Multicentre prospective validation of a urinary peptidome-based classifier for the diagnosis of type 2 diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is one of the major late complications of diabetes. Treatment aimed at slowing down the progression of DN is available but methods for early and definitive detection of DN progression are currently lacking. The 'Proteomic prediction and Renin angiotensin aldosterone system Inhibition prevention Of early diabetic nephRopathy In TYpe 2 diabetic patients with normoalbuminuria trial' (PRIORITY) aims to evaluate the early detection of DN in patients with type 2 diabetes (T2D) using a urinary proteome-based classifier (CKD273). METHODS: In this ancillary study of the recently initiated PRIORITY trial we aimed to validate for the first time the CKD273 classifier in a multicentre (9 different institutions providing samples from 165 T2D patients) prospective setting. In addition we also investigated the influence of sample containers, age and gender on the CKD273 classifier. RESULTS: We observed a high consistency of the CKD273 classification scores across the different centres with areas under the curves ranging from 0.95 to 1.00. The classifier was independent of age (range tested 16-89 years) and gender. Furthermore, the use of different urine storage containers did not affect the classification scores. Analysis of the distribution of the individual peptides of the classifier over the nine different centres showed that fragments of blood-derived and extracellular matrix proteins were the most consistently found. CONCLUSION: We provide for the first time validation of this urinary proteome-based classifier in a multicentre prospective setting and show the suitability of the CKD273 classifier to be used in the PRIORITY trial.

Molecular effects of C-Peptide in microvascular blood flow regulation.

C-Peptide is produced in beta-cells in the pancreas, and secreted into the blood stream in equimolar amounts with insulin. For a long time, C-peptide was considered as an important component in the biosynthesis of insulin, but otherwise believed to possess minimal biological activity. In the recent years, numerous studies demonstrated that lacking C-peptide in type 1 diabetic patients might exert an important role in the development of microvascular complications such as nephropathy or neuropathy. There is increasing evidence that the biological effects of C-peptide are, at least in part, mediated through the modulation of endothelial function and microvascular blood flow. In several tissues, an increase in microvascular and nutritional blood flow could be observed during substitution of physiological amounts of C-peptide. Recent studies confirmed that C-peptide stimulates endothelial NO release by the activation of Ca2+ calmodulin-regulated endothelial NO synthase. A restoration of Na+/K+-ATPase activity during C-peptide supplementation could be observed in erythrocytes and renal tubular cells. The improvement of erythrocyte Na+/K+-ATPase is associated with an increase in erythrocyte deformability, and improved rheological properties. In this article, we consider the role of C-peptide in the context of endothelial function and microvascular blood flow as pathophysiologic components in the development of microvascular complications in patients with diabetes mellitus and loss of beta-cell function.

C-peptide and its C-terminal fragments improve erythrocyte deformability in type 1 diabetes patients.

AIMS/HYPOTHESIS: Data now indicate that proinsulin C-peptide exerts important physiological effects and shows the characteristics of an endogenous peptide hormone. This study aimed to investigate the influence of C-peptide and fragments thereof on erythrocyte deformability and to elucidate the relevant signal transduction pathway. METHODS: Blood samples from 23 patients with type 1 diabetes and 15 matched healthy controls were incubated with 6.6 nM of either human C-peptide, C-terminal hexapeptide, C-terminal pentapeptide, a middle fragment comprising residues 11-19 of C-peptide, or randomly scrambled C-peptide. Furthermore, red blood cells from 7 patients were incubated with C-peptide, penta- and hexapeptides with/without addition of ouabain, EDTA, or pertussis toxin. Erythrocyte deformability was measured using a laser diffractoscope in the shear stress range 0.3-60 Pa. RESULTS: Erythrocyte deformability was impaired by 18-25% in type 1 diabetic patients compared to matched controls in the physiological shear stress range 0.6-12 Pa (P < .01-.001). C-peptide, penta- and hexapeptide all significantly improved the impaired erythrocyte deformability of type 1 diabetic patients, while the middle fragment and scrambled C-peptide had no detectable effect. Treatment of erythrocytes with ouabain or EDTA completely abolished the C-peptide, penta- and hexapeptide effects. Pertussis toxin in itself significantly increased erythrocyte deformability. CONCLUSION/INTERPRETATION: C-peptide and its C-terminal fragments are equally effective in improving erythrocyte deformability in type 1 diabetes. The C-terminal residues of C-peptide are causally involved in this effect. The signal transduction pathway is Ca(2+)-dependent and involves activation of red blood cell Na(+), K(+)-ATPase.

The beta-specific protein kinase C inhibitor ruboxistaurin (LY333531) suppresses glucose-induced adhesion of human monocytes to endothelial cells in vitro.

AIMS: Strong evidence shows that late diabetic complications in diabetes mellitus are substantially related to an increased synthesis of diacylglycerol with a subsequent activation of protein kinase C (PKC) beta. Several studies have shown that specific inhibition of the PKC isoform beta by ruboxistaurin is able to attenuate the development of microvascular complications under diabetic conditions. The aim of this in vitro study was to investigate the effect of ruboxistaurin on glucose-induced adhesion of monocytes to endothelial cells, representing one of the first pivotal steps in the course of atherogenesis. METHODS: Human umbilical venous endothelial cells were isolated and cultured to confluence in microtiter plates. After coincubation with monocytes in the presence of 0, 10, or 400 ng ruboxistaurin to achieve PKC beta-specific and -unspecific PKC inhibition, cells were fixed and monocyte adhesion was determined by means of a standardized chemiluminescence assay. Expression of adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin) was also measured by chemiluminescence methods. RESULTS: Adhesion of monocytes to endothelial cells cultured under hyperglycemic conditions (27.7 mM glucose) was increased by 30.9 +/- 5.1% (p < 0.001) versus endothelial cells cultured under normoglycemic (NG) conditions (5.5 mM). Pretreatment of endothelial cells with 10 nM (PKC beta-specific concentration) and 400 nM (PKC beta-unspecific concentration) led to a significant reduction of glucose-induced adhesion of monocytes to endothelial cells that was statistically not different from endothelial adhesion under NG conditions (-7.2 +/- 3.1 and -8.1 +/- 2.6%, respectively; not significant vs NG). A nonsignificant tendency to lower the expression of adhesion molecules was seen with 10 ng of ruboxistaurin. CONCLUSIONS: We conclude that monocyte adhesion to endothelial cells under hyperglycemic conditions is at least mediated by PKC beta activation. Ruboxistaurin is able to suppress this monocyte adhesion even in a PKC beta-specific concentration. Further studies should evaluate these potential effects of ruboxistaurin in vivo.

Intact and total proinsulin: new aspects for diagnosis and treatment of type 2 diabetes mellitus and insulin resistance.

Proinsulin, the precursor of insulin during physiological insulin production, has been demonstrated in the past to stimulate PAI-1 secretion and consecutively block fibrinolysis. Therefore, proinsulin is contributing as an independent factor to the increased cardiovascular risk of patients with type 2 diabetes. However, development of insulin resistance in the course of type 2 diabetes leads to increased insulin demands and finally to an impairment of beta-cell function in later disease stages. Appearance of intact proinsulin in the peripheral blood has been shown to be a good laboratory marker for this phenomenon since it indicates an exhaustion of the cleavage capacity of the intracellular processing enzymes. However, the close relation of the two pathophysiological entities also makes it a very specific marker for insulin resistance per se. During the past years, new immunoassays have been developed that are able to distinguish between intact proinsulin and its specific and unspecific cleavage products. Use of these assays in recent epidemiological and intervention studies has helped to get a better understanding about beta-cell dysfunction and its relation to insulin resistance and cardiovascular risk. In a large cross-sectional study with 4270 orally treated patients, elevation of fasting intact proinsulin was very closely related to insulin resistance, as assessed by iv glucose tolerance test in a subgroup, and by HOMA analysis in the entire patient population. Effective treatment of insulin resistance (e.g. with thiazolidindiones) led to a decrease in elevated proinsulin levels and to a decrease of the cardiovascular risk profile, while the levels remained high during sulfonylurea therapy. These results suggest to reconsider intact and total proinsulin as valuable diagnostic tools in diagnosis and treatment of type 2 diabetes. Based on the published data of the new specific immunoassays, patients with elevated intact proinsulin levels (> 10 pmol/L) should be regarded and treated as being insulin-resistant, while elevation of total proinsulin (>45 pmol/l) may help to identify the high cardiovascular risk patients. Both assays can thus be used to assess beta-cell function, to facilitate the selection of the most promising therapy, and may also serve to monitor treatment success in the further course of the disease.

Fasting intact proinsulin is a highly specific predictor of insulin resistance in type 2 diabetes.

OBJECTIVE: In later stages of type 2 diabetes, proinsulin and proinsulin-like molecules are secreted in increasing amounts with insulin. A recently introduced chemiluminescence assay is able to detect the uncleaved "intact" proinsulin and differentiate it from proinsulin-like molecules. This investigation explored the predictive value of intact proinsulin as an insulin resistance marker. RESEARCH DESIGN AND METHODS: In total, 48 patients with type 2 diabetes (20 women and 28 men, aged 60 +/- 9 years [means +/- SD], diabetes duration 5.1 +/- 3.8 years, BMI 31.2 +/- 4.8 kg/m2, and HbA1c 6.9 +/- 1.2%) were studied by means of an intravenous glucose tolerance test and determination of fasting values of intact proinsulin, insulin, resistin, adiponectin, and glucose. Insulin resistance was determined by means of minimal model analysis (MMA) (as the gold standard) and homeostatis model assessment (HOMA). RESULTS: There was a significant correlation between intact proinsulin values and insulin resistance (MMA P<0.05 and HOMA P<0.01). Elevation of intact proinsulin values above the reference range (>10 pmol/l) showed a very high specificity (MMA 100% and HOMA 92.9%) and a moderate sensitivity (MMA 48.6% and HOMA 47.1%) as marker for insulin resistance. Adiponectin values were slightly lower in the insulin resistant group, but no correlation to insulin resistance could be detected for resistin in the cross-sectional design. CONCLUSIONS: Elevated intact proinsulin seems to indicate an advanced stage of beta-cell exhaustion and is a highly specific marker for insulin resistance. It might be used as arbitrary marker for the therapeutic decision between secretagogue, sensitizer, or insulin therapy in type 2 diabetes.

Evaluation of human resistin assays with serum from patients with type 2 diabetes and different degrees of insulin resistance.

Resistin is a peptide hormone encoded at the RSTN gene that since its detection in mice is considered to be an important link between obesity and insulin resistance. However, the study reports and especially the human data are contradictory and require further investigation. The purpose of this study was to evaluate three commercially available resistin ELISAs with different target epitopes (Phoenix, Belmont, CA, USA (PH); Biovendor, Brno, Czech Republic (BV); and Immundiagnostik, Bensheim, Germany (ID)) from a laboratory and clinical perspective. All three assays successfully passed the standardized technical validation procedure, with an inter- and intra-assay variability below 10% and 15%, respectively. They proved to be different with regard to calibration and reference ranges, which may be linked to the different antibody specificities. The clinical evaluation was performed with fasting serum samples from 78 patients with type 2 diabetes (43 female, 35 male, age (mean +/- SD, range): 67 +/- 10, (41-86) years; BMI: 29.2 +/- 4.2 (21.6-41.9) kg/m2). Insulin resistance was calculated from the fasting insulin and glucose values by means of the HOMA analysis. Intact proinsulin served as comparative laboratory marker for insulin resistance. The mean resistin values of patients without insulin resistance were slightly higher (PH: 9.5 +/- 2.8 ng/ml; BV: 4.1 +/- 4.0 ng/ml; ID: 3.8 +/- 9.0 ng/ml) than the mean values of the resistant patients (PH: 9.0 +/- 1.7 ng/ml, n.s.; BV: 3.8 +/- 1.3 ng/ml, n.s.; ID: 0.8 +/- 1.0 ng/ml, p<0.05). Intact proinsulin levels correlated well with the HOMA score values (r = 0.64, p<0.001). No correlation was seen between any of the resistin assays and any of the other clinical or laboratory observation parameters collected, such as BMI, age, disease duration, triglycerides, LDL, HDL, insulin, glucose, or intact proinsulin. In conclusion, the resistin assays showed good technical quality, but the diagnostic value remains still unclear. It may, however, be concluded from this study that at least in cross-sectional epidemiological investigations, fasting human resistin concentrations are not significantly correlating with any clinical measure for insulin resistance.

Clinical and laboratory evaluation of specific chemiluminescence assays for intact and total proinsulin.

Measurement of proinsulin is an important tool in the assessment of pancreatic beta cell function in patients with type 2 diabetes. The goal of this study was to perform a technical and clinical evaluation of two specific chemiluminescence assays (CLIA) for the determination of intact and total proinsulin in comparison to a radioimmunoassay (RIA) method for the measurement of total proinsulin. A total of 191 serum samples from patients with type 2 diabetes were used to perform a regression analysis. The total proinsulin CLIA showed higher proinsulin levels than the two other proinsulin assays (mean +/- SD: 55.9 +/- 58.1 pmol/l, p < 0.001 in both cases). The intact proinsulin CLIA (22.5 +/- 20.9 pmol/l) gave lower values than the RIA for total proinsulin (31.9 +/- 25.4 pmol/l, p < 0.001 vs. CLIA, r = 0.948). The RIA has a 95% cross-reactivity to des31,32-proinsulin, which is secreted during the process of beta cell deterioration. The intact proinsulin CLIA has virtually no cross-reactivity with des31,32-proinsulin (1.4%) and is therefore more specific for intact proinsulin than the RIA. This test does not measure further degradation products, in contrast to the total proinsulin CLIA. The CLIA is, therefore, more specific for total proinsulin measurement than the RIA. Both CLIAs could be performed much faster (4 hours) than the RIA method (75 hours/ 4 days). In conclusion, the CLIA methods show improved qualitative outcomes, higher specificity and several technical advantages over the RIA method.

Stimulation of endothelial nitric oxide synthase by proinsulin C-peptide.

There is increasing evidence for biological functions of human C-peptide. Recently, we have described that proinsulin C-peptide increases nutritive capillary blood flow and restores erythrocyte deformability in type 1 diabetic patients, whereas it has no such effect in non-diabetic subjects. The aim of the current study was to elucidate cellular mechanisms of this vasodilator effect in vitro by measuring the nitric oxide (NO)-mediated increase of cGMP production in a RFL-6 reporter cell assay and by demonstrating endothelial calcium influx with the Fluo-3 technique. C-peptide increased the release of NO from endothelial NO synthase (eNOS) in bovine aortic endothelial cells in a concentration- and time-dependent manner. At physiological concentrations of C-peptide, endothelial NO production was more than doubled (208+/-12% vs control; p<0.001). The NO release was abolished by the inhibitor of NO synthase N(G)-nitro-L-arginine or when Ca(2+) was removed from the medium superfusing the endothelial cells. C-peptide stimulated the influx of Ca(2+) into endothelial cells. No change in Ser-1179 phosphorylation of eNOS was detected after 6.6nM C-peptide. C-peptide did not change eNOS mRNA levels after 1, 6 or 24h. These data indicate that C-peptide is likely to stimulate the activity of the Ca(2+)-sensitive eNOS by increasing the influx of Ca(2+) into endothelial cells. We suggest that this effect may contribute to the increase in skin and muscle blood flow previously demonstrated in human in vivo.