molecular resistance to insulin. The time course of events leading to tissue-specific resistance to insulin action in animal models of obesity have revealed that adipose is indeed the last tissue to become insulin resistant (23); this fact was confirmed in the present study. In the remaining tissues, after 2 wk of infliximab treatment, signal transduction through all elements studied were significantly improved, revealing that the metabolic outcomes of TNF-{alpha} inhibition were, at least in part, due to improved peripheral insulin action.

In the final part of the study, we demonstrate that the metabolic and molecular outcomes of infliximab treatment are accompanied by reduced molecular activation of JNK and reduced serine 307 phosphorylation of IRS1. In previous studies, the use of distinct approaches to inhibit TNF-{alpha} in cellular and animal models of insulin resistance promoted, to a variable degree, the modulation of signaling through JNK, which resulted in reduction of the inhibitory serine 307 phosphorylation of IRS1 (7, 10).

As a whole, the present study provides additional support for the role of TNF-{alpha} in the pathogenesis of insulin resistance and places the inhibition of TNF-{alpha} as an attractive approach for the treatment of diabetes mellitus and related disorders. Furthermore, the specific mechanism involved in infliximab-induced improvement of blood glucose levels was determined.


   Acknowledgments
 
We are indebted to Dr. N. Conran for English editing.


   Footnotes
 
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and Conselho Nacional de Pesquisa (CNPq).

Disclosure Statement: E.P.A., C.T.D.S., M.U., D.E.C., M.B.B., J.B.C., M.J.S., and L.A.V. have nothing to declare.

First Published Online August 30, 2007

Abbreviations: FOXO1, Forkhead box protein O1; IR, insulin receptor; IRS1, insulin receptor substrate-1; JNK, c-Jun N-terminal kinase; KITT, rate constant for glucose disappearance during an insulin tolerance test; LC, lean control; OC, obese control.

Received January 29, 2007.

Accepted for publication August 20, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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