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Suppression of Nuclear Factor-B and Stimulation of Inhibitor B by Troglitazone: Evidence for an Anti-inflammatory Effect and a Potential Antiatherosclerotic Effect in the Obese

Division of Endocrinology, Diabetes and Metabolism, State University of New York at Buffalo, and Kaleida Health, Buffalo, New York 14209

Address all correspondence and requests for reprints to: Paresh Dandona, M.D., Ph.D., Diabetes-Endocrinology Center of Western New York, 3 Gates Circle, Buffalo, New York. E-mail: pdandona{at}kaleidahealth.org

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
To elucidate whether troglitazone exerts an antiinflammatory effect in humans, in vivo, we investigated the suppression of nuclear factor B (NFB) in mononuclear cells (MNC) by this drug. We measured intranuclear NFB, total cellular NFB, inhibitor B (IB), reactive oxygen species (ROS) generation, and p47^phox subunit (a key component protein of nicotinamide adenine dinucleotide phosphate oxidase) in MNC. Plasma tumor necrosis factor (TNF)-, soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor type 1 (PAI-1), C-reactive protein (CRP), and interleukin (IL)-10 (antiinflammatory cytokine) concentrations were also measured as mediators of inflammatory activity that are regulated by the proinflammatory transcription factor NFB. Seven nondiabetic obese patients were given 400 mg troglitazone daily for 4 weeks. Blood samples were collected before and at weekly intervals thereafter. MNC were separated; and the levels of intranuclear NFB, total cellular NFB, IB, and p47 ^phox subunit and ROS generation were determined. Plasma was used to measure insulin glucose, TNF, sICAM, MCP-1, PAI-1, CRP, and IL-10. Plasma insulin concentrations fell significantly at week 1, from 31.2 ± 29.1 to 14.2 ± 11.4 mU/L (P < 0.01) and remained low throughout 4 weeks. Plasma glucose concentrations did not alter significantly. There was a fall in intranuclear NFB, total cellular NFB, and p47 ^phox subunit, with an increase in cellular IB at week 2, which persisted until week 4. There was a parallel fall in ROS generation by MNC at week 1; this progressed and persisted until week 4 (P < 0.001). Plasma TNF-, sICAM-1, MCP-1, and PAI-1 concentrations fell significantly at week 4. Plasma IL-10 concentration increased significantly, whereas plasma CRP concentrations decreased. We conclude that troglitazone has an antiinflammatory action that may contribute to its putative antiatherosclerotic effects.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
TROGLITAZONE IS A thiazolidinedione (TZD) that is used in the treatment of diabetes mellitus type 2 because it reduces insulin resistance through its binding to peroxisome proliferator-activated receptors- (PPAR; Ref. 1). There are recent data demonstrating that troglitazone may have antiinflammatory properties in experimental animals and in cellular systems, in vitro (2, 3). Because atherosclerosis is considered to be an inflammatory process (4), the potential antiinflammatory effect of troglitazone and other TZDs is of extreme importance. Type 2 diabetes and insulin resistance are strongly atherogenic (5). The antiinflammatory effect of troglitazone and other TZDs was initially believed to be attributable to their ability to bind to PPAR (6), whereas their glucose lowering effect is attributed to their ability to bind to the PPAR receptor (1). However, it has recently been shown that rosiglitazone, a pure PPAR agonist, exerts potent antiinflammatory effects in a mouse model of inflammatory bowel disease with experimental colitis induced with 4% dextran sodium sulfate (7).

In view of the above and the fact that troglitazone has been shown to reverse the intimal-medial thickness in the carotid artery (8), an index of atherosclerosis, we have now investigated whether troglitazone exerts an antiinflammatory effect. Inflammatory responses are now thought to be mediated by the activation of the transcription factor, nuclear factor B (NFB). NFB is normally bound to inhibitor B (IB) in the cytosol; this binding prevents its movement into the nucleus (9, 10). Proinflammatory stimuli induce the phosphorylation of IB, which releases NFB, and the latter translocates to the nucleus, where it induces the transcription of proinflammatory cytokines like tumor necrosis factor (TNF), interleukin (IL)-6, MCP-1, adhesion molecules like ICAM-1 and VCAM-1, and enzymes generating reactive oxygen species (ROS; Refs. 9 and 10). We have previously investigated the antiinflammatory effect of hydrocortisone using circulating mononuclear cells (MNC) as a model and have shown that hydrocortisone suppresses intranuclear and total cellular NFB and induces IB while suppressing ROS generation (11, 12). Using MNC as a target for the potential antiinflammatory action of troglitazone, we have now investigated the effect of this drug on NFB and IB in MNC. In addition, we measured plasma concentrations of TNF, MCP-1, sICAM-1, PAI-1, IL-10, and CRP as additional markers and mediators of inflammation. We also examined the effect of troglitazone on p47^phox subunit, the cardinal protein component of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme which converts molecular O₂ into superoxide (O^·₂^-) radical (13, 14), which is responsible for converting low-density lipoprotein (LDL) to the proinflammatory oxidized LDL (15).

These studies were undertaken before the removal of troglitazone from the formulary. Although this drug is no longer in clinical use, our observations are probably relevant to other TZD.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Seven obese subjects (age range, 32–52 yr; mean, 40.6 ± 8.0 yr), all with body mass index (BMI) levels greater than 37 kg/m² (BMI range, 37.0–60.9; mean, 46.1 ± 8.7 kg/m²), were included in this study (Table 1). All patients had a fasting venous plasma glucose of less than 100 mg/dL. None of the obese subjects were on vitamin E or C or any other antioxidant therapy. The subjects were not advised any special diet, and none of them were actively trying to lose weight during the period of the study. The Institutional Review Board of the Millard Fillmore Hospitals and the State University of New York at Buffalo approved the study. Written informed consent was obtained from all subjects.

MNC preparation (12, 16)

Blood samples were collected with EDTA as an anticoagulant. Three and a half milliliters of the anticoagulated blood sample were carefully layered over 3.5 mL polymorphonuclear leucocytes medium (Robbins Scientific Corp., Sunnyvale, CA). Samples were centrifuged at 450 x g for 30 min at 22 C. The MNC band was harvested, and cells were repeatedly washed with HBSS. This method provides yields greater than 95% pure MNC suspension.

MNC nuclear protein extract preparation and electrophoretic mobility shift assay

DNA-binding protein extracts were prepared from MNC by the method described by Andrews et al. (17). Total protein concentrations were determined using bicinchonic acid protein assay (Pierce Chemical Co., Rockland, IL). NFB gel retardation assay was performed using NFB-binding protein detection kit (Life Technologies, Inc., Long Island, NY). Briefly, the double-stranded oligonucleotide containing a tandem repeat of the consensus sequence for NFB binding site was radiolabeled with -P³²) by T₄ kinase. Then, 5 µg of the nuclear extract were mixed with the incubation buffer, and the mixture was preincubated at 4 C for 15 min. Labeled oligonucleotide (60,000 cpm) was added and the mixture was incubated at room temperature for 20 min. Samples were then applied to wells of 6% nondenaturing polyacrylamide gel. The gel was dried under vacuum and exposed to x-ray film. Densitometry was performed using Bio-Rad Laboratories, Inc. (Hercules, CA) molecular analyst software. These measurements were carried out at 0, 1, 2, 4, and 12 weeks.

NFB, IB, and p47 ^phox subunit Western blotting (11)

MNC cell lysates were prepared by adding 1 mL boiling lysis buffer (1% SDS, 1 mmol/L sodium orthovanadate, 10 mmol/L Tris, pH 7.4) to MNC pellets. Total protein concentrations were determined using bicinchonic acid protein assay (Pierce Chemical Co.). Sixty micrograms of total cell lysate were electrophoresed on 8% SDS polyacrylamide gels (SDS-PAGE) for NFB and p47 ^phox subunit and 12% for IB. The proteins were transferred to polyvinylidene difluoride membrane, blocked for 1 h in 5% nonfat dry milk, and then incubated for 1 h with polyclonal antibodies against NFB p65 (Rel A) or IB (Rockland Immunochemicals, Gilbertsville, PA) or monoclonal antibodies against p47 ^phox (Transduction Laboratories, Inc., San Diego, CA). Finally, the membrane was washed and developed using super signal chemiluminescence reagent (Pierce Chemical Co.). Densitometry was performed using Bio-Rad Laboratories, Inc. molecular analyst software. These measurements were carried out at 0, 1, 2, 4, and 12 weeks.

Plasma insulin and glucose measurement

Insulin was measured from fasting plasma samples at weeks 0, 1, 2, 4, and 12 using an enzyme-linked immunosorbent assay (ELISA) kit from Diagnostic Systems Laboratories, Inc. (Webster, TX). Glucose was measured by Hexokinase method (DADE PARAMAX). Plasma glucose concentrations were measured at all time points.

Plasma TNF-, sICAM-1, MCP-1, IL-10, PAI-1, and CRP measurements

TNF-, sICAM-1, and MCP-1 were assayed with ELISA kits from R&D Systems (Minneapolis, MN). IL-10 ELISA kit was purchased from Biosource International (Camarillo, CA). CRP ELISA kit was purchased from Diagnostic Systems Laboratories, Inc.. Plasma PAI-1 levels were measured using TintElize PAI-1 (Biopool International, Ventura, CA) kit. TNF-, sICAM-1, MCP-1, IL-10, PAI-1, and C-RP concentrations were measured at 0, 1, 4, and 12 weeks.

ROS generation assay (12, 16)

Respiratory burst activity of MNC was measured by detection of superoxide radical via chemiluminescence. Five hundred microliters of MNC (2 x 10⁵ cells) were delivered into a Lumi-aggregator (Chronolog, Malvern, PA) plastic flat-bottom cuvette, to which a spin bar was added. Fifteen microliters of 10-mmol/L luminol was then added, followed by 1 µL of 10-mmol/L formylmethionylleucinylphenylalanine. Chemiluminescence was recorded for 15 min (a protracted record after 15 min did not alter the relative amounts of chemiluminescence produced by various cell samples). Our method, developed independently, is similar to that published by Tosi and Hamedani (18). In this assay system, the release of superoxide radical, as measured by chemiluminescence, has been shown to be linearly correlated with that measured by the ferricytochrome C method. We further established that, in our assay system, there is a dose-dependent inhibition of chemiluminescence by superoxide dismutase and catalase, as well as diphenyleneiodionium (DPI, data not shown), a specific inhibitor of NADPH oxidase, the enzyme responsible for the production of superoxide radicals. The specific inhibitory effect of DPI on NADPH oxidase has been established by Hancock and Jones (19). Our assay system is exquisitely sensitive to DPI-induced inhibition at nanomolar concentrations.

Statistical analysis

The data on the densitometry of Western blots and electrophoretic mobility shift assay were normalized to a baseline of 100%. Data for ROS generation by MNC were also normalized to a baseline of 100%; sequential effects were analyzed by Kruskal-Wallis one-way ANOVA on ranks. Similar normalization of data, to a baseline of 100%, was also carried out for the indices measured in plasma TNF-, sICAM-1, MCP-1, IL-10, PAI-1, and CRP.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Fasting plasma glucose, insulin, and serum lipid concentrations

There was no significant change in fasting plasma glucose concentrations during the period of the study (week 0, 5.28 ± 0.83 mmol/L; week 1, 5.17 ± 0.49 mmol/L; week 2, 5.20 ± 0.59 mmol/L; week 3, 4.83 ± 0.42 mmol/L; week 4, 5.13 ± 0.62; and week 12, 5.1 ± 0.66 mmol/L. Plasma insulin concentrations fell significantly from 31.2 ± 26.9 µU/L at baseline to 14.2 ± 10.5 mU/L at week 1, 6.9 ± 2.8 mU/L at week 2, 7.3 ± 4.9 mU/L at week 4, and 10.5 ± 8.9 mU/L at week 12 (P < 0.001).

Serum lipid concentrations were: triglycerides, 1.35 ± 0.84 mmol/L; cholesterol, 4.91 ± 0.81 mmol/L; high-density lipoprotein, 1.2 ± 0.29 mmol/L; and LDL, 3.12 ± 0.73 mmol/L. Two patients had elevated triglyceride concentrations (>1.71 mmol/L). The mean concentrations of these indices remained unchanged after troglitazone. Triglyceride concentrations fell in five patients, whereas they increased in two. The increase occurred in patients whose triglyceride concentrations were normal.

Intranuclear NFB

Nuclear protein extracts from MNC showed a decrease in nuclear NFB quantities. This decrease, which started as early as the first week, persisted until week 4 and returned to basal level after the cessation of troglitazone. The densitometric quantitation of the shifted bands showed a fall to about 95.6 ± 9.3%, 80.9 ± 5.9%, and 55.1 ± 7.8% of the baseline at weeks 1, 2, and 4, respectively (P < 0.01), and 126.2 ± 26.0% after 8 weeks of troglitazone withdrawal (Fig. 1).

View larger version (35K): [in this window] [in a new window]   Figure 1. A, A representative gel shift assay showing the decreased NFB binding activity to the double-stranded oligonucleotide containing NFB binding site. Lane 1, 0 week; lane 2, 1 week; lane 3, 2 weeks; lane 4, 4 weeks after troglitozone intake. Lane 5, 8 weeks after troglitazone withdrawal. The sequence specificity of the protein-DNA interaction was determined using a specific unlabeled competitor oligonucleotide for NFkB binding site. Lane 6 shows the inhibition of NFkB binding of sample in lane 1 by the competitor. Lane 7 represents HeLa protein nuclear extract as a positive control, and lane 8 represents the radiolabeled oligonuceotide without nuclear extract. This gel is a representative of n = 7. B, Densitometry results of intranuclear NFB (*, P < 0.05).

Total NFB (p65) and IB protein

Total NFB (p65) fell significantly at week 1 (54 ± 26% of basal level) and progressed further at week 4, when it fell to 33 ± 15% of the basal level (P < 0.001 (Fig. 2). On the other hand, IB was induced in three subjects at week 1 and in the rest at week 2, when it increased by about 48 ± 47%, with no further change at week 4 (Fig. 3). The increase in IB was significant at week 2 and persisted until week 4 (P < 0.05). Both NFB and IB protein levels returned to baseline levels at week 12.

View larger version (25K): [in this window] [in a new window]   Figure 2. A, A representative Western blot showing the decease in NFB subunit quantity in MNC homogenates after troglitazone intake. This blot is representative of n = 7. B, Densitometry results of total NFB in MNC homogenates (*, P < 0.05).

View larger version (24K): [in this window] [in a new window]   Figure 3. A, A representative Western blot showing the increase in IB quantity in MNC homogenate after troglitazone intake. This blot is representative of n = 6. B, Densitometry results (*, P < 0.05).

The NADPH oxidase, p47 ^phox subunit, protein quantity in MNC homogenates fell significantly at week 1 and progressed until week 4 in a time-dependent fashion. Densitometry was performed on these blots and showed a fall to about 59 ± 22%, 50 ± 12%, and 35 ± 16% of the basal level at weeks 1, 2 , and 4 respectively (P < 0.001 (Fig. 4). p47 ^phox subunit protein levels returned to basal levels at 12 weeks. There was a significant correlation between ROS generation and p47^phox subunit expression.

View larger version (24K): [in this window] [in a new window]   Figure 4. A, A representative Western blot showing the decrease in p47phox subunit protein quantity in MNC homogenate after troglitazone intake. This blot is representative of n = 4. B, Densitometry of p47phox protein quantity after troglitazone administration (*, P < 0.05).

There was a fall in ROS generation to 60 ± 16% of the basal (100%) by week 1, which progressed and persisted until week 4, where it fell to 35 ± 18% of the basal. This fall was statistically significant at all time points from weeks 1–4 (P < 0.05; Fig. 5).

View larger version (20K): [in this window] [in a new window]   Figure 5. ROS generation by MNC after 400 mg/day troglitazone, decreased significantly at week 1 and onward until week 4 (*, P < 0.05). ROS generation returned thereafter, after troglitazone withdrawal.

Plasma TNF-, sICAM-1, MCP-1, PAI-1, CRP, and IL-10 concentrations

Plasma TNF- and sICAM-1 concentrations were inhibited significantly (P < 0.05) when expressed as a percent change over basal level (100%). TNF- and sICAM-1 concentrations increased after troglitazone withdrawal, toward the baseline (Table 2). MCP-1 concentrations decreased significantly (P < 0.05) at 4 weeks, with a return to baseline at week 12 (Table 2). PAI-1 and CRP concentrations also fell significantly after troglitazone intake, returning toward the baseline at week 12 (Table 2). Plasma IL-10 concentrations increased slightly, but significantly, when expressed as a percent of the basal level (Table 2).

View this table: [in this window] [in a new window]   Table 2. Plasma TNF-, sICAM-1, MCP-1, IL-10, PAI-1, and CRP after troglitazone intake

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

NFB is a transcription factor that regulates the expression of proinflammatory cytokines and the proteins/enzymes involved in ROS generation (9, 10). Thus, it modulates the molecular and cellular mechanisms involved in inflammation. The entry of NFB from the cytosol to the nucleus is regulated by IB, whose induction and binding to NFB prevents the translocation of NFB into the nucleus (9, 20). This reduces the expression of proinflammatory cytokines and ROS generation and thus inhibits inflammation. Indeed, glucocorticoids, known to be antiinflammatory, induce an increase in IB; this is now believed to be a major mechanism underlying the antiinflammatory effects of glucocorticoids (10, 11, 20, 21). We have recently demonstrated the induction of IB, a fall in intranuclear NFB, and also a fall in total cellular NFB content after a single injection of a modest dose of hydrocortisone in normal human subjects (11). There are previous data demonstrating that aspirin, which also has an antiinflammatory effect, also inhibits NFB (22) through the induction of IB. Thus, the role of NFB and its inhibition are central to the occurrence of inflammation and the mode of action of antiinflammatory drugs.

The reduction in ROS generation and NFB in MNC, after troglitazone, may allow a greater bioavailability of NO, which combines with O^·₂^- under conditions of increased ROS generation. In conditions associated with increased O^·₂^- generation, NO bioavailability may be diminished, thus reducing the ability of the blood vessels to dilate. An increase in the vasodilatory potential, in patients with atherosclerosis, by troglitazone (23) may be through a reduction of O^·₂^- generation and by the restoration of the bioavailability of NO. This may account for the beneficial effects of this drug in vasospastic angina and our recent observation that troglitazone improves postischemic endothelium-mediated vasodilation of the brachial artery (24).

Consistent with the dramatic effects of troglitazone on cellular mediators of inflammation are our observations on the plasma markers of inflammation. CRP, sICAM-1, MCP-1, and PAI-1 fell significantly during the short treatment period. These markers not only prognosticate for coronary heart disease but are also involved in the pathogenesis of atherosclerosis and inflammation. CRP binds to FC-RIIa on monocyte/macrophages and may trigger inflammation (25). ICAM-1 is an adhesion molecule, which promotes the attachment of leukocytes to the endothelium (26, 27), whereas MCP-1 is a chemokine attracting monocytes to the site of inflammation (28). Troglitazone caused a small, but significant, increase in IL-10. IL-10 is antiinflammatory, is induced by glucocorticoids (29), and may have a specific antiatherosclerotic effect (30, 31). The magnitude of inhibition of intranuclear NFB and total cellular NFB by troglitazone and the inhibition of ROS generation by MNC suggest that troglitazone may have a potent antiinflammatory effect. This is relevant to the process of atherosclerosis because it is a process of chronic inflammation characterized by an increase in proinflammatory cytokines and adhesion molecules and the invasion of the arterial intima by the inflammatory monocyte-macrophage (4). Increased expression of NFB is known to occur in atherosclerotic lesions (4). The inhibition of NFB in the circulating MNC is thus relevant to the process of atherogenesis. Macrophages in human atherosclerotic lesions, interestingly, have been shown to express PPAR (32). Indeed, troglitazone has been shown, in preliminary studies, to reverse atherosclerosis (8).

There is recent evidence showing that aspirin and other nonsteroidal antiinflammatory drugs restore insulin sensitivity through the inhibition of IB kinase (IKK) in cells overexpressing IKKß. Insulin resistance was defined as diminished tyrosine phosphorylation of IRS-1 and IRS-2 (33). It has also been shown that, in three Zucker rats, the administration of 120 mg/kg aspirin, daily, resulted in a fall in glucose concentrations (34). These observations also raise the possibility that the mechanisms underlying inflammation may contribute to insulin resistance.

The mechanism underlying the inhibition of NFB is not clear and will require further investigation. It is noteworthy that vitamin E has previously been shown to reduce intranuclear NFB in MNC, in vitro (35). It is possible that the -tocopherol moiety of troglitazone contributes to the NFB suppression shown by us. However, the total amount of -tocopherol in the dose of troglitazone given is relatively small. Indeed, the magnitude of the effect of troglitazone on ROS generation by MNC was similar to that observed by us with 800 IU vitamin E (36). The quantity of -tocopherol in troglitazone (400 mg) is less than a quarter of that contained in the above-mentioned dose of vitamin E. Clearly, therefore, there is an effect of troglitazone that is independent of -tocopherol.

Obesity is associated with an increase in tissue expression and plasma concentrations of TNF- (37, 38, 39, 40) and with an increase in the indices of oxidative damage (41); this cytokine has been implicated in the pathogenesis of insulin resistance. Furthermore, the activation of NFB is known to be associated with an increase in TNF expression and secretion (9). It is probable that the TZD moiety of troglitazone is responsible for this effect, because the circulating monocyte has been shown to have PPAR (2, 3). We may need to further resolve the question of whether this effect is mediated through PPAR alone, PPAR alone, or a combination of both. Whereas troglitazone and Pioglitazone have both PPAR and PPAR agonist properties (1), rosiglitazone seems to act exclusively through PPAR (42). There is evidence from animal and in vitro studies that both PPAR and PPAR agonists may have suppressive effects on macrophage function and NFB action (3). Indeed, a very recent report shows that, in a model of experimental colitis induced by dextran sodium sulfate administration in mice, troglitazone and rosiglitazone inhibited inflammation (7). However, the report did not attempt to demonstrate any effect of these drugs on NFB in the mouse model, in vivo. It is of interest that Maggi et al. have recently shown that troglitazone has an inhibitory effect on cytokine (lipopolysaccharides and -interferon)-induced IL-1 secretion, iNOS expression, and ROS generation by monocyte–macrophage cell lines, in vitro. Troglitazone-induced effects were similar to those observed with 15-d-^{12, 14}-PGJ₂, the putative natural, endogenous ligand of PPAR (43).

As mentioned above, troglitazone is no longer in clinical use. However, our findings are probably relevant to other TZDs, rosiglitazone and Pioglitazone, currently in clinical use and possibly other non-TZD agonists of PPAR.

In conclusion, troglitazone administration to the obese leads to a rapid diminution in intranuclear and total cellular NFB, ROS generation by MNC, and p47^phox subunit, in association with an increase in IB. In addition, troglitazone causes a fall in plasma concentrations of TNF, sICAM-1, MCP-1, CRP, and PAI-1. These effects, described for the first time, may be cardinal in the inhibition of inflammation, atherogenesis, and oxidative injury.

Received July 26, 2000.

Revised November 6, 2000.

Accepted November 14, 2000.

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