Husam Ghanim

Circulating Mononuclear Cells in the Obese Are in a Proinflammatory State

Background—In view of the increase in plasma concentrations of proinflammatory mediators tumor necrosis factor-&agr; (TNF-&agr;), 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/m2) and normal-weight control (n=16; BMI=23.8±1.9 kg/m2) subjects. Nuclear factor &kgr;B (NF-&kgr;B) binding to DNA in nuclear extracts was elevated (P<0.05) and the inhibitor of NF&kgr;B-&bgr; (I&kgr;B-&bgr;) was significantly lower (P<0.001) in the obese group. Reverse transcription–polymerase chain reaction revealed elevated levels of migration inhibitor factor (MIF), IL-6, TNF-&agr;, and matrix metalloproteinase-9 (MMP-9) mRNA expression in the obese subjects (P<0.05). Plasma concentrations of MIF, IL-6, TNF-&agr;, MMP-9, and CRP were also significantly higher. Plasma glucose, insulin, and free fatty acids (FFAs) were measured, and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. Plasma FFA concentration related significantly to BMI, IL-6, and TNF-&agr; mRNA expression and plasma CRP levels but not to HOMA-IR. On the other hand, the inflammatory mediators were significantly related to BMI and HOMA-IR. Conclusions—These data show (1) for the first time that MNC in obesity are in a proinflammatory state with an increase in intranuclear NF-&kgr;B binding, a decrease in I&kgr;B-&bgr;, and an increase in the transcription of proinflammatory genes regulated by NF-&kgr;B; (2) that plasma FFAs are a modulator of inflammation; and (3) that insulin resistance is a function of inflammatory mediators.

Increase in Plasma Endotoxin Concentrations and the Expression of Toll-Like Receptors and Suppressor of Cytokine Signaling-3 in Mononuclear Cells After a High-Fat, High-Carbohydrate Meal: Implications for insulin resistance

OBJECTIVE To compare the effect of a high-fat, high-carbohydrate meal (HFHC) with that of a high-fiber and fruit meal on the concentrations of endotoxin (lipopolysaccharide [LPS]), LPS-binding protein (LBP), the expression of toll-like receptors (TLRs), and the suppressor of cytokine signaling-3 (SOCS-3) in mononuclear cells. RESEARCH DESIGN AND METHODS Healthy lean subjects were given 910 calories of either an HFHC meal (n = 10) or an American Heart Association (AHA)-recommended meal rich in fiber and fruit (n = 10) after an overnight fast. Blood was collected before and at 1, 2, and 3 h after the meal. Cellular indexes of oxidative and inflammatory stress; the expression of SOCS-3, TLR2, and TLR4 in mononuclear cells; and plasma concentrations of LPS and LBP were measured. RESULTS HFHC meal intake induced an increase in plasma LPS concentration and the expression of SOCS-3, TLR2, and TLR4 protein, reactive oxygen species generation, and nuclear factor-κB binding activity (P < 0.05 for all). These increases were totally absent after the AHA meal rich in fiber and fruit. CONCLUSIONS The novel changes described after the HFHC meal elucidate further the mechanisms underlying postprandial inflammation and also provide the first evidence explaining the pathogenesis of insulin and leptin resistance mediated by SOCS-3 after such meals. In contrast, an AHA meal does not induce these effects.

Anti-Inflammatory and Profibrinolytic Effect of Insulin in Acute ST-Segment–Elevation Myocardial Infarction

Background—The clinical benefits of insulin previously observed in acute ST-segment–elevation myocardial infarction (STEMI) may be partially explained by an anti-inflammatory effect. We assessed this potential effect of insulin in STEMI patients treated with fibrinolytics. Methods and Results—Thirty-two patients receiving reteplase were randomly assigned infusions of either insulin at 2.5 U/h, dextrose, and potassium (GIK) or normal saline and potassium (C) for 48 hours. Plasma concentrations of high-sensitivity C-reactive protein (CRP), serum amyloid A (SAA), plasminogen activator inhibitor-1 (PAI-1), creatine kinase (CK), and CK-MB were measured at baseline and sequentially for 48 hours. Total p47phox protein in mononuclear cells was measured in a subgroup of 13 subjects. Baseline CRP and SAA were significantly increased (2- to 4-fold) at 24 and 48 hours in each group (P <0.01). However, in the insulin group, there was a significant (P <0.05) attenuation of the absolute rise in concentration of CRP and SAA from baseline. The absolute increase of CRP and SAA was reduced by 40% (CRP) and 50% (SAA) at 24 hours and at 48 hours compared with the control group. The absolute increase in PAI-1 from baseline and the percentage increase in p47phox over 48 hours were significantly (P <0.05) lower in the insulin-treated group. CK-MB peaked earlier and tended to be lower in insulin-treated subjects, especially in patients with inferior MI. Conclusions—Insulin has an anti-inflammatory and profibrinolytic effect in patients with acute MI. These effects may contribute to the clinical benefits of insulin in STEMI.

Orange juice neutralizes the proinflammatory effect of a high-fat, high-carbohydrate meal and prevents endotoxin increase and Toll-like receptor expression

BACKGROUND The intake of glucose or a high-fat, high-carbohydrate (HFHC) meal, but not orange juice, induces an increase in inflammation and oxidative stress in circulating mononuclear cells (MNCs) of normal-weight subjects. OBJECTIVE We investigated the effect of orange juice on HFHC meal-induced inflammation and oxidative stress and the expression of plasma endotoxin and Toll-like receptors (TLRs). DESIGN Three groups (10 subjects in each group) of normal, healthy subjects were asked to drink water or 300 kcal glucose or orange juice in combination with a 900-kcal HFHC meal. Blood samples were obtained before and 1, 3, and 5 h after the drinks and meal combinations were consumed. RESULTS Protein expression of the NADPH oxidase subunit p47(phox), phosphorylated and total p38 mitogen-activated protein kinase, and suppressor of cytokine signaling-3; TLR2 and TLR4 messenger RNA (mRNA) and protein expression; mRNA expression of matrix metalloproteinase (MMP)-9 in MNCs; and plasma concentrations of endotoxin and MMP-9 increased significantly after glucose or water were consumed with the meal but not when orange juice was consumed with the meal. The generation of reactive oxygen species by polymorphonuclear cells was significantly lower when orange juice was added to the meal than when water or glucose was added to the meal. CONCLUSIONS The combination of glucose or water and the HFHC meal induced oxidative and inflammatory stress and an increase in TLR expression and plasma endotoxin concentrations. In contrast, orange juice intake with the HFHC meal prevented meal-induced oxidative and inflammatory stress, including the increase in endotoxin and TLR expression. These observations may help explain the mechanisms underlying postprandial oxidative stress and inflammation, pathogenesis of insulin resistance, and atherosclerosis.

Testosterone concentrations in diabetic and nondiabetic obese men.

OBJECTIVE To determine the prevalence of subnormal testosterone concentrations in patients with obesity and with type 2 diabetes in a primary care clinic population. RESEARCH DESIGN AND METHODS Free testosterone concentrations of 1,849 men (1,451 nondiabetic and 398 diabetic) in the Hypogonadism In Males (HIM) study were analyzed. The HIM study was a U.S.-based cross-sectional study designed to define the prevalence of hypogonadism in men aged >45 years. Free testosterone was measured by equilibrium dialysis. RESULTS The prevalence of subnormal free testosterone concentrations in lean, overweight, and obese nondiabetic men was 26% (n = 275), 29% (n = 687), and 40% (n = 489), respectively (P < 0.001 for trend), and 44% (n = 36), 44% (n = 135), and 50% (n = 227), respectively, in diabetic men (P = 0.46 for trend within group and P < 0.05 compared with nondiabetic men). The mean free testosterone concentration of diabetic men was significantly lower than that of nondiabetic men. Free testosterone concentrations were negatively and significantly (P < 0.001) related to age (r = −0.37), BMI (r = −0.18), and sex hormone–binding globulin (r = −0.11) in multiple regression analysis. The average decline of free testosterone concentrations was 7.8 pg/ml per decade in nondiabetic men and 8.4 pg/ml per decade in diabetic men. CONCLUSIONS Forty percent of obese nondiabetic men and 50% of obese diabetic men aged ≥45 years have subnormal free testosterone concentrations. In view of its high prevalence, obesity is probably the condition most frequently associated with subnormal free testosterone concentrations in males. The concomitant presence of diabetes is associated with an additional increase in the prevalence of subnormal free testosterone concentrations.

A Resveratrol and Polyphenol Preparation Suppresses Oxidative and Inflammatory Stress Response to a High-Fat, High-Carbohydrate Meal

BACKGROUND High-fat, high-carbohydrate (HFHC) meals are known to induce oxidative and inflammatory stress, an increase in plasma endotoxin concentrations, and an increase in the expression of suppressor of cytokine signaling-3 (SOCS-3). HYPOTHESIS The intake of a nutritional supplement containing resveratrol and muscadine grape polyphenols reduces HFHC meal-induced oxidative and inflammatory stress and stimulates the activity of the antioxidant transcription factor, NF-E2-related factor-2 (Nrf-2), and its downstream targets. METHODS Ten normal, healthy subjects were given a 930-kcal HFHC meal either with placebo or with the supplement. Indices of oxidative stress, inflammation, Nrf-2 binding activity, the concentrations of endotoxin (lipopolysaccharide) and lipoprotein binding protein (LBP), and the expression of toll-like receptor 4 (TLR-4), CD14, IL-1β, TNFα, SOCS-3, Keap-1, NAD(P)H:quinone oxidoreductase-1 (NQO-1), and GST-P1 were measured. RESULTS The intake of the supplement suppressed the meal-induced elevations of plasma endotoxin and LBP concentrations, the expression of p47(phox), TLR-4, CD14, SOCS-3, IL-1β, and Keap-1, while enhancing Nrf-2 binding activity and the expression of NQO-1 and GST-P1 genes. CONCLUSION A supplement containing resveratrol and muscadine polyphenols suppresses the increase in oxidative stress, lipopolysaccharide and LBP concentrations, and expression of TLR-4, CD14, IL-1β and SOCS-3 in mononuclear cells after an HFHC meal. It also stimulates specific Nrf-2 activity and induces the expression of the related antioxidant genes, NQO-1 and GST-P1. These results demonstrate the acute antioxidant and antiinflammatory effects of resveratrol and polyphenolic compounds in humans in the postprandial state.

Differential Effects of Cream, Glucose and Orange Juice on Inflammation, Endotoxin and the Expression of Toll Like Receptor-4 and Suppressor of Cytokine Signaling-3

OBJECTIVE We have recently shown that a high-fat high-carbohydrate (HFHC) meal induces an increase in plasma concentrations of endotoxin (lipopolysaccharide [LPS]) and the expression of Toll-like receptor-4 (TLR-4) and suppresser of cytokine signaling-3 (SOCS3) in mononuclear cells (MNCs) in addition to oxidative stress and cellular inflammation. Saturated fat and carbohydrates, components of the HFHC meal, known to induce oxidative stress and inflammation, also induce an increase in LPS, TLR-4, and SOCS3. RESEARCH DESIGN AND METHODS Fasting normal subjects were given 300-calorie drinks of either glucose, saturated fat as cream, orange juice, or only water to ingest. Blood samples were obtained at 0, 1, 3, and 5 h for analysis. RESULTS Indexes of inflammation including nuclear factor-κB (NF-κB) binding, and the expression of SOCS3, tumor necrosis factor-α (TNF-α), and interleukin (IL)-1β in MNCs, increased significantly after glucose and cream intake, but TLR-4 expression and plasma LPS concentrations increased only after cream intake. The intake of orange juice or water did not induce any change in any of the indexes measured. CONCLUSIONS Although both glucose and cream induce NF-κB binding and an increase in the expression of SOCS3, TNF-α, and IL-1β in MNCs, only cream caused an increase in LPS concentration and TLR-4 expression. Equicaloric amounts of orange juice or water did not induce a change in any of these indexes. These changes are relevant to the pathogenesis of atherosclerosis and insulin resistance.

Troglitazone Reduces Reactive Oxygen Species Generation by Leukocytes and Lipid Peroxidation and Improves Flow-Mediated Vasodilatation in Obese Subjects

Because troglitazone has been shown to have antioxidant properties, we investigated whether troglitazone administration to obese subjects causes a reduction in (1) reactive oxygen species (ROS) generation by polymorphonuclear leukocytes (PMNLs) and mononuclear cells (MNCs) and (2) lipid peroxidation as reflected in the plasma concentrations of 9-hydroxyoctadecadienoic acid (9-HODE) and 13-hydroxyoctadecadienoic acid (13-HODE). Seven obese subjects were given 400 mg/d troglitazone for 4 weeks. Blood samples were obtained before troglitazone administration and at weekly intervals thereafter. Insulin concentrations fell significantly at week 1 and remained low at weeks 2 and 4 (P <0.001). ROS generation by PMNLs fell to 77.6±25.1% of the basal at week 1 and 47.9±41.1% at week 4 (P <0.001). ROS generation by MNCs fell to 59.8±15.7% of the basal at week 1 and 35.1±17.6% at week 4 (P <0.001). 9-HODE and 13-HODE concentrations fell significantly from 787.4±52.4 and 713.1±44.7 pg/mL to 720.4±66.7 (P <0.004) and 675.2±65.0 pg/mL (P <0.01) after 4 weeks, respectively. Postischemic dilatation of the brachial artery was measured by ultrasonography. The mean percent dilatation after forearm ischemia before and after troglitazone was 5.5±3.01% and 8.75±3.37% (P <0.02), respectively. The percent increase in diameter after nitroglycerin was 17.08±1.18% before troglitazone, whereas it was 18.9±1.91% (P <0.02) after troglitazone. We conclude that troglitazone has a potent and rapid biological inhibitory effect on ROS generation by PMNLs and MNCs and that it inhibits lipid peroxidation significantly. These changes are associated with a significant improvement in postischemic flow-mediated vasodilation in the brachial artery over a relatively short period of 4 weeks.

Carvedilol Inhibits Reactive Oxygen Species Generation by Leukocytes and Oxidative Damage to Amino Acids

BACKGROUND The purpose of this study was to test whether carvedilol has an antioxidant effect in humans in vivo. METHODS AND RESULTS We administered 3.125 mg of carvedilol twice daily to normal subjects for 1 week. ROS generation by polymorphonuclear leukocytes and mononuclear cells fell from 314+/-183.43 and 303+/-116 mV to 185+/-157 and 189+/-63 mV (P<0.025), respectively. m-Tyrosine fell from 4.24+/-0.99 to 4.03+/-0.97 ng/mL (P=0.01), and o-tyrosine fell from 4.59+/-1.10 to 4.24+/-0.99 ng/mL (P=0.004) in the absence of a change in phenylalanine concentrations. CONCLUSIONS We conclude that carvedilol significantly inhibits ROS generation by leukocytes and oxidative conversion of phenylalanine to m- and o-tyrosine.

Sitagliptin exerts an antinflammatory action.

CONTEXT Sitagliptin is an inhibitor of the enzyme dipeptidyl peptidase-IV (DPP-IV), which degrades the incretins, glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide, and thus, sitagliptin increases their bioavailability. The stimulation of insulin and the suppression of glucagon secretion that follow exert a glucose lowering effect and hence its use as an antidiabetic drug. Because DPP-IV is expressed as CD26 on cell membranes and because CD26 mediates proinflammatory signals, we hypothesized that sitagliptin may exert an antiinflammatory effect. PATIENTS AND METHODS Twenty-two patients with type 2 diabetes were randomized to receive either 100 mg daily of sitagliptin or placebo for 12 wk. Fasting blood samples were obtained at baseline and at 2, 4, and 6 hours after a single dose of sitagliptin and at 2, 4, 8, and 12 wk of treatment. RESULTS Glycosylated hemoglobin fell significantly from 7.6 ± 0.4 to 6.9 ± 3% in patients treated with sitagliptin. Fasting glucagon-like peptide-1 concentrations increased significantly, whereas the mRNA expression in mononuclear cell of CD26, the proinflammatory cytokine, TNFα, the receptor for endotoxin, Toll-like receptor (TLR)-4, TLR-2, and proinflammatory kinases, c-Jun N-terminal kinase-1 and inhibitory-κB kinase (IKKβ), and that of the chemokine receptor CCR-2 fell significantly after 12 wk of sitagliptin. TLR-2, IKKβ, CCR-2, and CD26 expression and nuclear factor-κB binding also fell after a single dose of sitagliptin. There was a fall in protein expression of c-Jun N-terminal kinase-1, IKKβ, and TLR-4 and in plasma concentrations of C-reactive protein, IL-6, and free fatty acids after 12 wk of sitagliptin. CONCLUSIONS These effects are consistent with a potent and rapid antiinflammatory effect of sitagliptin and may potentially contribute to the inhibition of atherosclerosis. The suppression of CD26 expression suggests that sitagliptin may inhibit the synthesis of DPP-IV in addition to inhibiting its action.