Neutralization of tumor necrosis factor-{alpha} reverses insulin resistance in skeletal muscle but not adipose tissue

Stephen E. Borst,1,2 Youngil Lee,1 Christine F. Conover,2 Eugene W. Shek,2 and Gregory J. Bagby3

1Department of Applied Physiology and Kinesiology, University of Florida, Gainesville 32611; 2Malcom Randall Veterans Affairs Medical Center, Gainesville, Florida 32608; and 3Department of Physiology, Louisiana State University Health Science Center, New Orleans, Louisiana 70112

Submitted 3 February 2004 ; accepted in final form 15 June 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We examined the possible role of tumor necrosis factor-{alpha} (TNF-{alpha}) as a mediator of insulin resistance in maturing male Sprague-Dawley rats. Rats were treated either with goat anti-murine TNF-{alpha} IgG (anti-TNF-{alpha}) or goat nonimmune IgG (NI) for 7 days. Vascular catheters were implanted, and rats were fasted overnight before hyperinsulinemic euglycemic clamp (HUC) studies were performed. TNF-{alpha} neutralization increased the rate of glucose infusion required to maintain euglycemia by 68%. Insulin-stimulated glucose transport into individual tissues was measured after bolus administration of 2-deoxy-[14C]glucose during HUC. Anti-TNF-{alpha} administration increased glucose transport in muscles composed predominantly of fast-twitch fibers: white gastrocnemius muscle (68% increase) and tibialis anterior muscle (64% increase). There were nonsignificant trends for increased glucose transport in the slow-twitch soleus muscle and in the mixed-fiber red gastrocnemius muscle. Glucose transport was unchanged in visceral and subcutaneous fat. Anti-TNF treatment did not alter body weight, muscle mass, or fat mass. Anti-TNF-{alpha} did not alter the distribution of the 17-kDa and 26-kDa forms of TNF-{alpha} in either muscle or fat. However, anti-TNF-{alpha} treatment caused an ~50% reduction in the secretion of TNF-{alpha} bioactivity in vitro by explants of visceral and subcutaneous fat. We conclude that TNF-{alpha} neutralization reversed insulin resistance substantially in fast-twitch muscle and may have done so in other muscles, while having little effect in fat. TNF-{alpha} neutralization was accompanied by reduced TNF-{alpha} bioactivity without tissue depletion of TNF-{alpha} protein.

glucose transport; muscle fiber type; soluble tumor necrosis factor-{alpha}; membrane tumor necrosis factor-{alpha}


MATURING Sprague-Dawley (S-D) rats become obese and develop impaired insulin responses in both liver (8) and skeletal muscle (6, 14). A large body of evidence suggests that TNF-{alpha} plays a role in the development of insulin resistance. Infusion of TNF-{alpha} results in an insulin-resistant state (13, 24). TNF-{alpha} is known to impair insulin signaling in cultured cells by three separate mechanisms (3). First, TNF-{alpha} has been shown to cause phosphorylation of Ser307 on insulin receptor substrate (IRS)-1 in murine adipocytes (11), rat hepatoma cells (25), and C2C12 muscle cells (9). This alteration makes IRS-1 resistant to subsequent insulin-stimulated tyrosine phosphorylation, resulting in reduced docking of phosphatidylinositol 3-kinase and in impaired insulin-stimulated glucose transport (11, 20, 26). Second, TNF-{alpha} phosphorylates and activates the protein tyrosine phosphatase SH-PTPase, which terminates insulin action by removing tyrosine phosphates from IRS-1 (1). Third, TNF-{alpha} phosphorylates and inactivates the protein phosphatase PP-1, preventing insulin activation of glycogen storage (9, 26). Hotamisligil (18) has shown that TNF-{alpha} receptor knockout in ob/ob mice improves glucose tolerance. We (4) and others (8, 21) have reported that the neutralization of TNF-{alpha} reverses insulin resistance in rodents. We found that administration of anti-TNF-{alpha} to S-D rats restores muscle insulin responsiveness, as assessed in isolated strips of soleus muscle (4).

The main purpose of the present study was to determine whether administration of anti-TNF-{alpha} could restore insulin responses in skeletal muscle, visceral fat, and subcutaneous fat. Reported overexpression of TNF-{alpha} in adipose tissue of insulin-resistant rodents (21) has led to the hypothesis that TNF-{alpha} of adipose origin causes systemic insulin resistance. However, we have recently reviewed other evidence suggesting that muscle insulin resistance is caused by locally produced TNF-{alpha} (3). A secondary purpose was to determine the effect of antibody administration on TNF-{alpha} expression in muscle and fat.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and experimental design. S-D male rats that were barrier raised and viral pathogen free were obtained from Zivic-Miller Laboratories (Zellenople, PA). Experimental procedures conformed to the Institute of Laboratory Animal Resources Guide to the Care and Use of Experimental Animals and were approved by the Institutional Animal Care and Use Committee at the Gainesville Veterans Affairs (VA) Medical Center. Rats weighing ~420 g were injected daily, subcutaneously, for 7 days with 8 mg of goat anti-murine TNF IgG (anti-TNF-{alpha}) or goat nonimmune IgG (NI). Polyclonal goat anti-murine TNF-{alpha} IgGs were prepared, as we have previously reported (4, 10), by using the Ribi adjuvant system containing 0.5 mg each of monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton in 0.2% Tween 80 (Ribi ImmunoChemistry Research, Hamilton, MT). The serum IgG fraction was obtained by polyethylene glycol precipitation and column chromatography by use of DEAE Bio-Gel A (Bio-Rad Laboratories, Richmond, CA). The neutralizing capacity of the anti-TNF-{alpha} IgG fraction in the L929 cytotoxicity assay was 6.5 and 9.0 x 105 50% neutralizing U/mg IgG against recombinant TNF-{alpha} and serum TNF-{alpha} from LPS-treated rats, respectively. Nonimmune goat IgG was prepared in the same way and had no detectable TNF-{alpha}-neutralizing activity. Neither IgG preparation demonstrated effective binding to LPS, IL-1, or interferon-{gamma} in an ELISA protocol.

Insulin responsiveness. Glucose transport into individual tissues was measured in vivo during hyperinsulinemic euglycemic clamps (HUC), as previously described by Lang and colleagues (22, 23). Briefly, vascular catheters were implanted, and rats were fasted overnight before HUC were performed. A dose of 100 mU of human insulin was administered by bolus injection followed by infusion at a rate of 100 mU/h. We have previously shown that this rate of insulin infusion produces maximal rates of glucose transport into individual skeletal muscles (22, 23) and elevates serum insulin above postprandial concentrations in S-D rats (28) and above the highest concentrations observed during glucose tolerance testing of S-D rats (7). D-Glucose (30%) was infused at an initial rate of 185 µmol·kg–1·min–1. Blood glucose was measured every 15 min and the rate of glucose infusion adjusted to maintain euglycemia. After HUC had been established for 140 min, an intravenous bolus tracer of 8 µCi 2-deoxy-[14C]glucose was administered. After an additional 40 min, the rat was euthanized and tissues were excised. The integrated specific activity of 2-deoxyglucose is calculated by measuring blood radioactivity periodically over the course of 40 min. The rate of whole body glucose utilization was calculated by dividing the glucose infusion rate by body weight and is expressed in micromoles per kilogram per minute. Glucose transport into individual tissues was estimated by measuring tissue content of phosphorylated 2-deoxy-[14H]glucose (22, 23). Tissue homogenates were precipitated using Somogyi reagents, and phosphorylated 2-deoxy-[14H]glucose was estimated as the difference in radioactivity before and after precipitation. Transport rates were corrected using a lump constant that accounts for competition for transport between glucose and 2-deoxyglucose (22, 23). Serum insulin was assayed by radioimmunoassay (Linco Research, St. Charles, MO).

Secretion of TNF-{alpha} protein and TNF-{alpha} bioactivity in fat explants. As previously described (24), 1 g of visceral or subcutaneous fat was minced and suspended in enriched culture medium (DMEM with glutamine, private, 4,500 mg/l glucose, Sigma nonessential amino acids, and 0.5% BSA). After incubation at 37°C for 60 min, medium was removed and centrifuged for 5 min at 13,000 g. TNF-{alpha} protein was measured using an ELISA that is specific for rat TNF-{alpha} and has a sensitivity of 0.7 pg/ml (BioSource International, Camarillo, CA). TNF-{alpha} bioactivity was measured using the WEHI 164 subclone 13 cytotoxicity assay (12). Briefly, 5 x 104 cells in RPMI 1640 containing 10% FCS, 1 mM L-glutamine, and 0.5 mg/ml actinomycin D were added to serially diluted samples in microtiter plates and incubated for 20 h at 37°C and 5% CO2. Cytotoxicity was assessed by measuring MTT-tetrazolium (3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion to formazan at 550 nm (16). TNF-{alpha} concentrations (pg/ml) were calculated using standard recombinant muTNF-{alpha} (Genentech, South San Francisco, CA). Goat anti-muTNF-{alpha} IgG reduces plasma TNF-{alpha} activity to undetectable levels.

Western analysis of tissue TNF-{alpha}. Tissue extracts were prepared as previously described (5). Tissue samples (0.2 g muscle or 0.5 g fat) were homogenized in buffer containing 0.9% NaCl, 10 mM Na2HPO4, 1 mmol PMSF, 1 mg/l each of pepstatin, aprotinin, and leupeptin, 0.5% Triton X-100, and 0.05% sodium azide, pH = 7.2. Samples were subjected to 1 freeze/thaw cycle, probe sonicated briefly, and centrifuged at 100,000 g for 1 h. Tissue extracts (~5 µg of protein) were subjected to 15% polyacrylamide gel electrophoresis, and proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were blocked with Starting Block (Pierce, Rockford, IL), incubated first with 1° antibody (1:1,400, rabbit anti-rat, BioSource, Camarillo, CA) and then with 2° antibody [1:2,800, goat F(ab)2 anti-rabbit, BioSource]. Bands were detected with enhanced chemiluminescence plus reagents (Amersham, Piscataway, NJ) and quantitated using phosphor-imaging and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Rat 17-kDa TNF-{alpha} (BioSource) was used as a positive control.

Statistical analysis. Differences among means were tested by ANOVA, with P < 0.05 defined as the threshold of significance. Post hoc analysis was performed using Fisher's exact least significant differences test. Values are reported as means ± SE.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Body composition. Active anti-TNF-{alpha} IgG was consistently detected in plasma samples taken after 2 and 4 days of antibody treatment. Administration of anti-TNF-{alpha} had no effect on food intake or body composition (Table 1). Likewise, weight gain, muscle mass, and fat mass, either visceral or subcutaneous, did not differ between nonimmune IgG and anti-TNF-{alpha}-treated animals (Table 1).


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Table 1. Effect of 7 days of treatment with anti-TNF-{alpha} on body composition

 
Insulin responsiveness. During HUC studies, the glucose infusion rate was increased 68% in rats treated with anti-TNF-{alpha} (128.7 ± 15.9 µmol·kg–1·min–1 for anti-TNF-{alpha} vs. 76.7 ± 5.9 µmol·kg–1·min–1 for NI; P < 0.05, n = 6–8; Fig. 1A). The serum insulin concentration during clamps was 19.2 ± 6.9 ng/ml, a concentration that produces maximal glucose transport (22, 23). TNF-{alpha} neutralization also increased glucose transport in two muscles composed predominantly of fast-twitch fibers (see Fig. 1B). In white gastrocnemius muscle, treatment with anti-TNF-{alpha} caused a 68% increase in glucose transport (74.6 ± 21.7 nmol·g–1·min–1 for anti-TNF-{alpha} vs. 46.6 ± 14.5 nmol·g–1·min–1 for NI, P < 0.05). In tibialis anterior muscle, anti-TNF-{alpha} caused a 64% increase in transport (61.4 ± 9.34 nmol·g–1·min–1 for anti-TNF-{alpha} vs. 37.4 ± 5.7 nmol·g–1·min–1 for NI, P < 0.05). There were near-significant trends for increased glucose transport in a slow-twitch muscle (soleus: 164 ± 26.6 nmol·g–1·min–1 for anti-TNF-{alpha} vs. 121 ± 29.5 nmol·g–1·min–1 for NI) and in a mixed fiber muscle (red gastrocnemius: 74.6 ± 21.7 nmol·g–1·min–1 for anti-TNF-{alpha} vs. 46.6 ± 14.5 nmol·g–1·min–1 for NI). Glucose transport was virtually unchanged in visceral fat (13.8 ± 5.2 nmol·g–1·min–1 for anti-TNF-{alpha} vs. 10.4 ± 1.5 nmol·g–1·min–1 for NI) and in subcutaneous fat (13.0 ± 2.8 nmol·g–1·min–1 for anti-TNF-{alpha} vs. 15.3 ± 2.4 nmol·g–1·min–1 for NI).



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Fig. 1. Anti-murine TNF-{alpha} IgG (anti-TNF-{alpha}) treatment increases glucose disposal during hyperinsulinemic euglycemic clamp (HUC; A) and insulin-stimulated glucose transport in muscle (B). Treatment with anti-TNF-{alpha} for 7 days caused a 68% increase in the rate of glucose infusion required to maintain euglycemia. HUC and in vivo administration of 2-deoxy-[14C]glucose were performed as described in MATERIALS AND METHODS. Anti-TNF-{alpha} treatment increased glucose transport in tibialis anterior (TA) and white gastrocnemius (WG) muscles, produced trends toward improved transport in soleus (sol) and red gastrocnemius (RG) muscles, and had no effect on transport in inguinal (IWAT) or retroperitoneal (RPWAT) white adipose tissue. Values are means ± SE; n = 6–8. *P < 0.05 (A); *P < 0.05 vs. NI, (*) P < 0.10 vs. NI (B).

 
TNF-{alpha} expression. Western analysis revealed that both the 17-kDa and 26-kDa forms of TNF-{alpha} are relatively abundant in fat, whereas in muscle, the 17-kDa form predominates (see Fig. 2). Anti-TNF-{alpha} did not alter tissue distribution of 17-kDa and 26-kDa forms of TNF-{alpha} in either muscle or fat. After incubation of fat explants in DMEM for 60 min at 37°C, TNF-{alpha} protein and TNF-{alpha} bioactivity present in conditioned media were measured, as described in MATERIALS AND METHODS (Table 2). Anti-TNF-{alpha} treatment had no effect on secretion of TNF-{alpha} protein by either visceral or subcutaneous fat (Table 2). However, there was significant overall reduction in secretion of TNF-{alpha} bioactivity by fat explants (P < 0.05) and a specific 59% reduction in secretion by visceral fat (P < 0.05) (Fig. 3).



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Fig. 2. Effect of anti-TNF-{alpha} administration on tissue distribution of 17-kDa and 26-kDa forms of TNF-{alpha}. Tissue extracts were prepared and Western analysis was performed as described in MATERIALS AND METHODS. A typical blot is shown (bottom). The lane at far left is ng rat TNF positive control. Each of the remaining lanes is from the tissue listed directly above. Values are means ± SE; n + 6–8. *P > 0.05 vs. nonimmune IgG (NI).

 

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Table 2. Effect of anti-TNF treatment on in vitro TNF secretion in fat explants

 


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Fig. 3. Anti-TNF-{alpha} reduces in vitro secretion of TNF-{alpha} bioactivity in fat explants. Minced samples of IWAT and RPWAT were incubated for 1 h at 37°C, and TNF bioactivity in conditioned medium was measured by WEHI cytotoxicity assay, as described in MATERIALS AND METHODS. Values are means ± SE; n = 6–8. *P < 0.05 vs. NI.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutralization of TNF-{alpha} is known to reverse hepatic insulin responsiveness in Zucker fatty rats. Hotamisligil et al. (21) first demonstrated in 1993 that administration of a TNF-{alpha} receptor-IgG chimeric protein construct could increase insulin-stimulated glucose disposal in Zucker fatty rats. Cheung et al. (8) administered a gene construct coding for a similar TNF-{alpha} inhibitory peptide to Zucker rats and observed a restoration of insulin suppression of hepatic glucose output without a reduction in adiposity. We have previously reported that administration of anti-TNF-{alpha} antibody to maturing S-D rats reverses insulin resistance in isolated strips of soleus muscle. The present study demonstrates that neutralization of TNF-{alpha} restores insulin responsiveness in a variety of skeletal muscles, but not in visceral or subcutaneous fat. Glycolytic muscle fibers have a greater dependence on glucose transport. Therefore, insulin resistance may have greater consequences in glycolytic muscles. We chose to study muscles that are relatively homogeneous for each of the major fiber types: soleus for type I slow oxidative fibers, tibialis anterior and white gastrocnemius for type II fast glycolytic fibers, and red gastrocnemius for type II fast oxidative fibers. We found increased insulin responsiveness in postural (soleus) and activity (gastrocnemius) muscles and probably also in muscles composed primarily of slow fibers (soleus), fast fibers (tibialis anterior), and mixed fibers (gastrocnemius).

We found that TNF neutralization increased insulin responsiveness in muscle, but not in adipose tissue. The reason for this differential effect is not clear. During maturation, S-D rats develop a reduced response to insulin in muscle (7, 14), but it is not known whether the response in fat is also reduced during the same period. One would predict that TNF neutralization would increase the response to insulin only in those tissues where that response was impaired to begin with. It is also possible that anti-TNF did not increase insulin responsiveness in fat because the antibody did not have sufficient access to fat tissue. However, anti-TNF did reduce secretion of TNF bioactivity in fat explants, which probably indicates that it was present in fat tissue.

Although neutralization of TNF-{alpha} reverses insulin resistance, it is not clear that TNF-{alpha} is elevated in insulin resistance. Several laboratories have reported that expression of TNF-{alpha} mRNA is elevated in adipose tissue of insulin-resistant rodents (17, 21) and humans (2, 15, 19). Saghizadeh et al. (27) found that TNF-{alpha} mRNA is elevated sixfold in muscle biopsies obtained from type 2 diabetic subjects. These reports have led to the hypothesis that TNF-{alpha} of adipose origin reaches the circulation, causing systemic insulin resistance. However, two observations argue against this hypothesis. First, we have shown that tissue levels of TNF-{alpha} protein are several orders of magnitude higher than circulating levels (5), indicating that the role of TNF-{alpha} in insulin resistance may be paracrine, rather than endocrine. Second, changes in TNF-{alpha} message do not always reflect changes in TNF-{alpha} protein, and recent evidence suggests that, whereas TNF-{alpha} mRNA is elevated in insulin resistance, TNF-{alpha} protein is not. We recently reported that TNF-{alpha} protein is decreased in visceral fat, subcutaneous fat, and glycolytic muscle of S-D rats during the onset of age-induced insulin resistance (5). In fat explants, we found that in vitro secretion of TNF-{alpha} protein is decreased with age, but secretion of TNF-{alpha} bioactivity is unchanged or increased (5). TNF-{alpha} bioactivity was measured by the WEHI cytotoxicity assay and reflects the actions both of TNF-{alpha} and of its endogenous inhibitor, putatively a soluble form of the TNF-{alpha} receptor. Thus it is possible that TNF-{alpha} action may be increased in insulin-resistant tissues despite the decrease in tissue content of TNF-{alpha} protein. Support for this concept is found in a report by Morin et al. (24), who found that, in fat explants obtained from aged Fischer 344 rats, in vitro secretion of TNF-{alpha} protein was decreased, whereas secretion of TNF-{alpha} bioactivity was markedly increased (24).

Tissue TNF-{alpha} is expressed in two forms, a 17-kDa soluble form (sTNF-{alpha}) and a 26-kDa membrane form (mTNF-{alpha}) (29). TNF-{alpha} is produced in the 26-kDa form and converted to the 17-kDa form by the action of TNF-{alpha}-converting enzyme (29). In visceral and subcutaneous fat from S-D rats, we found that both sTNF-{alpha} and mTNF-{alpha} were relatively abundant. In muscle, sTNF-{alpha} was the predominant form. Treatment with anti-TNF-{alpha} did not alter TNF-{alpha} expression. In Wistar rats, we observed a similar pattern, in which both mTNF-{alpha} and sTNF-{alpha} are abundant in fat and sTNF-{alpha} is predominant in muscle and liver (S. E. Borst, unpublished observation). We also found that high-fat feeding of Wistar rats increased the expression of sTNF-{alpha} in visceral fat and muscle (S. E. Borst, unpublished observation).

Our results shown in Fig. 2 indicate an apparent lack of effect by anti-TNF-{alpha} on tissue expression of TNF-{alpha} protein. In muscle and fat, we detected similar amounts of both mTNF-{alpha} and sTNF-{alpha}, whether rats were treated with neutralizing antibody or with nonimmune IgG. We interpret this finding to mean that, in all probability, the administered antibody effectively neutralized the biological activity of TNF-{alpha} within these tissues but that TNF-{alpha} remained present in the tissue and remained detectable by Western analysis. This interpretation is supported by the findings in Table 2 and Fig. 3. Explants of fat obtained from anti-TNF-{alpha}-treated and NI-treated rats secreted similar amounts of TNF-{alpha} protein in vitro, as assessed by ELISA (Table 2). However, TNF-{alpha} bioactivity, as assessed by WEHI cytotoxicity, was 50% lower in the anti-TNF-{alpha}-treated group. We interpret the lower bioactivity to reflect the presence of antibody within fat tissue. Therefore, we think that neutralizing antibody was present in both muscle and fat but caused enhanced insulin responsiveness in muscle only.

In the present study, neutralization of TNF-{alpha} restored muscle insulin responsiveness, indicating that TNF-{alpha} plays a role in the pathophysiology of insulin resistance. The fact that TNF-{alpha} is virtually undetectable in the blood of insulin-resistant S-D rats (5) is consistent with TNF-{alpha} functioning in a paracrine fashion to decrease insulin responsiveness in skeletal muscle. S-D rats display both muscle (7, 14) and hepatic insulin resistance (8). In this study, whole body glucose disposal was significantly increased, with the increase apparently predominating in skeletal muscle after a 7-day period of reduced TNF-{alpha} activity. It is not clear whether increased muscle glucose transport was sufficient in magnitude to account for all of the improvement in glucose disposal.


    ACKNOWLEDGMENTS
 
This research was supported by a Veterans Administration Merit Award to S. E. Borst.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. Borst, VA Medical Center, GRECC-182, 1601 SW Archer Rd., Gainesville, FL 32608-1197 (E-mail: seborst{at}ufl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
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