Nuclear factor-kappaB suppressive and inhibitor-kappaB stimulatory effects of troglitazone in obese patients with type 2 diabetes: evidence of an antiinflammatory action?

It has been shown recently that troglitazone exerts an anti-inflammatory effect, in vitro, and in experimental animals. To test these properties in humans, we investigated the effect of troglitazone on the proinflammatory transcription factor nuclear factor-kappaB and its inhibitory protein IkappaB in mononuclear cells (MNC) and plasma soluble intracellular adhesion molecule-1, monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, and C-reactive protein. We also examined the effect of troglitazone on reactive oxygen species generation, p47(phox) subunit expression, 9-hydroxyoctadecadienoic acid (9-HODE), 13-HODE, o-tyrosine, and m-tyrosine in obese patients with type 2 diabetes. Seven obese patients with type 2 diabetes were treated with troglitazone (400 mg/day) for 4 weeks. Blood samples were obtained at weekly intervals. Nuclear factor-kappaB binding activity in MNC nuclear extracts was significantly inhibited after troglitazone treatment at week 1 and continued to be inhibited up to week 4. On the other hand, IkappaB protein levels increased significantly after troglitazone treatment at week 1, and this increase persisted throughout the study. Plasma monocyte chemoattractant protein-1 and soluble intracellular adhesion molecule-1 concentrations did not decrease significantly after troglitazone treatment, although there was a trend toward inhibition. Reactive oxygen species generation by polymorphonuclear cells and MNC, p47(phox) subunit protein quantities, plasminogen activator inhibitor-1, and C-reactive protein levels decreased significantly after troglitazone intake. 13-HODE/linoleic acid and 9-HODE/linoleic acid ratios also decreased after troglitazone intake. However, o-tyrosine/phenylalanine and m-tyrosine/phenylalanine ratios did not change significantly. These data show that troglitazone has profound antiinflammatory effects in addition to antioxidant effects in obese type 2 diabetics; these effects may be relevant to the recently described beneficial antiatherosclerotic effects of troglitazone at the vascular level.

Insulin inhibits intranuclear nuclear factor kappaB and stimulates IkappaB in mononuclear cells in obese subjects: evidence for an anti-inflammatory effect?

In view of the fact that insulin resistance is associated with atherogenesis and that troglitazone, an insulin sensitizer, has anti-inflammatory effects, which may be potentially antiatherogenic in the long term, we have now investigated whether insulin has potential anti-inflammatory effects. We infused 2.0 to 2.5 IU/h in 5% dextrose (100 mL/h) iv into 10 obese subjects for 4 h followed by 5% dextrose alone for 2 h. The rate of insulin infusion was varied to maintain glucose concentrations as close to the baseline as possible. Blood samples were obtained before and at 2, 4, and 6 h. Subjects were also infused with 5% dextrose without insulin and with saline on separate occasions. Intranuclear nuclear factor kappaB (NFkappaB) in mononuclear cells fell at 2 and further at 4 h, reverting toward the baseline at 6 h (P < 0.05). IkappaB increased significantly at 2 h, increasing further at 4 h and remaining elevated at 6 h (P < 0.001). Reactive oxygen species (ROS) generation by mononuclear cells fell significantly at 2 h and fell further at 4 h; it partially reverted to baseline at 6 h (P < 0.005). p47(phox) subunit, the key protein of nicotinamide adenine dinucleotide phosphate oxidase also fell at 2 h and 4 h, reverting toward the baseline at 6 h (P < 0.05). In addition, soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) fell significantly following insulin infusion. Glucose or saline infusions without insulin caused no alteration in NFkappaB, IkappaB, ROS generation, p47(phox) subunit, sICAM-1, MCP-1, or PAI-1. We conclude that insulin has a potent acute anti-inflammatory effect including a reduction in intranuclear NFkappaB, an increase in IkappaB, and decreases in ROS generation, p47(phox) subunit, plasma soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1. This acute anti-inflammatory effect, if demonstrated in the long term, may have implications for atherosclerosis and its complications.

Suppression of nuclear factor-kappaB and stimulation of inhibitor kappaB by troglitazone: evidence for an anti-inflammatory effect and a potential antiatherosclerotic effect in the obese.

To elucidate whether troglitazone exerts an antiinflammatory effect in humans, in vivo, we investigated the suppression of nuclear factor kappaB (NFkappaB) in mononuclear cells (MNC) by this drug. We measured intranuclear NFkappaB, total cellular NFkappaB, inhibitor kappaB (IkappaB)alpha, 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)-alpha, 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 NFkappaB. 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 NFkappaB, total cellular NFkappaB, IkappaBalpha, and p47 (phox) subunit and ROS generation were determined. Plasma was used to measure insulin glucose, TNFalpha, 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 NFkappaB, total cellular NFkappaB, and p47 (phox) subunit, with an increase in cellular IkappaBalpha 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-alpha, 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.

Insulin inhibits the expression of intercellular adhesion molecule-1 by human aortic endothelial cells through stimulation of nitric oxide.

Intercellular adhesion molecule-1 (ICAM-1) is expressed by endothelial and other cell types and participates in inflammation and atherosclerosis. It serves as a ligand for leukocyte function-associated antigen-1 on leukocytes and is partially responsible for the adhesion of lymphocytes, granulocytes, and monocytes to cytokine-stimulated endothelial cells and the subsequent transendothelial migration. Its expression on endothelial cells is increased in inflammation and atherosclerosis. As it has been suggested that insulin and hyperinsulinemia may have a role in atherogenesis, we have now investigated whether insulin has an effect on the expression of ICAM-1 on human aortic endothelial cells (HAEC). HAEC were prepared from human aortas by collagenase digestion and were grown in culture. Insulin (100 and 1000 microU/mL) caused a decrease in the expression of ICAM-1 (messenger ribonucleic acid and protein) by these cells in a dose-dependent manner after incubation for 2 days. This decrease was associated with a concomitant increase in endothelial nitric oxide synthase (NOS) expression also induced by insulin. To examine whether the insulin-induced inhibition of ICAM-1 was mediated by nitric oxide (NO) from increased endothelial NOS, HAEC were treated with N(omega)-nitro-L-arginine, a NOS inhibitor. N(omega)-Nitro-L-arginine inhibited the insulin-induced decrease in ICAM-1 expression in HAEC at the messenger ribonucleic acid and protein levels. Thus, the inhibitory effect of insulin on ICAM-1 expression is mediated by NO. We conclude that insulin reduces the expression of the proinflammatory adhesion molecule ICAM-1 through an increase in the expression of NOS and NO generation and that insulin may have a potential antiinflammatory and antiatherosclerotic effect rather than a proatherosclerotic effect.

Increased IkappaB expression and diminished nuclear NF-kappaB in human mononuclear cells following hydrocortisone injection.

We have recently demonstrated that hydrocortisone and other glucocorticoids inhibit reactive oxygen species (ROS) generation by mononuclear (MNC) and polymorphonuclear leucocytes (PMNL). Since NF-kappaB/IkappaB system regulates the transcription of proinflammatory genes, including those responsible for ROS generation, we tested the hypothesis that hydrocortisone may stimulate IkappaB production thus inhibiting NF-kappaB translocation from the cytosol into the nucleus in MNC, in vivo. One hundred milligram of hydrocortisone was injected intravenously into 4 normal subjects. Blood samples were obtained prior to the injection and at 1, 2, 4, 8 and 24 hr after the injection. Nuclear extracts and total cell lysates were prepared from MNC by standard techniques. IkappaB levels in MNC homogenates increased at 1 hr, peaked at 2-4 hr, started to decrease at 8 hr, and returned to baseline levels at 24 hr. NF-kappaB in MNC nuclear extracts decreased at 1 hr, reached a nadir at 4 hr, gradually increased at 8 hr and returned back to baseline levels at 24 hr. The total protein content of NF-kappaB subunit (P65) in MNC lysates also showed a decrease following hydrocortisone injection. This decrease was observed at 2 hr, reached a nadir at 4 hr, and returned to baseline levels at 24 hr. ROS generation inhibition paralleled NF-kappaB levels in the nucleus. It was inhibited at 1 hr, reached a nadir at 2-4 hr, started to increase at 8 hr, and returned to basal levels at 24 hr. Our data demonstrate that hydrocortisone induces IkappaB and suppresses NF-kappaB expression in MNC in parallel. IkappaB further reduces the translocation of NF-kappaB into the nucleus thus preventing the expression of proinflammatory genes.