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Received June 28, 2004; revision received August 26, 2004; accepted October 15, 2004. The original description of the metabolic syndrome by Reaven1 consisted of obesity, insulin resistance, hypertension, impaired glucose tolerance or diabetes, hyperinsulinemia and dyslipidemia characterized by elevated triglyceride, and low HDL concentrations. All of the features described above are risk factors for atherosclerosis, and thus, metabolic syndrome constituted a significant risk for coronary heart disease2–5 (Table). The features of obesity/overweight and insulin resistance also provided a significant risk for developing type 2 diabetes.5,6 The risks for coronary heart disease and diabetes with metabolic syndrome are greater than those for simple obesity alone, and therefore, an understanding of the pathogenesis and through it, a rational approach to its therapy are of prime importance. View this table: Classic Biological Effects of Insulin and Classic Metabolic Syndrome Based on Resistance to the Metabolic Effects of Insulin As our understanding of the action of insulin evolves to comprehensively include the recent discoveries,7 we can better see that insulin resistance is the basis of most if not all of the features of this syndrome. The original conceptualization of this syndrome was on the basis of resistance to the metabolic actions of insulin. Thus, hyperinsulinemia, glucose intolerance, type 2 diabetes, hypertriglyceridemia, and low HDL concentrations could be accounted for by resistance to the actions of insulin on carbohydrate and lipid metabolism. Although the features described above would to some extent explain the atherogenesis, Reaven has maintained that hyperinsulinemia itself contributes to atherogenicity, and thus, insulin is atherogenic, leading to the coronary heart disease and cerebrovascular disease associated with this syndrome. Obesity probably leads to hypertension through (1) increased vascular tone created by a reduced bioavailability of NO because of increased oxidative stress,8 (2) increased asymmetric dimethylarginine (ADMA) concentrations,9 (3) increased sympathetic …

Background: Glucose intake has been shown to cause an increase in intranuclear nuclear factor-B and a decrease in inhibitor B that are consistent with a proinflammatory effect. We investigated the effect of glucose intake on 2 other proinflammatory transcription factors, activator protein 1 (AP-1) and early growth response 1 (Egr-1), and on the genes regulated by them, ie, the genes for matrix metalloproteinases 2 (MMP-2 )a nd 9( MMP-9) and tissue factor (TF), respectively. Objective: The objective of the study was to ascertain whether the intake of 75 g glucose induces an increase in AP-1, Egr-1, and the genes regulated by them. Design: Eight healthy subjects were given 75 g glucose dissolved in 300 mL water to drink. Blood samples were collected before and 1, 2, and 3 h after glucose intake. Four weeks later, the same subjects were given 300 mL water sweetened with saccharine, and blood samples were collected at the same time points. Mononuclear cells (MNCs) were separated, and nuclear fractions were isolated. Results: AP-1 and Egr-1 binding activities were significantly higher 1 and 2 h after glucose intake and then decreased toward the baseline by 3 h. The expression of MMP-2 and TF in MNC homogenates also was significantly higher at 2 and 3 h. Plasma concentrations of MMP-2 were significantly higher at 3 h, whereas those of MMP-9 were significantly higher at 1, 2, and 3 h. In addition, TF was significantly higher at 2 and 3 h. Intake of saccharine-sweetened water had no significant effect on the inflammatory mediators measured in this study. Conclusion: Glucose induces proinflammatory changes, including increases in AP-1, Egr-1, MMPs, and TF, the factors that regulate processes that are potentially relevant to atherosclerotic plaque rupture and thrombosis. Am J Clin Nutr 2004;80:51–7.

Background In view of the stimulatory effect of glucose on reactive oxygen species (ROS) generation, we investigated the possibility that a mixed meal stimulates ROS generation and possibly induces concomitant proinflammatory changes. Objective The objective was to determine whether the intake of a 900-kcal mixed meal induces an increase in ROS generation by leukocytes and an inflammatory response at the cellular level. Design Nine normal-weight subjects were given a 900-kcal mixed meal, and 8 normal-weight subjects were given 300 mL water after an overnight fast. Blood samples were collected at 0, 1, 2, and 3 h. ROS generation by mononuclear cells and polymorphonuclear leukocytes and the expression of p47(phox) subunit were measured. Intranuclear nuclear factor kappaB (NF-kappaB) binding and the expression of inhibitor kappaBalpha (IkappaBalpha), IkappaB kinase alpha (IKKalpha), and IkappaB kinase beta (IKKbeta) were measured. Plasma concentrations of C-reactive protein (CRP) and soluble intercellular adhesion molecule were also measured. Results ROS generation by mononuclear cells and polymorphonuclear leukocytes and p47(phox) expression increased significantly. The expression of IKKalpha and IKKbeta and DNA-binding activity of NF-kappaB increased significantly, whereas IkappaBalpha expression decreased. Plasma CRP concentrations increased. The intake of 300 mL water did not induce a change in any of the above indexes. Conclusions These data show that the intake of a mixed meal results in significant inflammatory changes characterized by a decrease in IkappaBalpha and an increase in NF-kappaB binding, plasma CRP, and the expression of IKKalpha, IKKbeta, and p47(phox) subunit. These proinflammatory changes are probably relevant to the state of chronic hypertension and obesity and to its association with atherosclerosis.

It has previously been shown that oral intake of 300 calories of glucose (75 g), lipid, or protein increases reactive oxygen species (ROS) generation by polymorphonuclear cells (PMNL) and mononuclear cells (MNCs). We investigated the effects of 75 g glucose on proinflammatory transcription factor, nuclear factor-kappaB (NFkappaB), in mononuclear cells. To further investigate whether the effects of macronutrient-induced oxidative stress are due to consumption of calories or are nutrient specific, we investigated the effects of acute oral challenge of equicaloric amounts of alcohol (300 calories) on ROS generation and NF-kappaB activation in MNCs and PMNL and compared them with those of glucose and water (control). Sixteen normal healthy adult volunteers were given either vodka (10 subjects), glucose solution (10 subjects), or 300 mL water (7 subjects). Vodka and glucose drinks were equivalent to 300 calories. We measured ROS generation and intranuclear NF-kappaB activation by PMNL cells and MNCs at 1 hour, 2 hours, and 3 hours following ingestion. ROS generation by both MNC and PMNL increased significantly (P

Endothelial dysfunction is increasingly recognised as a key event in the pathogenesis of atherosclerosis, which occurs in association with insulin resistance early in the course of type 2 diabetes mellitus (T2DM). Thiazolidinediones (TZDs), such as rosiglitazone, are a class of oral antidiabetic agents that act primarily as insulin sensitisers, reducing insulin resistance with associated improvements in glycemic control. Available data indicate that thiozolidinediones also have beneficial effects on numerous markers of endothelial function and profound antiinflammatory activity, indicative of potential antiatherogenic activity. These effects may be of considerable clinical significance if sustained during long-term therapy, given the morbidity and mortality associated with atherosclerosis in T2DM patients.

In view of the pro-oxidant and proinflammatory effects of angiotensin II, we have tested the hypothesis that valsartan, an angiotensin receptor blocker, may exert a suppressive action on reactive oxygen species (ROS) generation, nuclear factor kappa B (NF-kappa B) in mononuclear cells. Four groups of eight normal subjects were given 1) 160 mg daily of valsartan, 2) 80 mg daily of simvastatin, 3) 40 mg quinapril, or 4) no treatment. Fasting blood samples were obtained before treatment and at d 1, 8, and 14 (7 d after the cessation of the drug). After valsartan, ROS generation by polymorphonuclear cells and mononuclear cells fell significantly by more than 40% (P0.01). NF-kappa B binding activity and the expression of total cellular p65, a protein component of NF-kappa B, fell significantly (P0.01). The expression of inhibitor kappa B (I kappa B) increased significantly (P0.05). Plasma C-reactive protein (CRP) concentration fell significantly (P0.01). All indices, except I kappa B, reverted toward baseline, 7 d after the cessation of the drug. I kappa B persisted in an elevated state. Neither quinapril nor simvastatin given for 7 d produced a suppression of ROS generation, intranuclear NF-kappa B, p65, or CRP, and these two agents did not alter cellular I kappa B either. The untreated controls also did not demonstrate a change in their ROS generation or NF-kappa B binding activity or plasma CRP concentration. We conclude that valsartan at a modest dose exerts a profound and rapid ROS and inflammation-suppressive effect that may be relevant to its potential beneficial effects in atherosclerosis, diabetes, and congestive cardiac failure. In contrast, quinapril and simvastatin produced no similar effect over the period of 1 wk. Our observations may also have implications to clinical situations in which a rapid antiinflammatory effect is required.

The normal endothelium produces a number of vasodilator substances such as nitric oxide (NO) and prostacyclin (PGI2) that regulate vasomotor tone, reduce platelet aggregation, and inhibit the recruitment and activity of inflammatory cells. The functions of vascular endothelial cells are disturbed in diabetic patients. The major cause for mortality and a great percent of morbidity in patients with diabetes mellitus is atherosclerosis. Insulin has recently been shown to stimulate NO release and the expression of NO synthase by the endothelium. Insulin is thus a vasodilator, has anti-platelet activity, and now has been shown to be anti-inflammatory and thus, potentially anti-atherogenic. Similar anti-inflammatory effects of thiazolidenediones (TZDs), troglitazone, and rosiglitazone suggest that they too may have potential anti-atherogenic effects. These effects of insulin and TZDs are of importance since the two major states of insulin resistance, obesity and type 2 diabetes, are associated with a marked increase in atherosclerosis, coronary heart disease, and stroke. These recent observations have extremely important implications for the understanding of the pathogenesis of atherosclerosis in insulin-resistant states and for a rational approach to their comprehensive treatment, including the prevention of atherosclerosis and its complications. This review challenges the previously proposed hypothesis that hyperinsulinemia represents a common pathophysiological pathway of diabetic complications and advances our hypothesis that insulin, through its effect on the endothelium, leucocytes, and platelets, has anti-inflammatory and thus potentially anti-atherogenic properties. Furthermore, through its anti-inflammatory effects, its use improves clinical outcomes in at least two clinical states characterized by profound inflammation-acute myocardial infarction and sepsis.

While acute myocardial infarction and patients admitted to intensive care units carry a serious risk of mortality and morbidity, the concomitant occurrence of hyperglycemia enhances this risk. This has been established through various studies over the past decade (1–3). More recently, it has been shown that the treatment of these conditions with intravenous infusions of insulin results in marked improvements in clinical outcomes (3–5). While the normalization of blood glucose concentration may be one factor in the improvement of these outcomes, insulin may exert benefits of its own, independently of the plasma glucose concentrations. Both septicemia and acute myocardial infarction are inflammatory states with markedly enhanced catabolism. There may be hyperglycemia and an increase in plasma free fatty acid (FFA) concentrations in both of these conditions. Increased FFA concentrations are known to be associated with a deterioration of clinical outcomes and may have toxic effects of their own on vascular reactivity and on the myocardium (6–9). FFAs may have effects on endothelial nitric oxide (NO) production (6), prostacyclin production (10), and the stability of prostacyclin in plasma (11). FFAs may also induce a state of insulin resistance through the induction of protein kinase C and inflammation through the suppression of inhibitor κB (IκB) in the skeletal muscle (12). Proinflammatory changes are also induced in the mononuclear cells by FFAs as reflected in an increase in nuclear factor-κB (NF-κB), the major proinflammatory transcription factor, an increase in reactive oxygen species (ROS) generation, and an increase in p47 phox (13). Thus, the normalization of FFA concentrations by insulin may produce a benefit. Similarly, it can be argued that insulin administration may facilitate the uptake of glucose by insulin-responsive organs, including the myocardium, thus providing for their metabolic needs. These mechanisms are based on our understanding of …

Type 2 diabetes and obesity are major risk factors for the development of coronary artery disease (CAD) and premature atherosclerosis. Both conditions are associated with insulin resistance, oxidative stress, and inflammation. Inflammatory mediators, including plasma interleukin 6, tumor necrosis factor alpha, and tumor necrosis factor R are elevated in these individuals. The elevations of inflammatory mediators may contribute to the pathogenesis of atherosclerosis, because atherosclerosis is an inflammation of the arterial wall. There is evidence that the thiazolidinedione (TZD) class of drugs may alleviate some of the adverse atherosclerotic effects common in patients with type 2 diabetes. Considerable recent data suggest that the TZDs possess anti-inflammatory properties and exert an effect on the atherogenic process, including effects on endothelial function, monocyte/macrophage function, lipid abnormalities, smooth muscle cell migration, and fibrinolysis, all functions that are abnormal in the presence of insulin resistance. These actions of TZDs are consistent with the recently described anti-inflammatory effects of insulin. The use of TZDs as potent anti-inflammatory agents in patients with type 2 diabetes is an approach that would normalize glucose levels, as well as potentially alleviate the long-term risk of atherosclerosis.

Insulin exerts a vasodilatory effect through the release of nitric oxide (NO) from the endothelium. We have recently demonstrated that insulin also inhibits the expression of intracellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1), 2 major proinflammatory mediators, by human aortic endothelial cells (HAEC) and the proinflammatory mediator, nuclear factor (NF-kappa B), in the nucleus in parallel with an increase in endothelial nitric oxide synthase (e-NOS) expression. The inhibition of ICAM-1 by insulin is NO dependent. Because tumor necrosis factor-alpha (TNF-a ) is proinflammatory and may thus inhibit the action of insulin at the endothelial cell level, we have now investigated whether TNF-a affects (1) insulin receptor content; (2) insulin receptor (IR) autophosphorylation induced by insulin, and (3) e-NOS expression by the endothelial cells. TNF-alpha (1 to 5 ng/mL) caused e-NOS inhibition in a dose-dependent fashion as measured by Western blotting. This inhibition was reduced with insulin addition. TNF-alpha also inhibited tyrosine autophosphorylation of the IR in HAEC induced by insulin and reduced IR beta-subunit protein expression in HAEC. These effects of insulin and TNF-alpha were independent of cell proliferation, as cell counts did not change with insulin or TNF-alpha. Our data demonstrate that TNF-alpha may exert its effect by inhibiting IR autophosphorylation in HAEC and also by reducing IR protein (IRP) expression. Although the inhibition of IR autophosphorylation by TNF-alpha is known to occur at the adipocyte level, the data on the inhibitory effect of TNF-alpha on insulin-induced e-NOS expression and IRP contents are novel.

BACKGROUND It was recently shown that glucose challenge leads to increased generation of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNs) and mononuclear cells (MNCs). OBJECTIVE To further elucidate the relation between nutrition and ROS generation, we investigated the effect of lipid and protein challenges on ROS generation by leukocytes. DESIGN After having fasted overnight, one group of healthy subjects consumed a carbohydrate- and protein-free cream preparation (1257 kJ) and another group of healthy subjects consumed an equienergetic pure preparation of casein. Sequential blood samples were obtained after the intake of cream and casein. ROS were measured by chemiluminescence after stimulation by N-formyl-methionyl-leucinyl-phenylalanine. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) and alpha-tocopherol was measured by HPLC. RESULTS ROS generation by MNCs and PMNs increased significantly 1, 2, and 3 h after cream intake and 1 h after protein intake. Cholesterol concentrations did not change significantly, whereas triacylglycerol concentrations increased significantly 2 h after cream intake. Total TBARS concentrations increased 1 h after cream intake and remained elevated 3 h after intake, but the increase was not significant when corrected for changes in triacylglycerol. After casein intake, total cholesterol, triacylglycerol, and TBARS concentrations did not change significantly. alpha-Tocopherol concentrations did not change significantly after either cream or casein intake. CONCLUSIONS Both fat and protein intakes stimulate ROS generation. The increase in ROS generation lasted 3 h after cream intake and 1 h after protein intake. Cream intake also caused a significant and prolonged increase in lipid peroxidation. These data are important because increased ROS generation and lipid peroxidation are key events in atherogenesis.

Aim: The purpose of this study was to assess the response of diabetics to scaling and root planing treatment and subgingival oral irrigation as adjunctive therapy. Method: A total of 52 type 1 and 2 diabetics (mean age 51.3±14) with adult periodontitis were randomized to two groups. Treatment included ultrasonic scaling and scaling and root planing in both groups (control and test) plus subgingival water irrigation 2× daily for the test group. Assessments were made prior to and at 6 and 12 weeks after treatment. Parameters measured were modified gingival index (MGI), probing pocket depth (PPD), plaque index (PI), clinical attachment level (CAL), and bleeding on probing (BOP). Systemic measurement of Reactive Oxygen Species (ROS) generation, cytokines (TNF-α, IL-1β, IL-10, and PGE2), and glycated hemoglobin (HbA1C). Results: After treatment, analysis of data showed that both groups had clinical and systemic improvement. The test group had a statistically significant reduction for MGI, PI, and BOP compared to controls (p

A selected set of substituted pyridone-annelated isoindigos 3a–f has been synthesized via interaction of 5- and 6-substituted oxindoles 2a–f with 6-ethyl-1,2,9-trioxopyrrolo[3,2-f]quinoline-8-carboxylic acid (1) in acetic acid at reflux. Among these isoindigos, the 5'-chloro and 5'-bromo derivatives 3b and 3d show strong and selective antiproliferative activities against a panel of human hematological and solid tumor cell-lines, but not against noncancerous cells, suggesting their potential use as anticancer agents. In all the tested cell lines, compound 3b was a 25%–50% more potent inhibitor of cell growth than 3d, suggesting the critical role of the substitution at 5'-position of the benzo-ring E. The IC50 values after 48 hours incubation with the 5'-chloro compound 3b were 6.60 µM in K562, 8.21 µM in THP-1, 8.97 µM in HepG2, 11.94 µM in MCF-7 and 14.59 µM in Caco-2 cancer cells, while the IC50 values in noncancerous HEK-293 and L-929 were 30.65 µM and 40.40 µM, respectively. In addition, compound 3b induced higher levels apoptosis in K562 cells than 3d, as determined by annexin V/7-AAD flowcytometry analysis. Therefore, further characterization of the antitproliferative mechanisms of compounds 3b and 3d may provide a novel chemotherapeutic agents.

Background The essential oil (EO) of Artemisia vulgaris L. has been traditionally used worldwide for treating a large number of diseases. Although major components in A. vulgaris EO have been shown to inhibit growth of different cancer cells, as pure compounds or part of other plants extracted oil, no information is known about its anti-proliferative activities. Therefore, the current investigation has evaluated the toxicity of the plant extracted oil from buds (AVO-b) and leaves (AVO-l) and characterized their growth inhibitory effects on cancer cells. Methods AVO-b and AVO-l from A. vulgaris L. were extracted by hydrodistillation, and their effect on the viability of human HL-60 promyelocytic leukemia and various other cancer cell lines was tested using MTT assay. Flow cytometric analysis of apoptosis, DNA fragmentation assay, caspases enzymatic activities and Western blotting were used to determine the apoptotic pathway triggered by their action on HL-60 cells. Results Low concentrations of AVO-b and AVO-l inhibited the growth of HL-60 cells in a dose- and time-dependent manner. Employing flow cytometric, DNA fragmentation and caspase activation analyses, demonstrated that the cytotoxic effect of the oils is mediated by a caspase-dependent apoptosis. Kinetic studies in the presence and absence specific caspase inhibitors showed that activation of caspase-8 was dependent and subsequent to the activation of caspases-9 and -3. In addition, the essential oil caused a disruption of the mitochondrial transmembrane potential (ΔΨm), increased the release of cytochrome c to the cytosol, and altered the expression of certain members of Bcl-2 family (Bcl-2, Bax and Bid), Apaf-1 and XIAP. Interestingly, low doses of AVO-b and AVO-1 also induced apoptosis in various cancer cell lines, but not in noncancerous cells. Conclusions The results demonstrate that the EO-induced apoptosis in HL-60 cells is mediated by caspase-dependent pathways, involving caspases-3, -9, and -8, which are initiated by Bcl-2/Bax/Bid-dependent loss of ΔΨm leading to release of cytochrome c to the cytoplasm to activate the caspase cascade. The finding that AVO-b and AVO-l are more efficient to induce apoptosis in different cancer cell lines than noncancerous cells, suggests that A. vulgaris might be a promising source for new anticancer agents.

We have recently demonstrated that troglitazone exerts an anti-inflammatory effect in the insulin resistant obese in vivo in parallel with its insulin-sensitizing effect. Because these effects are thought to be mediated through peroxisome proliferator-activated receptors α and γ (PPARα and PPARγ), we have now examined the possibility that troglitazone may modulate the expression of PPARα and PPARγ. Seven obese hyperinsulinemic subjects were administered 400 mg troglitazone daily for 4 weeks. Fasting blood samples were obtained before and during troglitazone therapy at 1, 2, and 4 weeks. Fasting insulin concentrations fell at week 1 and persisted at lower levels till 4 weeks. PPARγ expression fell significantly at week 1 and fell further at weeks 2 and 4. In contrast, PPARα expression increased significantly at week 2 and further at week 4. 9- and 13-hydroxyoctadecanoic acid, products of linoleic acid peroxidation and agonists of PPARγ, decreased during troglitazone therapy. We conclude that troglitazone, ...

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 κB (NFκB) in mononuclear cells fell at 2 and further at 4 h, reverting toward the baseline at 6 h (P < 0.05). IκB 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). p47phox 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 NFκB, IκB, ROS generation, p47phox subunit, sICAM-1, MCP-1, or PAI-1. We conclude that insulin has a potent acute anti-inflammatory effect including a reduction in intranuclear NFκB, an increase in IκB, and decreases in ROS generation, p47phox 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.

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-κB and its inhibitory protein IκB 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, p47phox 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-κB 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 ...

Since glucose intake acutely increases reactive oxygen species (ROS) generation by polymorphonuclear leucocytes (PMN) and mononuclear cells (MNC), we have now investigated whether a fast over a period of 48h reduces ROS generation by these cells. Eight normal subjects were fasted for 48h. Blood samples were obtained at 0, 24h and 48h. ROS generation by PMN fell significantly at 24h (66.1 ± 19.5% of basal) and further at 48h (45.9 ± 23.0 % of basal; p < 0.001). ROS generation by MNC fell to 62.4 ± 16.5% at 24h and by 48.4 ± 16.5% (p < 0.001) by 48h. The level of p47phox subunit, an index of NADPH oxidase, the enzyme converting molecular oxygen to superoxide (O˙2−) radical, also fell in parallel. Plasma o-tyrosine/phenylalanine ratio fell significantly from 0.326 ± 0.053 mmol/mol to 0.303 ± 0.055 mmol/mol at 48h and m-tyrosine/phenylalanine ratio fell from 0.363 ± 0.063 mmol/mol to 0.340 ± 0.064 mmol/mol (p < 0.05). Thus, a 48h fast may reduce ROS generation, total oxidative load and oxidative dama...

We have recently demonstrated that reactive oxygen species (ROS) generation by polymorphonuclear leukocytes (PMNL) and mononuclear cells (MNC) is inhibited following the intravenous administration of hydrocortisone. This is associated with a parallel decrease in intranuclear NFkappaB, known to modulate inflammatory responses including ROS generation. We have also shown that the plasma levels of interleukin-10 (IL-10), an anti-inflammatory and immunosuppressive cytokine produced by TH2 cells, are also increased after hydrocortisone administration. In this study, we have investigated the effect of hydrocortisone on p47(phox) subunit, a key component of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, in MNC and the pharmacodynamics of this effect with ROS generation and plasma IL-10 levels. p47(phox) subunit protein levels in MNC showed a progressive decrease after hydrocortisone administration. It reached a nadir at 4 hours and increased thereafter to a baseline level at 24 hours. ROS generation also decreased, reached a nadir between 2 and 4 hours, and returned to a baseline level at 24 hours. IL-10 concentrations increased, peaked at 4 hours, and reverted to the baseline levels at 24 hours. In conclusion, p47(phox) subunit suppression may contribute to the inhibition of ROS generation in MNC after hydrocortisone administration. This suppression occurs in parallel with the suppression of NFkappaB and an increase in IL-10 plasma levels. Therefore, it would appear that the decrease in intranuclear NFkappaB and an increase in IL-10 may cause the inhibitory modulation on p47(phox) subunit and ROS generation by MNC following hydrocortisone and other glucocorticoids.

In view of our recent demonstration that insulin inhibits the expression of intercellular adhesion molecule-1 (ICAM-1) and the fact that ICAM-1 expression is known to be modulated by nuclear factor-kappaB (NFkappaB), we have now investigated whether insulin inhibits intranuclear NFkappaB binding activity. We have also investigated whether insulin inhibits the pro-inflammatory chemokine, monocyte chemoattractant protein-1 (MCP-1), which attracts leucocytes to the inflamed sites and is also regulated by NFkappaB. Insulin was incubated with cultured human aortic endothelial cells (HAEC) at 0, 100 and 1000 microU/mL. Intranuclear NFkappaB binding activity was suppressed by approximately 45% at 100 microU/mL and by 60% at 1000 microU/mL (p0.05). MCP-1 mRNA expression was also suppressed by 47% at 100 microU/mL and by 79% at 1000 microU/mL (p0.05). We conclude that insulin at physiologically relevant concentrations exerts an inhibitory effect on the cardinal pro-inflammatory transcription factor NFkappaB and the pro-inflammatory chemokine MCP-1; these effects suggest an anti-inflammatory and potential anti-atherogenic effects of insulin.

Increased reactive oxygen species generation by the leukocytes of the obese may be responsible for increased oxidative injury to lipids and proteins and, hence, atherosclerosis. We have investigated whether reactive oxygen species generation by leukocytes and other indexes of oxidative damage in the body fall with short-term dietary restriction and weight loss. Nine nondiabetic obese subjects (body mass index, 32.5-64.4 kg/m(2)), not taking any antioxidants, were put on a 1000-Cal diet. Fasting blood samples were taken at 0, 1, 2, 3, and 4 weeks and at 12 weeks after the cessation of dietary restriction. Blood samples were also obtained at 1 and 2 h after administration of 75 g oral glucose at 0 and 4 weeks. Mononuclear cells (MNC) and polymorphonuclear leukocytes (PMN) were isolated, and reactive oxygen species generation was measured. Plasma concentrations of thiobarbituric acid-reactive species (TBARS), 13-hydroxyoctadecadienoic acid (13-HODE), 9-hydroxyoctadecadienoic acid (9-HODE), carbonylated proteins, o-tyrosine, and m-tyrosine as indexes of oxidative damage to lipids, proteins and amino acids, respectively, were measured. Antioxidant vitamins were measured as indexes of antioxidant reserves. Plasma tumor necrosis factor-alpha concentrations were also measured. Mean weight loss was 2.4 +/- 0.6 kg at week 1, 2.5 +/- 1.7 kg at week 2, 3.9 +/- 0.8 kg at week 3, and 4.5 +/- 2.8 kg at week 4 (P < 0.05). Reactive oxygen species generation by PMN fell from 236.4 +/- 95.8 to 150.9 +/- 69.0, 125.9 +/- 24.3, 96.0 +/- 39.9, and 103.1 +/- 35.7 mV at weeks 1, 2, 3, and 4, respectively (P < 0.001). It increased 3 months after the cessation of dietary restriction to 270.0 +/- 274.3 mV. Reactive oxygen species generation by MNC fell from 187.8 +/- 75.0 to 101.7 +/- 64.5, 86.9 +/- 42.8, 63.8 +/- 14.3, and 75.1 +/- 32.2 mV and increased thereafter to 302.0 +/- 175.5 mV at 1, 2, 3, 4, and 16 weeks, respectively (P < 0.005). Reactive oxygen species generation by PMN and MNC increased in response to glucose; the relative increase was greater at 4 weeks than that at week 0 due to a fall in the basal levels of reactive oxygen species generation. Consistent with the fall in reactive oxygen species generation, there was a reduction in plasma TBARS from 1.68 +/- 0.17 micromol/L at week 0 to 1.47 micromol/L at 4 weeks (P < 0.05). The 13-HODE to linoleic acid ratio fell from a baseline of 100% to 56.4 +/- 36.1% at 4 weeks (P < 0.05), and the 9-HODE to linoleic acid ratio fell from a baseline of 100% to 60.5 +/- 37.7% at 4 weeks (P < 0.05). Carbonylated proteins fell from 1.39 +/- 0.27 microgram/mg protein at week 0 to 1.17 +/- 0.12 microgram/mg protein at week 4 (P < 0.05); o-tyrosine fell from 0.42 +/- 0.03 mmol/mol phenylalanine at week 0 to 0.36 +/- 0.02 mmol/mol phenylalanine at 4 weeks (P < 0.005), and m-tyrosine fell from 0.45 +/- 0.04 mmol/mol phenylalanine at week 0 to 0.40 +/- 0.03 mmol/mol phenylalanine at 4 weeks (P < 0.05). The basal concentrations of TBARS, 9-HODE, 13-HODE, carbonylated proteins, o-tyrosine, and m-tyrosine in the obese were significantly greater than those in normal subjects. On the other hand, tumor necrosis factor-alpha concentrations did not change during this 4-week period, nor was there any change in antioxidant vitamins. This is the first demonstration of 1) an increase in reactive oxygen species-induced damage in lipids, proteins, and amino acids in the obese compared with normal subjects; and 2) a decrease in reactive oxygen species generation by leukocytes and oxidative damage to lipids, proteins, and amino acids after dietary restriction and weight loss in the obese over a short period.

Diabetes mellitus is associated with increased ROS generation, oxidative injury and obesity. To elucidate the relationship between nutrition and ROS generation, we have investigated the effect of glucose challenge on ROS generation by leucocytes, p47phox protein, a key protein in the enzyme NADPH oxidase and alpha-tocopherol levels. Blood samples were drawn from 14 normal subjects prior to, at 1, 2 and 3 h following ingestion of 75 g glucose. ROS generation by polymorphonuclear leucocytes (PMNL) and mononuclear cells (MNC) increased to a peak of 244 +/- 42% and 233 +/- 34% of the basal respectively at 2h. The levels of p47phox in MNC homogenates increased significantly at 2 h and 3 h after glucose intake. alpha-Tocopherol levels decreased significantly at 1 h, 2 h and 3 h. We conclude that glucose intake stimulates ROS generation and p417phox of NADPH oxidase; increases oxidative load and causes a fall in alpha-tocopherol concentration.

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 μU/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 Nω-nitro-l-arginine, a NOS inhibitor. Nω-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.

We have examined the effect of short-term triiodothyronine (T3) administration on reactive oxygen species (ROS) generation by leukocytes in 9 euthyroid subjects. At a dose of 60 microg/d orally for 7 days, T3 induced a significant increase in ROS generation by mononuclear cells (MNCs) from 183 +/- 102 mV at baseline to 313 +/- 111 mV on the seventh day (P.02), and by polymorphonuclear leukocytes (PMNLs) from 195 +/- 94 mV at baseline to 302 +/- 104 mV on the seventh day (P.02). There was also a significant increase in meta-tyrosine (P.001) and ortho-tyrosine (P.001), known indices of oxidative damage to proteins and amino acids. However, there was no increase in plasma thiobarbituric acid-reactive substances (TBARS), an index of oxidative damage to lipids, and in the level of carbonylated proteins, a less sensitive index to assess protein oxidation. There was no decrease in the level of antioxidants such as alpha-tocopherol, vitamin A, beta-carotene, lycopene, and lutein/zeaxanthin. The stimulatory effect on ROS generation may reflect a generalized increase in metabolic activity or may be a specific effect on NADPH oxidase in leukocyte membranes. The absence of a significant change in TBARS, carbonylated proteins, alpha-tocopherol, vitamin A, beta-carotene, lycopene, and lutein/zeaxanthin may reflect the short duration of the increased ROS load.

To examine the hypothesis that heparin may affect leukocyte function and that it may have anti-inflammatory properties, we investigated the effect of heparin on reactive oxygen species (ROS) generation by leucocytes. Heparin was injected intravenously at a dose of 10000 units into eight normal subjects. Blood samples were collected from the antecubital vein sequentially, prior to and following heparin at 0, 0.5, 1, 2, and 4 hours. ROS generation was inhibited significantly by polymorphonuclear cells (PMNL) at 0.5, 1, and 2 hours and returned to baseline level at 4 hours. Similarly, ROS generation was inhibited markedly by mononuclear cells (MNC) at 0.5 hours, with a peak inhibition at 1 hour; it returned to baseline level by 4 hours. The maximum inhibition of ROS generation by PMNL was 57.3±19% of the basal, while that by MNC was 56.4±11% of the basal. Since ROS are proinflammatory and cause tissue damage, it is possible that heparin may have an anti-inflammatory effect in vivo, apart from its antithrombotic effect. Since ROS also bind to nitric oxide (NO) and reduce the bioavailability of NO, heparin may indirectly increase the bioavailability of NO and thus act as a vasodilator. This effect of heparin may be of particular relevance to its use in unstable angina and following thrombolysis in acute myocardial infarction in preventing reperfusion injury.

After the demonstration that hydrocortisone inhibits reactive oxygen species (ROS) generation by leukocytes in vivo in a highly predictable manner, we investigated the effect of dexamethasone at a dose of 4 mg, which is thought to be roughly equivalent to 100 mg hydrocortisone. We also tested the hypothesis that dexamethasone may increase the plasma concentration of interleukin-10 (IL-10), an immunomodulatory cytokine that inhibits T(H)1 cells. Dexamethasone (4 mg given intravenously) markedly inhibited ROS generation by mononuclear cells and polymorphonuclear leukocytes. The onset of the effect on polymorphonuclear leukocytes occurred at 1 hour (76.3% +/- 9.3% of basal value), and the peak effect occurred at 4 hours (22.9% +/- 6.4% of basal value), with a significant inhibition still persistent at 8 hours (51.3% +/- 14.3% of basal value; F = 66.7; P < .001). ROS generation was restored to baseline at 24 hours (97.6% +/- 9.5%). The inhibitory effect of dexamethasone on mononuclear cells was 78.3% +/- 9.5% of baseline at 1 hour, 11.4% +/- 6.6% at 4 hours, 30.3% +/- 14.1% at 8 hours, and 102.3% +/- 18% at 24 hours (F = 66.5; P < .001). The peak inhibitory effect of dexamethasone on mononuclear cells (11.4% +/- 6.6%) was significantly greater (P < .05) than that on polymorphonuclear leukocytes (22.9% +/- 6.4%). Plasma IL-10 concentrations increased consistently from 4.8 +/- 1.8 pg/mL within 1 hour of dexamethasone injection and peaked at 4 hours (8.8 +/- 2.3 pg/mL), declining to baseline at 8 hours (F = 4.26; P < .004). Dexamethasone (and possibly other glucocorticoids) therefore exerts its immunosuppressive and anti-inflammatory effects by inhibiting ROS generation by leukocytes and by increasing the plasma concentrations of IL-10.

In view of the fact that glucocorticoids have an immunosuppressive effect and the fact that interleukin-10 (IL-10) is inhibitory to T helper cell function, we have now investigated the effect of hydrocortisone on plasma IL-10 concentrations. Seven normal subjects were injected with 100 mg hydrocortisone intravenously between 8 and 9:00 a.m. Sequential blood samples were obtained prior to and 1, 2, 4, 8 and 24 h after the injection. Plasma IL-10 concentrations increased significantly and consistently following the injection in all subjects. The peak increase of IL-10 occurred at 4 h and the restoration to baseline by 8 h. The sequential values were (mean+/-SD): 3.0+/-1.3 pg/ml at 2 h, 9+/-4.2 pg/ml at 4 h, 3.7+/-1.8 pg/ml at 8 h and 3.7+/-1.4 pg/ml at 24 h. The magnitude of increase was 436% of the basal at peak effect. This effect of hydrocortisone (and possibly other glucocorticoids) may contribute to the immunosuppressive effect of this drug. IL-10 may also be potentially useful in the assessment of Cushing's Syndrome as a marker of end organ effect of glucocorticoids.

To test the hypothesis that oxidative damage associated with tissue hypoxia plays a role in neovascularization, a lipid hydroperoxide (LHP) was injected into the vitreous of rabbits. Single injections of LHP (50-600 microg) caused a sustained retinal neovascularization visualized clinically by ophthalmoscopy and confirmed by microscopy. Vasodilators, i.e. histamine and nitric oxide, peaked at 6h and 7 days, respectively. The levels of both tumor necrosis factor-alpha and interleukin-1alpha peaked at 12h and dropped to basal levels by 24h. Expression of vascular endothelial growth factor (VEGF) and transforming growth factor-beta peaked at 24h and were sustained throughout the following 3 weeks, and platelet-derived growth factor was also elevated throughout the same period. Upregulation of these five angiogenic cytokines, but not basic fibroblast growth factor, occurred prior to the appearance of neovascularization. Leakage of fluorescein at the tips of new vessels was demonstrated by fluorescein angiography. Linoleic hydroperoxide induced neovascularization, but saturated or unsaturated native C-18 fatty acids had no effect. The cascade of multiple, angiogenic cytokines induced by LHP may interact to promote sustained neovascularization.

Background Non-insulin-dependent diabetes mellitus (NIDDM) and hyperhomocysteinemia are both associated with increased lipid peroxidation (oxidative stress). This may contribute to the accelerated vascular disease associated with these conditions. It is not known whether the coexistence of elevated homocysteine levels will stimulate oxidative stress further than that caused by diabetes alone. Methods Plasma concentrations of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, were measured in patients with NIDDM who had previously had a methionine load test; some of the patients had hyperhomocysteinemia. Results Plasma TBARS concentrations were elevated in diabetics with vascular disease. The additional presence of hyperhomocysteinemia was not associated with a further increase in plasma TBARS concentrations. Conclusions Lipid peroxidation is increased in patients with diabetes mellitus and macrovascular disease and is not further elevated by the coexistence of elevated homocysteine levels. It is possible that diabetes maximally stimulates oxidative stress and any further acceleration of vascular disease in patients who have coexistent hyperhomocysteinemia is mediated through mechanisms other than lipid peroxidation.

Stress-Induced Premature Senescence (SIPS), a senescence-like state achieved as a result of a variety of stresses, is associated with relative inability to undergo apoptosis. Sirtuins are a family of histone deacetylases known primarily for their implication in the aging process; however their role in cancer, an aging associated disease, is still poorly understood. In the present study, we investigated whether alterations in sirtuins expression may be associated with aggressive tumor behavior. We have compared expression profiles of sirtuins between drug sensitive and resistant cancer cells MCF7, SaOS-2, A2780 and HL-60. Expression levels of Sirtuin 1-6 varied among cell lines; however Sirtuin-7 was significantly reduced in all chemoresistant cells tested. Knockdown of Sirt7 expression in human breast MCF7 cell line by RNAi induced senescence-associated β-galactosidase activity, reduced cell proliferation rate, induced drug resistance and increased cell migration, suggesting that this gene may play an active role in regulating cancer cell response to stress. Thus, Sirt7 may represent a compelling target for anti-cancer interventions

As procalcitonin concentrations have been shown to be elevated in patients with septicemia and gram-negative infections in particular, we proceeded to investigate the effect of endotoxin, a product of gram-negative bacteria, on procalcitonin concentrations in normal human volunteers. Endotoxin from Escherichia coli 0113:H10:k, was injected i.v. at a dose of 4 mg/kg BW into these healthy volunteers. Blood samples were obtained before and 1, 2, 4, 6, 8, and 24 h after injection of the endotoxin. Each patient's cardiovascular and overall clinical status was monitored over this period. The patients developed chills and rigors, myalgia, and fever between 1-3 h. Tumor necrosis factor-alpha levels increased sharply at 1 h and peaked at 90 min, reaching the baseline concentration thereafter by 6 h. Interleukin-6 levels increased more gradually, peaking at 3 h and reaching the baseline concentration at 8 h. The procalcitonin concentration, which was undetectable (10 pg/mL) at 0, 1, and 2 h, was detectable at 4 h and peaked at 6 h, maintaining a plateau through 8 and 24 h (4 ng/mL). There was no elevation of calcitonin concentrations, which remained below 10 pg/mL, the lowest sensitivity of the assay. Procalcitonin was measured by a two-antibody immunoradiometric assay specific for this peptide, with no cross-reactivity with calcitonin, katacalcin, or calcitonin gene-related peptide. We conclude that endotoxin induces the release of procalcitonin systemically, that this increase is not associated with an increase in calcitonin, and that the increase in procalcitonin associated with septicemia in patients may be mediated through the effect of endotoxin described here. Whether procalcitonin participates in the mechanisms underlying inflammation remains to be investigated.

The sirtuins are NAD+-dependent histone/protein deacetylases that are similar to Saccharomyces cerevisiae silent information regulator 2 (Sir2). Sirtuins regulate various normal and abnormal cellular and metabolic processes, including tumorigenesis, neurodegeneration, and processes associated with type 2 diabetes and obesity. Several age-related diseases, such as Alzheimer's disease, and longevity have also been linked to the functions of sirtuins. A thorough understanding of the mechanisms of action of the sirtuins may therefore yield novel therapeutic strategies targeting these processes; several small-molecule and naturally occurring inhibitors and activators of these enzymes have been identified. This review describes the mechanisms regulating sirtuin activity, as well as how these modes of regulation may be exploited to manipulate activity in the context of various pathological states (ie, metabolic diseases, cancer and neurodegenerative diseases). The possible metabolic outcomes of the pharmacological manipulation of sirtuins are also discussed.

The major cause of heart attack and stroke is atherosclerosis. Because the atherosclerotic lesion contains lipid, and because elevated cholesterol levels are epidemiologically associated with heart attack, the major thrust of dietary research in relation to heart disease has been toward fats in the diet. Decades of research in this area have led to the clear conclusions that (1) an increase in low-density lipoprotein cholesterol (LDL-C) predicts the risk of coronary heart disease (CHD) and stroke; (2) the risk of CHD is inversely related to the circulating level of high-density lipoprotein cholesterol (HDL-C); and that (3) the level of very-low-density lipoprotein cholesterol (VLDL-C) does not directly predict heart-attack risk but that an increase in this cholesterol fraction appears to enhance the risk posed by LDL-C. Additionally, the subclass of lipid known as small dense LDL-C has, over the past decade, been shown to predict pathology related to atherosclerosis. Prospective, randomized studies of the beneficial effect of lowering blood cholesterol levels through the use of a variety of drugs have conclusively shown that reducing the level of LDL-C markedly lowers the risk of CHD morbidity and mortality. A recent study directed at increasing the circulating level of HDL has also shown an improved outcome in CHD,1 and another study aimed at reducing triglycerides and raising the level of HDL in diabetic subjects has also shown improved outcome.2 Clearly, therefore, decreasing LDL-C and increasing HDL-C with or without a concomitant decrease in triglycerides improves cardiovascular outcomes.

Fasting hyperinsulinemia is associated with an increased risk of atherosclerotic complications of heart attack and stroke. This has resulted in the concept that insulin may promote atherosclerosis in spite of the absence of any evidence that insulin is atherogenic either in the human or in experimental models. Recent evidence shows that insulin exerts vasodilatory, anti-platelet and anti-inflammatory effects at the cellular level in vitro and in the human in vivo. Since atherosclerosis is a chronic inflammatory process of the arterial wall, insulin may be potentially anti-atherosclerotic in the long term. More recent data on experimental atherosclerosis in the mouse shows that (1) insulin administration reduces the number and the size of atherosclerotic lesions in apo E null mice and (2) in IRS-2 null mice, the interruption in insulin signal transduction results in enhanced atherogenicity. Finally, the use of a low dose of insulin infusion in patients with acute myocardial infarction has been shown to markedly improve clinical outcomes, both in diabetic and nondiabetic patients. Our own most recent data show that a low dose infusion of insulin in patients with acute myocardial infarction induces a reduction in inflammation (C-reactive protein and serum amyloid A) and oxidative stress, and promotes fibrinolysis. We conclude that insulin is anti-inflammatory and potentially antiatherogenic and may be of use in the treatment of cardiovascular inflammatory conditions.

The metabolic syndrome, as described by Reaven, is a combination of obesity, insulin resistance, hypertension, hypertriglyceridemia, low HDL cholesterol, and hyperinsulinemia. The syndrome was recognized as a very high pro-atherogenic risk causing coronary heart disease. More recently, other features like an elevated plasma PAI -1 and CRP have been added to the syndrome. In view of the recent data demonstrating that insulin exerts an anti-inflammatory effect while macronutrients exert a pro-inflammatory effect, we are in a better position to explain not only why an insulin resistant state like the metabolic syndrome is pro-inflammatory but also to explain how it develops. This review discusses the relevance of these recent observations and puts into perspective the pathogenesis of various features of the metabolic syndrome and also predicts some features which may get incorporated into it in the future.

Insulin has a free fatty acid (FFA)-suppressive effect, vascular endothelial growth factor (VEGF)- and matrix metalloproteinase (MMP)-lowering effect, and a potential myocardial-protective effect. Whether low-dose insulin exerts these effects in patients with acute myocardial infarction (MI) was investigated. Thirty-two patients administered thrombolytics and heparin were randomly assigned to a modified glucose-insulin-potassium (GIK) regimen (insulin 2.5 U/hour, dextrose and potassium titrated to prevent hyperglycemia) or normal saline solution and potassium (controls) for 48 hours. Plasma FFA, serum VEGF, pro-MMP-1, and myoglobin were measured at baseline and sequentially for 48 hours. FFA concentrations were increased at baseline; increased further in the first 4 hours in controls (por=0.008), but not in the GIK group, and were higher at 4 hours in controls compared with the GIK group (p=0.0009). VEGF decreased to 7% of baseline at 2 hours and remained suppressed in both groups (p=0.0008). Pro-MMP-1 decreased in both groups (p0.005), but this decrease was seen earlier at 2 hours in the GIK group compared with 4 hours in controls. There was no significant increase in myoglobin at 2 hours in the GIK group, whereas there was a significant increase in controls. Mean blood glucose was 131 mg/dl in controls and 124 mg/dl in the GIK group (p=NS). In conclusion, this modified GIK regimen attenuated the increase in FFA, but did not suppress it to less than the threshold for myocardial FFA uptake. It suppressed pro-MMP-1 rapidly and decreased myoglobin, whereas heparin suppressed VEGF in patients with acute MI. This provided additional rationale for conducting a trial to assess the clinical benefits of this modified GIK regimen in patients with acute MI.

Background: We have previously demonstrated an early and potent antiinflammatory effect of troglitazone and rosiglitazone. Hypothesis: Because inflammatory mediators interfere with insulin signal transduction, we have now hypothesized that rosiglitazone exerts an initial antiinflammatory effect independently of its metabolic actions including the suppression of the plasma concentration of free fatty acids (FFAs), insulin, and glucose after which insulin sensitization occurs. Patient and Methods: Fourteen patients with type 2 diabetes were included in the study. Eight patients were given 2 mg daily of rosiglitazone for 6 wk, whereas the other six patients were given a placebo for the same period. Results: After a 2-mg dose of rosiglitazone, plasma FFAs, insulin, and glucose concentrations and homeostasis model assessment of insulin resistance did not change. Plasma C-reactive protein, serum amyloid A, and matrix metalloproteinase concentrations fell significantly at wk 1 and continued to be significantly lower than the baseline levels by 25, 29, and 24%, respectively, at wk 6. Leukocyte count was significantly lower at wk 6 after rosiglitazone, whereas there was no change in the control group. Plasma adiponectin concentrations increased significantly at wk 2 and continued to increase during the treatment period with rosiglitazone. Resistin concentrations fell significantly by 10% at wk 6 only. There were no changes in any of these indices in the placebo group. Conclusions: A low dose of rosiglitazone exerts an early and potent antiinflammatory effect with an increase in adiponectin and a fall in resistin concentrations without causing any metabolic changes (fall in plasma glucose, FFAs, and insulin concentrations) over a 6-wk period. The increase in adiponectin and the decrease in resistin after rosiglitazone are thus related primarily to its antiinflammatory effects rather than its metabolic actions. These observations have implications in relation to the mode of action of this drug as an insulin-sensitizing agent and also its use as a potential antiinflammatory and antiatherogenic drug in the future.

Purpose: We identified the temporal expression of activator protein-1 (AP-1) and matrix metalloproteinases (MMPs) after linoleic acid hydroperoxide (LHP) induction of retinal neovascularization. Methods: After injection of LHP into the vitreous of rabbits, samples were collected for AP-1 binding activity and mRNA for MMP-9 and MMPs activity. AP-1 binding activity was measured by eletrophoretic mobility shift assay. MMP-9 activity was measured by zymography and mRNA by quantitative RT-PCR. Results: AP-1 binding activity was increased at 1–3 hr. MMP-9 mRNA levels were increased at 3 hr in the neural retina and by 12 hr in the retinal pigment epithelium (RPE) layer. MMP-9 proteolytic activity was elevated within the neural retina and within the vitreous and in the RPE-interphotoreceptor matrix (IPM) at 12 hr and peaked at 24 hr or 4 days. Conclusions: LHP increases the transcription factor AP-1 which in turn may regulate retinal MMP-9 synthesis during neovascularization.

Because hyperglycemia is a major detrimental factor in the prognosis of acute cardiovascular conditions such as acute myocardial infarction (AMI) and stroke, and because an acute glucose challenge in healthy subjects has been shown to induce oxidative stress in mononuclear cells (MNCs), we have now investigated whether glucose induces inflammatory stress at the cellular and molecular level. Glucose ingestion (75 g in 300 mL water) in healthy human subjects resulted in an increase in intranuclear nuclear factor kappaB (NF-kappaB) binding, the reduction of inhibitor kappaB alpha (IkappaBalpha) protein, and an increase in the activity of inhibitor kappaB kinase (IKK) and the expression of IKKalpha and IKKbeta, the enzymes that phosphorylate IkappaBalpha, in MNCs. Glucose intake caused an increase in NF-kappaB binding to NF-kappaB2, NF-kappaB2a, and NF-kappaB3 sequences in the promoter site of tumor necrosis factor alpha (TNF-alpha) gene along with an increase in the expression of TNF-alpha messenger RNA in MNCs. Membranous p47(phox) subunit, an index of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression and activation, also increased after glucose intake. We conclude that glucose intake induces an immediate increase in intranuclear NF-kappaB binding, a fall in IkappaBalpha, an increase in IKKalpha, IKKbeta, IKK activity, and messenger RNA expression of TNF-alpha in MNCs in healthy subjects. These data are consistent with profound acute pro-inflammatory changes in MNCs after glucose intake.

The vascular endothelium controls vasomotor tone and microvascular flow and regulates trafficking of nutrients and biologically active molecules. When endothelial activation is excessive, compromised microcirculation and subsequent cellular hypoxia contribute to the risk of organ failure. We hypothesized that strict blood glucose control with insulin during critical illness protects the endothelium, mediating prevention of organ failure and death. In this preplanned subanalysis of a large, randomized controlled study, intensive insulin therapy lowered circulating levels of ICAM-1 and tended to reduce E-selectin levels in patients with prolonged critical illness, which reflects reduced endothelial activation. This effect was not brought about by altered levels of endothelial stimuli, such as cytokines or VEGF, or by upregulation of eNOS. In contrast, prevention of hyperglycemia by intensive insulin therapy suppressed iNOS gene expression in postmortem liver and skeletal muscle, possibly in part via reduced NF-kappaB activation, and lowered the elevated circulating NO levels in both survivors and nonsurvivors. These effects on the endothelium statistically explained a significant part of the improved patient outcome with intensive insulin therapy. In conclusion, maintaining normoglycemia with intensive insulin therapy during critical illness protects the endothelium, likely in part via inhibition of excessive iNOS-induced NO release, and thereby contributes to prevention of organ failure and death.

Objective: Elevation of free fatty acids (FFAs) by the infusion of triglyceride-heparin emulsion infusion (TG-Hep) causes insulin resistance (IR). We examined the effect of insulin sensitizer (rosiglitazone) on FFA-induced IR. Design: Nine obese subjects underwent a 6-h infusion of TG-Hep before and after 6 wk of rosiglitazone (8 mg/d) treatment. Hyperinsulinemic euglycemic clamps were performed during 0–2 and 4–6 h of TG-Hep. Results: After rosiglitazone for 6 wk, fasting FFA concentration fell, but not significantly (489 ± 63 at 0 wk; 397 ± 58 μmol/liter at 6 wk; P = 0.16), whereas C-reactive protein (4.26 ± 0.95 at 0 wk; 2.03 ± 0.45 μg/ml at 6 wk) and serum amyloid A (17.36 ± 4.63 at 0 wk; 8.77 ± 1.63 μg/ml at 6 wk) decreased significantly. At 0 wk, TG-Hep infusion caused a decrease in glucose infusion rate (GIR) from 4.49 ± 0.95 mg/kg·min to 3.02 ± 0.59 mg/kg·min (P = 0.018). Rosiglitazone treatment resulted in an increase in baseline GIR to 6.29 ± 0.81 mg/kg·min (P = 0.03 vs. 0 wk), which decreased to 4.52 ± 0.53 mg/kg·min (P = 0.001) after 6 h of TG-Hep infusion. The decrease in GIR induced by TG-Hep infusion was similar before and after rosiglitazone therapy [1.47 ± 0.50 vs. 1.77 0.3 mg/kg·min (28.9 ± 6.5 vs. 26.4 ± 3.7%); P = 0.51]. The rise in FFAs and triglycerides after TG-Hep infusion was significantly lower at 6 wk (P = 0.006 for FFAs; P = 0.024 for triglycerides). Conclusions: We conclude that rosiglitazone: 1) causes a significant increase in GIR; 2) induces a decrease in inflammatory mediators, C-reactive protein, and serum amyloid A; 3) decreases the rise in FFAs and triglycerides after TG-Hep infusion; and 4) does not prevent FFA-induced IR.

The normal endothelium performs the function of vasodilation, platelet inhibition, and the suppression of inflammation through the secretion of nitric oxide (NO) and prostacyclin (PGI2). Endothelial damage through diabetes, hypercholesterolemia, and other atherosclerotic risk factors results in the reduction of NO and PGI2 synthesis and secretion; and these changes turn the blood vessel into a proconstrictor, proaggregatory, and proinflammatory state. Because atherosclerosis is a chronic inflammation of the arterial wall, abnormal endothelial function would predispose the artery to atherogenesis and to a prothrombotic state. Recent studies have demonstrated that insulin exerts an anti-inflammatory effect in addition to inhibiting platelet aggregation and the expression of other prothrombotic factors. These facts challenge the conventional view that insulin is the mediator of atherogenesis in insulin-resistant states of obesity and type 2 diabetes, which are characterized by hyperinsulinemia. Furthermore, insulin has been shown to be anti-inflammatory, antioxidant, and profibrinolytic and cardioprotective in patients with acute myocardial infarction. Insulin is required as an anti-inflammatory hormone with potential antiatherosclerotic effects.

The objective of the study was to determine whether plasma migration inhibitor factor (MIF) concentration and mononuclear cell (MNC) mRNA are elevated in obesity and whether treatment with metformin reduces plasma MIF concentration. Forty obese subjects [body mass index (BMI), 37.5 +/- 4.9 kg/m(2)] and 40 nonobese healthy subjects (BMI, 22.6 +/- 3.4 kg/m(2)) had their plasma MIF, glucose, insulin, free fatty acids (FFAs) and C-reactive protein (CRP) concentrations measured. Sixteen obese patients and 16 nonobese healthy subjects had RNA prepared from MNCs. Eight obese subjects with normal glucose concentration were treated with metformin 1 g (Glucophage XR; 1000 mg twice daily) twice daily for 6 wk. Eight obese subjects were used as controls. Plasma concentration of glucose, insulin, FFAs, and MIF was measured by appropriate assays. mRNA for MIF was measured by real-time PCR. Forty obese subjects had a fasting concentration of MIF of 2.8 +/- 2.0 ng/ml, whereas 40 nonobese subjects had a fasting MIF concentration of 1.2 +/- 0.6 ng/ml (P < 0.001). Plasma MIF concentrations were significantly related to BMI (r = 0.52; P < 0.001). mRNA for MIF was correlated to plasma FFAs (r = 0.40; P < 0.05) and plasma CRP (r = 0.42; P < 0.05) concentrations. Eight obese subjects had their fasting blood samples taken before and after taking a slow-release preparation of metformin at 1, 2, 4, and 6 wk. The mean plasma concentration fell from 2.3 +/- 1.4 to 1.6 +/- 1.2 ng/ml at 6 wk (P < 0.05). Obese subjects not on treatment with metformin showed no change. During the period of treatment with metformin, the body weight did not change and the plasma concentration of glucose, insulin, and FFAs did not alter. We conclude that: 1) plasma MIF concentrations and MIF mRNA expression in the MNCs are elevated in the obese, consistent with a proinflammatory state in obesity; 2) these increases in MIF are related to BMI, FFA concentrations, and CRP; 3) metformin suppresses plasma MIF concentrations in the obese, suggestive of an antiinflammatory effect of this drug; and 4) this action of metformin may contribute to a potential antiatherogenic effect, which may have implications for the reduced cardiovascular mortality observed with metformin therapy in type 2 diabetes mellitus.

Background— In view of the increase in plasma concentrations of proinflammatory mediators tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C-reactive protein (CRP) in obesity, we investigated whether peripheral blood mononuclear cells (MNC) from obese subjects are in a proinflammatory state. Methods and Results— MNC were prepared from fasting blood samples of obese (n=16; body mass index [BMI]=37.7±5.0 kg/m 2 ) and normal-weight control (n=16; BMI=23.8±1.9 kg/m 2 ) subjects. Nuclear factor κB (NF-κB) binding to DNA in nuclear extracts was elevated ( P P P Conclusions— These data show (1) for the first time that MNC in obesity are in a proinflammatory state with an increase in intranuclear NF-κB binding, a decrease in IκB-β, and an increase in the transcription of proinflammatory genes regulated by NF-κB; (2) that plasma FFAs are a modulator of inflammation; and (3) that insulin resistance is a function of inflammatory mediators.