Impaired fibrin clot lysis is a key abnormality in diabetes and complement C3 is one protein identified in blood clots. This work investigates the mechanistic pathways linking C3 and hypofibrinolysis in diabetes using ex vivo/in vitro studies.

Introduction and Aims: Hepcidin synthesis occurs in response to iron overload, inflammation and inhibition of erythropoiesis in patients with hemodialysis (HD).In this study, we investigated the early response of hepcidin-25 (hep) to administration of short-acting epoetin-beta (epo) and long-acting methoxy polyethylene glycol-epoetin-beta (PEG-epo) and whether these erythropoietin drugs (rHuEPO) make a difference in ferrokinetics of HD patients.Methods: The study included 9 patients with HD but no diabetes mellitus (4 men and 5 women with a mean age of 59 ± 7.0 years and a mean duration of HD of 120 ± 95 months).Serum levels of hep were measured before and 3, 6, 9 and 18 hours after intravenous injection with either epo or PEG-epo.The measurements of hep in response to epo or PEG-epo were performed using the same patients separately at an interval of 2 years.The hep level was measured using a high-throughput LC-MS/MS method.Serum levels of ferritin, haemoglobin (Hb), and CRP were measured at the start of dialysis.Data are expressed as mean ± SD.Statistical analysis was performed using the Mann-Whitney U test or Fisher`s exact probability test.Results: Serum ferritin level was 38.2 ± 50.3ng/mL when epo was given, which did not differ significantly from that (15.3 ± 11.1ng/mL) when PEG-epo was given.The hep level was significantly increased from the basal level of 6.8 ± 9.8ng/mL to 15.9 ± 20.9ng/mL at 6 hours after epo administration, and decreased significantly at 18 hours (4.6 ± 8.7 ng/mL).The hep level was significantly increased from the basal level of 10.9 ± 13.6 to 25.0 ± 29.3ng/mL at 6 hours after PEG-epo administration, and decreased at 18 hours (15.2 ± 18.1 ng/mL).There was no difference in the time-course effects of epo and PEG-epo on serum hep levels.Based on the basal ferritin level, we divided 18 samples into two groups; high ferritin group: 7 samples with serum fer level of > 15.0 ng/mL and lower ferritin group: 11 samples with lower limits of normal.In the lower ferritin group, the hep level was 5.0 ng/mL or less at all 5 measurement time points.However, in the high ferritin group, the hep level was significantly increased from the basal level of 19.8 ± 12.0 to 46.8 ± 19.8 ng/mL at 6 hours after rHuEPO administration.There was no difference in the number of samples taken from the patients receiving PEG-epo between the two groups.The Hb levels did not differ between the low ferritin group (10.6 ± 2.6g/dL) and the high ferritin group (11.2 ± 0.7g/dL).The values for CRP were < 0.1 mg/dl at all measurements.Conclusions: Our data suggest that up-regulation of hepcidin-25 in the early phase of rHuEPO-induced erythropoiesis, which occurs regardless of short-or long-acting agents, is dependent on serum ferritin levels in HD patients.

Healthy first-degree relatives of patients with Type 2 diabetes are at increased risk of Type 2 diabetes and cardiovascular disease (CVD). Alterations in plasma levels of inflammatory and thrombotic markers are associated with both Type 2 diabetes and CVD. Increased clot density with prolonged lysis times are associated with increased cardiovascular risk.[1-6] The aim of the present study was to investigate whether complement C3, C-reactive protein (CRP) and fibrin clot characteristics are altered in relatives of patients with Type 2 diabetes and are associated with a family history of diabetes. Patients with Type 2 diabetes, their first-degree relatives and healthy control subjects, age- and sex-matched to relatives, were recruited as described previously [7, 8]. All subjects were White European and gave informed consent according to protocols approved by the Leeds Teaching Hospitals Trust Research Ethics Committee. Metabolic and haemostatic factors were determined as described previously [7, 8]. The relatives underwent an oral glucose tolerance test to exclude the possibility of undiagnosed diabetes. Plasma levels of complement C3 and CRP were both measured by in-house ELISAs [9] (inter-assay coefficients of variance were 7.7% and 2.8% for the C3 and CRP ELISAs, respectively). Clot formation and lysis were measured by turbidimetric assays as described previously [10] (inter-assay coefficients of variance were 3.7% for lag time, 1.9% for maximum absorbance, and 3.3% for lysis time). Continuous variables were tested for normal distribution and analysed accordingly (normally distributed variables: one-way anova with post-hoc Scheffé analysis for pairwise comparison; not normally distributed variables: Kruskal–Wallis with Mann–Whitney U-test for pairwise comparison, with Bonferroni adjustment). The contribution of demographic, metabolic and hemostatic variables to group differences in inflammatory and turbidimetric variables was estimated by univariate general linear regression analyses. Independent associations with a family history of Type 2 diabetes were investigated by stepwise logistic regression analysis including the variables body mass index (BMI), glucose, HbA1c, triglycerides, total cholesterol, high-density lipoprotein (HDL), fibrinogen, plasminogen activator inhibitor-1 (PAI-1), C3, CRP and lysis time. All analyses were carried out using spss v12.0 (SPSS Inc., Chicago, IL, USA). The characteristics of first-degree relatives, healthy controls and Type 2 diabetes patients are shown in Table 1. The patients were older, but relatives and controls were age matched. The relatives showed intermediate levels between controls and patients for most parameters, with differences maintained after adjustment for age and gender (data not shown). C3 and CRP (Table 1) were significantly higher in relatives and patients compared to controls, however, C3 and CRP were not significantly different between relatives and patients. There were no significant differences in clot lag time (LagC) between the three groups, whereas significant trends across the groups were found for clot density (MaxAbsC), lysis time and area under the clot formation/lysis curve (AUC) (Table 1), with the differences remaining significant after adjustment for age and gender (data not shown). C3, CRP and lysis time significantly correlated with metabolic and hemostatic parameters (correlation coefficients ≥ 0.4) as follows: C3 correlated with BMI (controls, relatives and patients), insulin (relatives and patients), insulin resistance estimated by homeostasis model assessment (HOMA) (relatives), triglycerides (controls and patients), fibrinogen and lysis time (controls), and with CRP (in all three groups). CRP correlated with BMI (controls), insulin (patients) and fibrinogen (controls and relatives). Lysis time correlated with fibrinogen, C3 (controls) and PAI-1 (relatives). In linear regression analyses for C3 and CRP, including the variables age, gender, BMI, HOMA, cholesterol, triglycerides and HDL, independent predictors of C3 were BMI, insulin resistance (HOMA) and triglycerides accounting for 21%, 9.7%, and 3.2%, respectively, of variance. After accounting for these variables the group differences in C3 remained significant (P = 0.011). Independent predictors of CRP were BMI, HOMA and triglycerides accounting for 16%, 5.8% and 4.5%, respectively, of variance. These variables accounted for the observed between-group differences in CRP. In linear regression analysis for lysis time, including the variables age, gender, fibrinogen, factor (F)XIII, PAI-1, C3, CRP, BMI, HOMA, cholesterol, triglycerides, and HDL, independent predictors of lysis time were fibrinogen, FXIII, PAI-1, C3 and total cholesterol accounting for 6.7%, 5.8%, 5.1%, 1.5% and 2.0%, respectively, of variance. These variables accounted for the between-group differences in lysis time. In a stepwise logistic regression model comparing relatives and controls, including BMI, glucose, HbA1c, triglycerides, total cholesterol, HDL, fibrinogen, PAI-1, C3, CRP and lysis time, only PAI-1 (odds ratio for a 1SD increase: 1.81 (95% CI 1.17–2.80), P = 0.008) and C3 [odds ratio for a 1SD increase: 1.75 (1.13–2.71), P = 0.012] were independently associated with a family history of Type 2 diabetes. This is the first study to investigate fibrin clot formation and lysis in first-degree relatives of Type 2 diabetes patients. Similar to diabetes patients, hemostatic and metabolic factors clustered in relatives and contributed to an adverse fibrin phenotype, characterized by prolonged fibrinolysis times. This may in part explain the increased cardiovascular risk in relatives. Consistent with our previous results [10], we found that fibrinogen, FXIII and PAI-1 were significant determinants of lysis time, accounting for ∼ 17% of its variance. Interestingly, C3 was also an independent predictor of lysis time. We have recently identified C3 as a component of plasma clots and shown a direct influence of C3 on fibrin clot structure/function providing evidence for a functional link between elevated C3 levels and prolonged fibrinolysis [11]. These results therefore suggest that a proinflammatory phenotype in first-degree relatives may contribute to thrombotic risk. C3 and CRP were elevated in relatives and Type 2 diabetes patients and clustered with metabolic and hemostatic cardiovascular risk factors, which accounted for the increased CRP levels in the relatives. Metabolic and hemostatic cardiovascular risk factors did not fully account for elevated C3 in relatives and patients. In addition, elevated C3 was independently associated with a family history of diabetes. These data suggest that C3 may be more specifically related to the inflammatory processes contributing to the development of Type 2 diabetes and CVD than CRP, which appears to reflect non-specific inflammatory processes in the present study. The results are consistent with our previous studies in which C3, but not CRP, was independently associated with CVD after accounting for conventional cardiovascular risk factors [5, 9]. Our results contradict those of Kriketos et al. who reported no difference in C3 levels between relatives of Type 2 diabetes patients and controls [12]. This discrepancy most likely reflects the smaller group sizes (19 relatives and 22 controls) resulting in a lack of power to detect significant difference, despite a similar trend in that study. A potential limitation of the present study is the disparity in the ages of the patients compared with the relatives and matched controls. However, the similarity in inflammatory and hemostatic risk factor profiles in relatives and patients despite the disparity in age emphasises the importance of these observations which suggest abnormalities in inflammation and hemostasis predate the development of diabetes, perhaps at the time when insulin resistance achieves maximal levels, but compensatory hyperinsulinemia maintains euglycemia prior to the development of overt diabetes. In conclusion, healthy first-degree relatives of Type 2 diabetes patients showed a proinflammatory and hypofibrinolytic phenotype similar to that seen in their older relatives with diabetes. Moreover, complement C3 was both related to fibrinolytic activity and independently associated with a family history of Type 2 diabetes. This proinflammatory hypofibrinolytic phenotype may contribute to the increased risk of Type 2 diabetes and CVD in relatives and these data lend further support for exploring C3 as a specific marker for disease development. This study was supported by grants from the British Heart Foundation, the Northern and Yorkshire Regional Health Authority, and the United Leeds Teaching Hospitals Special Trustees. V. Schroeder is supported by fellowships from the Swiss National Science Foundation and the Novartis Jubilee Foundation. The authors state that they have no conflict of interest.

Objective: Neprilysin (NEP), a zinc metalloendopeptidase, has a role in blood pressure control and lipid metabolism. The present study tested the hypothesis that NEP is associated with insulin resistance and features of the metabolic syndrome (MetS) in a study of 318 healthy human subjects and in murine obesity, and investigated NEP production by adipocytes in-vitro . Methods and results: In 318 white European males, plasma NEP was elevated in the MetS and increased progressively with increasing MetS components. Plasma NEP activity correlated with insulin, homoeostasis model assessment and body mass index (BMI) in all subjects ( P <0.01). Quantitative reverse transcriptase PCR (RT–PCR) and western blotting showed that in human pre-adipocytes NEP expression is upregulated 25- to 30-fold during differentiation into adipocytes. Microarray analysis of mRNA from differentiated human adipocytes confirmed high-NEP expression comparable with adiponectin and plasminogen activator inhibitor-1. In a murine model of diet-induced insulin resistance, plasma NEP levels were significantly higher in high-fat diet (HFD)-fed compared with normal chow diet (NCD)-fed animals (1642±529 and 820±487 pg μl −1 , respectively; P <0.01). Tissue NEP was increased in mesenteric fat in HFD compared with NCD-fed mice ( P <0.05). NEP knockout mice did not display any changes in insulin resistance, glucose tolerance, or body and epididymal fat pad weight compared with wild-type mice. Conclusion: In humans, NEP activity correlated with BMI and measures of insulin resistance with increasing levels in subjects with multiple cardiovascular risk factors. NEP protein production in human adipocytes increased during cell differentiation and plasma and adipose tissue levels of NEP were increased in obese insulin-resistant mice. Our results indicate that NEP associates with cardiometabolic risk in the presence of insulin resistance and increases with obesity.

BACKGROUND:Alanine aminotransferase (ALT) predicts the development of Type 2 diabetes mellitus and cardiovascular disease in Caucasian subjects.OBJECTIVES:This study aimed to determine the incidence of an elevated ALT and its relationship to metabolic and atherothrombotic risk factors in a healthy British South Asian population.PATIENTS/METHODS:One hundred and forty-three participants from the West Yorkshire community were recruited randomly from general practice registers and were grouped according to whether their ALT was above or within the normal range (cut-off 35 IU L(-1)) and examined for differences in metabolic and atherothrombotic risk factors. All participants were originally from South Asia, with their grandparents being born in India, Pakistan, or Bangladesh.RESULTS:The incidence of a raised ALT was 24%. Those with a raised ALT had a more adverse metabolic profile, with significantly higher body mass index, waist/hip ratio, fasting insulin, glucose, homeostasis model assessment homeostasis model assessment insulin resistance (HOMA-IR), and triglycerides, and lower high-density lipoprotein (HDL) cholesterol. Fifty per cent had the metabolic syndrome [International Diabetes Federation (IDF) criteria]. They also had a more adverse atherothrombotic profile, with higher tissue-type plasminogen activator and plasminogen activator inhibitor-1 (PAI-1) antigen. In accordance, the group as a whole showed a positive correlation of ALT (age-adjusted) with waist/hip ratio, insulin, glucose, triglycerides, PAI-1 antigen, factor XIII B subunit, and FXII, and a negative correlation with HDL cholesterol.CONCLUSION:Raised ALT is common in apparently healthy British South Asians, and is significantly associated with an adverse metabolic and atherothrombotic risk profile.

Background: The metabolic syndrome is a cluster of atherothrombotic risk factors that are commonly associated with insulin resistance. Objectives: The aim of this study was to investigate ethnic differences in insulin resistance and non-traditional cardiovascular risk factors in relation to the International Diabetes Federation (IDF) definition of the metabolic syndrome. Patients and methods: A total of 245 healthy South Asians and 245 age- and sex-matched Caucasians were studied. C-reactive protein (CRP), complement C3, plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (t-PA) were measured and homeostasis model assessment-insulin resistance (HOMA-IR) was calculated from fasting plasma glucose and insulin levels. Results: Fifty Caucasian (20%) and 95 (39%) South Asian subjects had the metabolic syndrome as defined by the IDF. In South Asian subjects, HOMA-IR, CRP, C3, PAI-1 and t-PA were significantly higher in subjects with the metabolic syndrome. In contrast, in Caucasian individuals there was no difference in HOMA-IR or C3 levels and only CRP, PAI-1 and t-PA were higher in subjects with the metabolic syndrome. In a logistic regression model, plasma levels of CRP and PAI-1 were independent predictors of the metabolic syndrome in Caucasians, whereas plasma levels of C3 and t-PA as well as HOMA-IR were independent predictors of the metabolic syndrome in South Asian subjects. Conclusions: In the cohort of individuals studied, the IDF definition of the metabolic syndrome was associated with insulin resistance in the South Asian but not the Caucasian population. This work also showed ethnic differences in non-traditional cardiovascular risk factors in the presence of the metabolic syndrome.

OBJECTIVE:Accumulating evidence raises the hypothesis that dysregulation of intrinsic clock mechanisms are involved in the development of the metabolic syndrome, type 2 diabetes mellitus and cardiovascular disease. The aim of the present study was to investigate the relationship between three known common polymorphisms in the Clock gene and features of the metabolic syndrome in man.METHODS:Genotype and haplotype analysis was carried out in a cohort of 537 individuals from 89 families characterized for inflammatory, atherothrombotic and metabolic risk associated with insulin resistance.RESULTS:Heritability of the metabolic syndrome, defined according to International Diabetes Federation criteria, was 0.40. Haplotype analysis indicated three common haplotypes: CAT, TGT and CGC (rs4864548-rs3736544-rs1801260) with frequencies of 31, 33 and 28%, respectively. The CGC haplotype was less prevalent in subjects with the metabolic syndrome (P=0.0015) and was associated with lower waist circumference (P=0.007), lower hip circumference (P=0.023), lower body mass index (P=0.043) and lower leptin levels (P=0.028). The CAT haplotype was significantly associated with the presence of the metabolic syndrome (P=0.020).CONCLUSIONS:These findings suggest that the Clock gene CGC haplotype may be protective for the development of obesity and support the hypothesis that genetic variation in the Clock gene may play a role in the development of the metabolic syndrome, type 2 diabetes and cardiovascular disease.

The association of complement C3 genotype with coronary artery disease, markers of the metabolic syndrome and C3 plasma levels -

The aim of this study was to determine whether complement C3 is an indicator of coronary artery disease (CAD). We measured plasma C3 and CRP levels in 278 patients undergoing coronary angiography for typical symptoms of CAD and 269 healthy age and sex matched controls. C3 levels were significantly higher in patients compared with controls (1.15 g/l and 0.92 g/l respectively; p<0.001). In the patient group, C3 levels correlated with BMI, fasting glucose, HbA1c, fibrinogen, CRP and HDL in both men and women. CRP levels were also higher in patients compared with controls (1.14 mg/l and 0.86 mg/l respectively; p=0.005) and correlated with markers of the metabolic syndrome. In a logistic regression model including C3, smoking, hypertension, cholesterol and diabetes, C3 was independently associated with CAD with an odds ratio of 3.20 for a 1 SD increase in C3 levels. In contrast, CRP was not independently associated with CAD in a similar regression analysis. In conclusion, both C3 and CRP plasma levels are elevated in patients with symptoms of CAD. However, C3 seems to be a better indicator of CAD than CRP in this study, suggesting that C3 could be an additional marker for risk stratification in atherosclerosis.