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Background The FcγRs genotypes have been reported to play a key role in the defence against malaria parasites through both cellular and humoral immunity. This study aimed to investigate the possible correlation between FcγR (IIa, IIIa, and IIIb) genes polymorphism and the clinical outcome for anti‐malarial antibody response of Plasmodium falciparum infection among Saudi children. Methods A total of 600 volunteers were enrolled in this study, including 200 malaria-free control (MFC) subjects, 218 patients with uncomplicated malaria (UM) and 182 patients with severe malaria (SM). The FcγR genotypes were analysed using PCR amplification methods, and measurements of immunoglobulin were determined using enzyme-linked immunosorbent assay (ELISA) technique. Results The data revealed that the FcγRIIa-R/R131 showed a statistically significant association with SM patients when compared to UM patients. Furthermore, higher levels of IgG1, IgG2, and IgG4 were associated with the FcγRIIa-H/H131 genotype among UM patients. Although the FcγRIIa-F/V176 genotype was not associated with UM, it showed a significant association with severe malaria. Interestingly, the FcγRIIIa-V/V176 genotype offered protection against SM. Moreover, SM patients carrying the FcγRIIIa-F/F genotype showed higher levels of AMA-1-specific IgG2 and IgG4 antibodies. The FcγRIIIb-NA1/NA1 and FcγRIIIb-NA2/NA2 genotypes did not show significant differences between the UM and the MFC groups. However, the genotype FcγRIIIb-NA2/NA2 was statistically significantly associated with SM patients. Conclusions The data presented in this study suggest that the influence of the FcγRIIa-R/R131, FcγRIIIa-F/F176 and FcγRIIIb-NA2/NA2 genotypes are statistically significantly associated with SM patients. However, the FcγRIIa-H/H13 and FcγRIIIa-V/V176 genotypes have demonstrated a protective effect against SM when compared to UM patients. The impact of the FcyR (IIa, IIIa and IIIb) gene variants and anti-malaria IgG subclasses play an important role in susceptibility to malaria infection and disease outcome in Saudi children.

Awad Alshahrani,1–3 Mohammed AlDubayee,1–3 Mahmoud Zahra,3 Firas M Alsebayel,3 Nawaf Alammari,3 Faisal Alsudairy,3 Muath Almajed,3 Ahmad Aljada4 1Department of Medicine, Ministry of National Guard Health Affairs (MNG-HA), Riyadh, Kingdom of Saudi Arabia; 2King Abdullah International Medical Research Centre (KAIMRC), Riyadh, Kingdom of Saudi Arabia; 3College of Medicine, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Kingdom of Saudi Arabia; 4Department of Biochemistry and Molecular Medicine, College of Medicine, Alfaisal University, Riyadh, Kingdom of Saudi ArabiaCorrespondence: Ahmad AljadaDepartment of Biochemistry and Molecular Medicine, College of Medicine, Alfaisal University, P.O. Box 50927, Riyadh 11533, Kingdom of Saudi ArabiaTel +966 112158 834Email aaljada@alfaisal.eduBackground: Interactions between environmental factors, such as diet and lifestyle, and metabolic pathways are pivotal in understanding aging mechanisms. hNAA40, Nicotinamide phosphoribosyltransferase (NAMPT), and NAD-dependent protein deacetylase sirtuin-1 (SIRT-1) have been shown to exert important biological processes, including stress response and aging.Methods: hNAA40, NAMPT, and SIRT-1 mRNA expression in peripheral blood mononuclear cells (PBMC) were quantitated in 30 lean adult volunteers of normal weight, 30 obese, 20 drug-naïve obese Type 2 diabetes mellitus (T2DM), and 30 obese T2DM on Metformin. Similarly, hNAA40, NAMPT, and SIRT-1 expression in PBMC were quantitated in 36 normal healthy adults randomly assigned to three different groups (Glucose or Whey proteins or lipids; 300 kcal). Blood samples were obtained at 1, 2, and 3 hrs after the macronutrient intake.Results: There was an increase in hNAA40 and a decrease in NAMPT and SIRT-1 expression in PBMC from T2DM. Metformin treatment reverted hNAA40, NAMPT, and SIRT-1 expression levels to normal levels. Glucose intake resulted in a significant increase in expression of hNAA40 at 1 hr and decreased significantly at 3 hrs post intake. Lipid intake resulted in an increase in expression of hNAA40 at 2 hr post intake and returned to normal levels at 3 hrs. Neither glucose nor lipid intake resulted in a significant change in NAMPT or SIRT-1 expression. Whey proteins resulted in significantly lower expression of NAMPT at 3 hrs and did not alter the expression levels of SIRT-1 significantly.Conclusion: hNAA40, NAMPT, and SIRT-1 pathway could play a role in the determination of the healthy life-span. Metformin modulates this pathway.Keywords: hNAA40, nicotinamide phosphoribosyltransferase, NAMPT, sirtuin-1, T2DM, obesity, senescence

Obesity is associated with an increased risk of insulin resistance (IR) and type 2 diabetes mellitus (T2DM) which is a multi-factorial disease associated with a dysregulated metabolism and can be prevented in pre-diabetic individuals with impaired glucose tolerance. A metabolomic approach emphasizing metabolic pathways is critical to our understanding of this heterogeneous disease. This study aimed to characterize the serum metabolomic fingerprint and multi-metabolite signatures associated with IR and T2DM. Here, we have used untargeted high-performance chemical isotope labeling (CIL) liquid chromatography-mass spectrometry (LC-MS) to identify candidate biomarkers of IR and T2DM in sera from 30 adults of normal weight, 26 obese adults, and 16 adults newly diagnosed with T2DM. Among the 3633 peak pairs detected, 62% were either identified or matched. A group of 78 metabolites were up-regulated and 111 metabolites were down-regulated comparing obese to lean group while 459 metabolites were up-regulated and 166 metabolites were down-regulated comparing T2DM to obese groups. Several metabolites were identified as IR potential biomarkers, including amino acids (Asn, Gln, and His), methionine (Met) sulfoxide, 2-methyl-3-hydroxy-5-formylpyridine-4-carboxylate, serotonin,L-2-amino-3-oxobutanoic acid, and 4,6-dihydroxyquinoline. T2DM was associated with dysregulation of 42 metabolites, including amino acids, amino acids metabolites, and dipeptides. In conclusion, these pilot data have identified IR and T2DM metabolomics panels as potential novel biomarkers of IR and identified metabolites associated with T2DM, with possible diagnostic and therapeutic applications. Further studies to confirm these associations in prospective cohorts are warranted.

Background Although there is increasing evidence showing that cell senescence is increased in circulating PBMC in type 2 diabetes mellitus (T2DM), the data are contradictory. This study examined several senescence biomarkers, including LMNA/C transcript variants, p16INK4a, p53, and p21Cip1/WAF, in PBMC of T2DM patients and the effect of Metformin on these senescence markers. Methods Blood samples were obtained from 30 lean, 30 obese, 20 newly diagnosed type 2 diabetes mellitus (T2DM), and 30 T2DM on Metformin. PBMC were isolated and mRNA expression of the senescence biomarkers were quantified by RT-qPCR. The effect of ectopic expression of LMNA and LMNC in human monocytic cells lines (THP-1 and U937) on several inflammatory mediators were also examined. Results LMNA expression was significantly higher in PBMC of obese and T2DM patients. LMNC expression was significantly inhibited in T2DM patients. LMNAΔ10 and Progerin mRNA expression was not detected in PBMC of all groups. Expression of p16INK4a, p21Cip1/WAF and p53 were inhibited significantly in T2DM. Metformin treatment reverted LMNA, LMNC, and p53 expression levels to normal levels. Upregulation of LMNA in monocytic THP-1 and U937 cell lines induced CD68, TNFα, CCL2, IL-6 and NOS2. Conclusions These data support the notion that LMNA may mediate senescence in PBMCs of T2DM by upregulating inflammatory pathways. Metformin may exert its anti-inflammatory property by modulation of senescence mediator LMNA.

Background To evaluate sirtuin-7 (SirT7) mRNA expression status in breast cancer patients with different metastatic stages and survey SirT7 mRNA expression status in eight different types of cancer. Methods The expression of SirT7 in the commercially available TissueScan qPCR Breast Cancer Disease cDNA arrays containing 16 normal, 23 Stage I, 36 IIA, 22 IIB, 8 IIIA, 23 IIIA, 6 IIIB, 13 IIIC, and 5 IV were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) assay. Similar analysis was performed in TissueScan qPCR Cancer Survey cDNA array, which includes breast, colon, kidney, liver, lung, ovarian, prostate, and thyroid specimens. Results The mRNA expression levels of SirT7 were significantly higher in breast cancer samples compared to normal breast specimens ( P < 0.001). Stratification of patients into groups according to metastatic stages indicated statistically significantly higher levels of SirT7 mRNA in CS-I, CS-II, and CS-III when compared to normal breast tissue ( P < 0.05). Notably, SirT7 mRNA levels were higher in CS-I, CS-IIA, CS-IIB, and CS-IIIA ( P < 0.05). Additionally, there were significantly lower SirT7 mRNA levels in thyroid carcinoma when compared to their corresponding normal tissue ( P < 0.05). Conclusions Our results indicate an increase in the mRNA expression level of SirT7 in breast cancer, particularly in CS-I, CS-IIA, CS-IIB, and CS-IIIA. The relationship of altered SirT7 with breast cancer progression and patient survival should be prospectively explored in future studies.

Background: Although peripheral blood mononuclear cells (PBMC) have been demonstrated to be in a pro-inflammatory state in obesity and type 2 Diabetes Mellitus (T2DM), characterization of circulating PBMC phenotypes in the obese and T2DM and the effect of Metformin on these phenotypes in humans is still ill-defined and remains to be determined. Methods: Thirty normal healthy adult volunteers of normal weight, 30 obese subjects, 20 obese newly diagnosed diabetics and 30 obese diabetics on Metformin were recruited for the study. Fasting blood samples were collected and PBMC were isolated from whole blood. Polarization markers (CD86, IL-6, TNFα, iNOS, CD36, CD11c, CD169, CD206, CD163, CD68, CD11b, CD16 and CD14) were measured by RT-qPCR. Gene expression fold changes were calculated using the 2ˆ(–delta delta Ct) method for RT-qPCR. Results: Obesity and T2DM are associated an increased CD68 marker in PBMC. mRNA expression of CD11b, CD11c, CD169 and CD163 were significantly reduced in PBMC from T2DM subjects whereas CD11c was significantly inhibited in PBMC from obese subjects. On the other hand, macrophage M1-like phenotype was observed in T2DM circulation as demonstrated by increased mRNA expression of CD16, IL-6, iNOS, TNFα, and CD36. There were no significant changes in CD14 and CD86 in the obese and T2DM when compared to the lean subjects. Metformin treatment in T2DM reverted CD11c, CD169, IL-6, iNOS, TNFα, and CD36 to levels comparable to lean subjects. CD206 mRNA expression was significantly upregulated in PBMC of T2DM while Metformin treatment inhibited CD206 expression levels. Conclusions: These data support the notion that PBMC in circulation in T2DM express different pattern of phenotypic markers than the patterns typically present in M1 and M2 like cells. These phenotypic markers could be representative of metabolically activated macrophages (MMe)-like cells. Metformin, on the other hand, reduces MMe-like cells in circulation.

Background and Aim: Neonatal infection, including bacterial sepsis, is a major health care issue with an annual global mortality in excess of one million lives. Therefore, this study aimed to evaluate the potential diagnostic value of C-reactive protein (CRP), E-selectin, procalcitonin (PCT), interleukins-6 (IL-6), and tumor necrosis factor-α (TNF-α) both independently and in combination for the diagnosis of neonatal sepsis in its earliest stages. Materials and Methods: A total of 320 subjects were included in this study. A prospective cross-sectional study was conducted among neonates admitted to Neonatal Intensive Care Unit at King Abdulaziz Medical City, Riyadh, KSA during January 2013 to August 2015, the study based on three study groups categorized according to clinical symptoms and blood culture result. Study groups include healthy control neonates (n = 80), clinical sepsis (CS) group (n = 80) with clinical signs of sepsis but their blood culture was negative, and sepsis group with clinical signs of sepsis and their blood culture was positive. Results: The study observed significant difference in plasma levels of CRP, IL-6, TNF-α, E-selectin, and PCT in patients group when compared with control group (P < 0.001). Furthermore, the levels are significantly different between patient groups including CS and neonatal sepsis group. Moreover, result observed significant difference in CRP and IL-6 in early onset sepsis (EOS) when compared with late onset sepsis (LOS) neonates (P < 0.001 and 0.01), respectively, while there were no significant difference in TNF-α, E-selectin, and PCT between EOS and LOS (P = 0.44, 0.27 and 0.24), respectively. Regarding biomarkers accuracy, the result showed that CRP has the best diagnostic accuracy with cutoff value of 3.6 ng/ml (sensitivity 78% and specificity of 70%). The best combination is shown with CRP and IL-6 in which sensitivity increased to 89% and specificity to 79%. Conclusion: It was concluded that infected new-born babies have a higher E-selectin, PCT, IL-6, TNF-α, and CRP compared with the neonates with CS and control. IL-6, TNF-α, and CRP should be measured in combination for mare diagnostic accuracy in neonatal sepsis. Likewise, PCT should be investigated as a part of sepsis screening for all suspected neonates.

Purpose: This study examined the effect of permissive underfeeding compared to target feeding and intensive insulin therapy (IIT) compared to conventional insulin therapy (CIT) on the inflammatory mediators monocyte chemoattractant protein 1 (MCP-1), soluble intercellular adhesion molecule 1 (sICAM-1), and tissue factor (TF) in critically ill patients. Methodology: This was a substudy of a 2 &times, 2 factorial design randomized controlled trial in which intensive care unit (ICU) patients were randomized into permissive underfeeding compared to target feeding groups and into IIT compared to CIT groups (ISRCTN96294863). In this substudy, we included 91 patients with almost equal numbers across randomization groups. Blood samples were collected at baseline and at days 3, 5, and 7 of an ICU stay. Linear mixed models were used to assess the differences in MCP-1, sICAM-1, and TF across randomization groups over time. Results: Baseline characteristics were balanced across randomization groups. Daily caloric intake was significantly higher in the target feeding than in the permissive underfeeding groups (P-value &lt, 0.01), and the daily insulin dose was significantly higher in the IIT than in the CIT groups (P-value &lt, 0.01). MCP-1, sICAM-1, and TF did not show any significant difference between the randomization groups, while there was a time effect for MCP-1. Baseline sequential organ failure assessment (SOFA) score and platelets had a significant effect on sICAM-1 (P-value &lt, 0.01). For TF, there was a significant association with age (P-value &lt, 0.01). Conclusions: Although it has been previously demonstrated that insulin inhibits MCP-1, sICAM-1 in critically ill patients, and TF in non-critically ill patients, our study demonstrated that IIT in critically ill patients did not affect these inflammatory mediators. Similarly, caloric intake had a negligible effect on the inflammatory mediators studied.

Metformin has been shown to exert anti-neoplastic and chemopreventive activities in epidemiological and animal studies. This review article discusses the epidemiological studies and examines the possible mechanisms by which metformin exerts its anti-carcinogenic activities in breast, colon, ovarian, lung, and prostate cancers. We performed a systematic review of the clinical studies examining the anti-neoplastic activities of metformin and the potential mechanisms associated with these activities. Several observational and biological studies revealed a significant association between metformin and reduction in cancer incidence. The mechanisms by which metformin exerts these effects are unknown. This action may be mediated through activation of AMP-activated protein kinase (AMPK), inhibition of the mammalian target of rapamycin (mTOR) pathway, and inhibition of insulin like growth factors (IGFs), and many others. Further laboratory investigation and large, prospective population clinical trials are required to elucidate metformin anti-neoplastic and chemo-preventive actions.

Cardiovascular disease is a major cause of morbidity and mortality among people with type 2 diabetes mellitus. The peroxisome proliferator-activated receptor (PPAR) agonists have a significant role on glucose and fat metabolism. Thiazolidinediones (TZDs) are predominantly PPAR agonists, and their primary benefit appears to be the prevention of diabetic complications by improving glycemic control and lipid profile. Recently, the cardiovascular safety of rosiglitazone was brought to center stage following meta analyses and the interim analysis of the RECORD trial. Current evidence points to rosiglitazone having a greater risk of myocardial ischemic events than placebo, metformin, or sulfonylureas. This review article discusses the mechanism of action of PPAR agonists and correlates it with clinical and laboratory outcomes in the published literature. In addition, this review article attempts to discuss some of the molecular mechanisms regarding the association between TZDs therapy and the nontraditional cardiovascular risks.

BACKGROUND The mature human insulin receptor (INSR) has two isoforms: The A isoform and the B isoform. INSR upregulation has been suggested to play a role in cancer. OBJECTIVE To establish quantitative PCR method for INSR transcript variants and examine their differential expression as a diagnostic tumor marker in breast cancer. METHODS The differential expression of IR-A and IR-B were evaluated by TaqMan qRT-PCR assay in the commercially available Breast Cancer Disease cDNA and Cancer Survey cDNA arrays. RESULTS The mRNA expression levels of IR-A was statistically significantly higher in breast cancer when compared to normal breast tissue while IR-B mRNA expression was down regulated significantly in breast cancer. Stratification of patients into groups according to metastatic stages indicated statistically significantly higher levels of IR-A mRNA in clinical stage (CS)-IV, and lower IR-B levels in CS-IIA, CS-IIIB and CS-IIIC. However, IR-A:IR-B ratio showed a statistically significant increase in all stages. Cancer Survey cDNA array demonstrated lower levels of IR-B mRNA in breast adenocarcinoma, liver carcinoma and lung carcinoma only while IR-A expression was significantly altered in kidney carcinoma without any significant differences in IR-A:IR-B ratios. CONCLUSIONS The results demonstrate an increased IR-A:IR-B ratio in all clinical stages of breast cancer. Thus, IR-A:IR-B ratio may have a diagnostic biomarker utility in breast cancer.

Sirtuin (SirT), a family of conserved histone deacetylases and transferases, has been proposed to function in inflammatory, cancer, and metabolic diseases. However, it is unclear how SirT modulates these processes. In this study, the effect of metformin on senescence and antisenescence mediators (SirT1-7, p53, and p16(INK4a)) mRNA expression in white blood cells (WBCs) following lipopolysaccharides (LPS)-induced inflammation in mice was examined.C57BL/6 mice were treated with metformin in their drinking water (2 mg/mL) for 1 week followed by intraperitoneal injection of LPS from Escherichia coli serotype 0111:B4 at 2 mg/kg. Blood was collected at the basal level and 1, 2, and 3 hr after LPS injection. SirT1-7, p53, and p16(INK4a) mRNA expression in WBCs was measured by real-time quantitative polymerase chain reaction (RT-qPCR).SirT7 at 2 hr, SirT1 at 3 hr, and p16(INK4a) at 1 hr were inhibited significantly in WBCs following LPS injection. There were no significant changes in other SirT nor p53 mRNA expression in WBCs after LPS injection. Metformin inhibited SirT2 expression in WBCs significantly (P0.05) and did not induce any significant changes in other SirT forms and p53, whereas it induced p16(INK4a) mRNA expression in WBCs (P0.05) at the basal levels. Additionally, metformin treatment significantly inhibited SirT7, SirT1, and p16(INK4a) mRNA expression in WBCs at 1, 2, and 3 hr, whereas p53 was inhibited significantly at 2 hr after LPS injection.SirT7 and SirT1 are stress responsive proteins that may mediate inflammation. The data suggest that metformin may exert its potential antisenescence and anti-inflammatory effects by targeting SirT7 and SirT1 pathways. SirT7 inhibition may allow the healing process and prevention of tissue damage by enabling cells to survive through inhibition of cytokines and inflammatory mediators under severe stress conditions.

Obesity is associated with insulin resistance and inflammation. The circulating human mononuclear cell (MNC) has been shown to respond to low-dose insulin infusion. We have now investigated whether in obesity: (1) phosphorylated insulin receptor beta subunit (p-INSR-beta) is reduced in the MNC; (2) pro-inflammatory mediators including inhibitor of kappa light polypeptide gene enhancer in B cells-kinase beta (IKBKB), suppressor of cytokine signalling-3 (SOCS) and protein kinase C-beta 2 (PRKCB2) are increased and related to p-INSR-beta; and (3) the reduction in MNC p-INSR-beta is related to the reduction in insulin sensitivity.MNCs were prepared from fasting blood samples of 16 normal weight and 16 obese female subjects.Our data show that p-INSR-beta is reduced significantly in MNCs from obese subjects compared with that of normal controls. MNCs from obese subjects have higher IKBKB expression, increased nuclear factor kappa B (NFkappaB) binding and higher mRNA expression of TNFAIP1 and IL6 genes. NFkappaB binding, TNFAIP1 mRNA and plasma C-reactive protein are inversely related to p-INSR-beta. PRKCB2 mRNA and protein expression were significantly higher in the obese subjects and were related significantly to pro-inflammatory mediators but not to p-INSR-beta. SOCS3 mRNA expression was markedly elevated and positively related to pro-inflammatory mediators including IKBKB and PRKCB2 on the one hand and inversely related to p-INSR-beta on the other.We conclude that in obesity the MNC is characterised by reduced p-INSR-beta and increased inflammatory mediators including IKBKB, PRKCB2 and SOCS3. The increase in SOCS3 but not IKBKB or PRKCB2 is related inversely to p-INSR-beta and might mediate the inhibition of p-INSR-beta. These data elucidate the relationship between inflammation and insulin resistance using the MNC as a model.

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 …

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.

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.

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

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 ...

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.

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 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.

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.

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.

Although the net balance between vasoconstriction and vasodilation determines the tone of the blood vessel, the vasodilatory-vasoconstrictive response following a challenge may also be determined by the intrinsic mechanical and biological properties of the vascular smooth muscle. This article reviews the action of nitric oxide, acetylcholine,and insulin; the effects of hyperglycemia, increase in free fatty acids, diabetes, and obesity on the vasculature; treatment of hypertension in diabetes; the benefits of lowering blood pressure and intensity of blood pressure control; and compares specific antihypertensive therapies on cardiovascular outcomes. The effects of antihypertensive therapy on microvascular complications, choice of antihypertensive regimen, antioxidant and anti-inflammatory effects of antihypertensive drugs, and the potential role of antidiabetic drugs in the treatment of hypertension are also presented.

Fasting hyperinsulinemia is associated with an increased risk of atherosclerotic complications, namely heart attack and stroke, which has led to the concept that insulin may promote atherosclerosis despite the absence of any evidence that insulin is atherogenic either in humans or in experimental models. Recent evidence shows that insulin exerts vasodilatory, antiplatelet, and anti-inflammatory effects at the cellular level in vitro and in humans in vivo. Because atherosclerosis is an inflammatory process, insulin is probably antiatherosclerotic in the long-term. Recent data on experimental atherosclerosis in mice show that (a) insulin administration reduces the number and the size of atherosclerotic lesions in apolipoprotein E null mice; and (b) in insulin receptor substrate-2 null mice, the interruption in insulin signal transduction results in enhanced atherogenicity. The use of a low dose of insulin infusion in patients with acute myocardial infarction (AMI) has been shown to markedly improve clinical outcomes both in diabetic and nondiabetic patients. The authors' most recent data show that a low-dose infusion of insulin in patients with AMI induces a reduction in inflammation (C-reactive protein and serum amyloid A) and oxidative stress and may have a role in myocardial protection. The authors conclude that insulin is both anti-inflammatory and antiatherogenic and may be of use in the treatment of cardiovascular inflammatory conditions, including AMI.

Recent data have revealed that the plasma concentration of inflammatory mediators, such as tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), is increased in the insulin resistant states of obesity and type 2 diabetes, raising questions about the mechanisms underlying inflammation in these two conditions. It is also intriguing that an increase in inflammatory mediators or indices predicts the future development of obesity and diabetes. Two mechanisms might be involved in the pathogenesis of inflammation. Firstly, glucose and macronutrient intake causes oxidative stress and inflammatory changes. Chronic overnutrition (obesity) might thus be a proinflammatory state with oxidative stress. Secondly, the increased concentrations of TNF-alpha and IL-6, associated with obesity and type 2 diabetes, might interfere with insulin action by suppressing insulin signal transduction. This might interfere with the anti-inflammatory effect of insulin, which in turn might promote inflammation.

OBJECTIVE—We recently demonstrated a potent anti-inflammatory and thus a potential antiatherogenic effect of insulin in human aortic endothelial cells and mononuclear cells at physiologically relevant concentrations. We have now further investigated the anti-inflammatory suppressive action of insulin on vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9. VEGF and MMP-9 play a central regulatory role in angiogenesis, contribute to the pathogenesis of proliferative retinopathy, and have also been found to accelerate atherosclerosis. RESEARCH DESIGN AND METHODS—Insulin was infused (2 IU/h) in 5% dextrose (100 ml/h) and KCl (8 mmol/h) into 10 fasting, obese, nondiabetic subjects for 4 h. Subjects were also infused with 5% dextrose without insulin and with saline on two separate occasions. Blood samples were obtained at 0, 2, 4, and 6 h. RESULTS—Plasma insulin concentrations increased from a basal level of 12.5 ± 2.2 to 28.2 ± 3.3 μU/ml at 2 h and 24.4 ± 3.7 μU/ml at 4 h after insulin infusion. VEGF concentration decreased from 307.2 ± 163.8 pg/ml (100%) at 0 h to 73.5 ± 20.9% of the basal level at 2 h and 67.1 ± 23.2% at 4h. Plasma MMP-9 concentrations decreased from 375 ± 196.3 ng/ml (100%) at 0 h to 83 ± 22% of the basal level at 2 h and to 82 ± 21% of the basal level at 4 h (P < 0.05). Dextrose infusion alone did not change plasma VEGF concentration. However, plasma MMP-9 concentration increased significantly at 4 h following dextrose infusion alone (P < 0.05). Saline infusions without insulin caused no alteration in glucose, insulin, VEGF, or MMP-9. CONCLUSIONS—These observations may have implications for a potential antiretinopathic and antiatherosclerotic effect of insulin in the long term.

The rationale for the treatment of diabetes has hitherto been directed to the correction of hyperglycemia because hyperglycemia is known to be responsible for the symptoms of polyuria and polydipsia in the short term and microangiopathic complications of diabetes in the long term. The correction of hyperglycemia leads to immediate resolution of polydipsia and polyuria and the prevention of microvascular complications in the long term (1, 2). The relationship between the decline in glycosylated hemoglobin (HbA1c) and the reduction in the rate of complications is well established, and a fall in HbA1c can reasonably predict a fall in the rate of microangiopathic complications (1, 2). In view of this close relationship between glycemia and the microangiopathic complications of diabetes, it is now accepted that any means used to decrease blood glucose concentrations will result in a reduction in microangiopathic complications. Thus, glycemic control is the cardinal measure required to prevent microangiopathic complications. Whereas microangiopathic complications lead to blindness (retinopathy) and renal failure (nephropathy) and may contribute to neuropathy, the most important complication leading to mortality in type 2 diabetes is macroangiopathy or atherosclerosis (3). Hyperglycemia may contribute to macrovascular disease, but the evidence related to its prevention by the control of hyperglycemia is limited (2, 4, 5). Whereas observational studies demonstrate a relationship between HbA1c and cardiovascular events, prospective interventional studies do not demonstrate an impressive reduction in cardiovascular events with a fall in HbA1c (2, 4, 5). In both Kumamoto (5) and UKPDS studies (2), the reduction in cardiovascular events was just short of statistical significance. Although the Kumamoto study results and correlations are based on a fall in HbA1c by more than 2%, the number of patients included was small. On the other hand, the UKPDS study was large, but the overall HbA1c fall was less than 1% (0.9%; Ref. 2). The Diabetes Control and Complications Trial, which was carried out over a period of 10 yr, achieved a consistent reduction in HbA1c of 2% and showed a decrease in cardiovascular events, but the reduction was again short of being statistically significant (1). In this case, the relative youth of the patients with type 1 diabetes may have contributed to a low overall rate of macrovascular events. It would thus appear that at best, glycemia levels do contribute to atherogenic complications of heart attack and stroke but that their contribution is small. These considerations are important because we need to develop a rational strategy for the control/prevention of macrovascular complications. Macrovascular disease (atherosclerosis) accounts for 70% of the mortality in type 2 diabetes, making heart attacks and strokes two to four times more frequent in these patients when compared with controls (6). Thus, a type 2 diabetic patient without a history of coronary heart disease (CHD) carries the same risk of having a heart attack as a nondiabetic with a previous history of a heart attack (7). It is important that several epidemiological studies have shown that fasting hyperinsulinemia predicts cardiovascular events (8). This was initially interpreted as evidence that insulin is atherogenic. However, because hyperinsulinemia is a reflection of an insulin-resistant state, it is now increasingly accepted that insulin resistance rather than hyperinsulinemia is proatherogenic. Furthermore, there are recent data that insulin has a potent antiinflammatory effect that may inhibit atherogenesis in the long term (9). This action of insulin may also explain why insulin-resistant states may be proinflammatory and proatherogenic.

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 (P0.01) 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 (P0.001). 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.

Abstract —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 P P P P P P

We studied the effect of short-term nadolol administration on the reactive oxygen species (ROS) generation by polymorphonuclear leukocytes and mononuclear cells in 8 normal subjects. At a oral dose of 40 mg/day for 5 days, nadolol produced a decrease in the ROS generation by leukocytes. ROS generation by polymorphonuclear leukocytes decreased by 38% from 134 +/- 44 mV at baseline to 83 +/- 34 mV after 5 days (p = 0.005), and ROS generation by mononuclear cells decreased by 33% from 174 +/- 69 mV at baseline to 117 +/- 55 mV after 5 days (p = 0.015). There was also a significant reduction in linoleic acid oxidation as reflected by the lower levels of 9- and 13- hydroxy-octadecadienoic acid after 5 days. There was no change in the plasma thiobarbituric acid-reacting substances, a less sensitive index of oxidative damage to lipids. There was also no significant change in the levels of metatyrosine and orthotyrosine, which are known indexes of oxidative damage to amino acids and proteins. The absence of a significant change in metatyrosine, orthotyrosine, and thiobarbituric acid-reacting substances may reflect the short duration of nadolol administration and the decreased ROS load. Because ROS may induce lipid peroxidation, this inhibitory effect of nadolol on ROS generation by leukocytes and linoleic acid oxidation may inhibit low-density lipoprotein oxidation and thus atherogenesis. This effect may partly explain the favorable outcomes observed in patients with coronary artery disease on long-term beta-blocker therapy.

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 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.

To determine whether hydrocortisone given intravenously inhibits reactive oxygen species (ROS) generation by polymorphonuclear neutrophils (PMNLs) in vivo and, if so, to describe the pharmacodynamics of this effect.A prospective, open label study in normal subjects.A clinical research unit of a tertiary referral center for diabetes and endocrinology.Eight normal subjects (age range, 2450 yrs).An indwelling cannula was inserted into the antecubital vein. Sequential blood samples were obtained from the cannula just before, and after, the intravenous injection of hydrocortisone (100 mg) at 1, 2, 4, 8, and 24 hrs.ROS generation by PMNLs and mononuclear cells (MNCs) was assayed as previously observed in a chemiluminometer. ROS generation by PMNLs and MNCs was inhibited by hydrocortisone at 1 hr; this effect peaked at 2 hrs and began to recover by 4 hrs; ROS generation had recovered to the baseline by 24 hrs. Although the pharmacodynamic effect of hydrocortisone on PMNLs and MNCs was similar, the peak inhibition was significantly greater for PMNLs (26% of basal vs. 43% of basal, p.02) than MNCs.There is a marked, consistent, inhibition of ROS generation by PMNLs, which parallels that of MNCs after intravenous hydrocortisone. The pharmacodynamics of this effect are consistent with our current clinical practices.

Purpose: To evaluate epiretinal membranes in proliferative eye disease for the presence of vascular endothelial growth factor (VEGF) and tumor necrosis factor α (TNF-α). Methods: Membranes were surgically removed from 66 patients with proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR) and macular pucker (MP). Cytokine concentrations were determined by ELISA (VEGF) and bioassay (TNF-α). Results: VEGF was detected in all 66 membranes investigated. The highest VEGF values were found in patients with type I diabetes (mean = 5,994 pg/mg protein). In patients with type II diabetes, the values were at a mean of 1,242 pg/mg protein. When coagulation therapy was performed for longer than 3 months prior to surgery, VEGF was significantly (p < 0.05) reduced. Intermediate levels of VEGF were found in PVR membranes (mean = 1,417 pg/mg protein). The lowest activity was found in MP (mean = 216 pg/mg protein). In contrast, TNF-α was present in 16 PDR membranes, 9 PVR membranes and 8 MP membranes. Conclusion: The presence of VEGF in all membranes investigated indicates that this cytokine plays an important role in angiogenesis in ischemic retinal disease and in membrane growth in proliferative disorders.