Thomas Kunt

Biological activity of C-peptide on the skin microcirculation in patients with insulin-dependent diabetes mellitus.

19 insulin-dependent diabetes mellitus (IDDM) patients participated in a randomized double-blind crossover investigation to investigate the impact of human C-peptide on skin microvascular blood flow. The investigation was also carried out with 10 healthy volunteers. Blood pressure, heart rate, blood sugar, and C-peptide levels were monitored during a 60-min intravenous infusion period of C-peptide (8 pmol kg-1 min-1) or saline solution (154 mmol liter-1 NaCl), and 30 min after stopping the infusion. During the same time period, capillary blood cell velocity (CBV), laser Doppler flux (LDF), and skin temperature were assessed in the feet. In the verum arm, C-peptide levels increased after starting infusion to reach a maximum of 2.3+/-0.2 nmol liter-1 after 45 min, but remained below 0. 15 nmol liter-1 during the saline treatment. Baseline CBV was lower in diabetic patients compared with healthy subjects (147+/-3.6 vs. 162+/-4.2 micron s-1; P < 0.01). During C-peptide administration, CBV in IDDM patients increased progressively from 147+/-3.6 to 167+/-3.7 micron s-1; P < 0.001), whereas no significant change occurred during saline infusion or in healthy subjects. In contrast to the CBV measurements, the investigation of LDF, skin temperature, blood pressure, heart rate, or blood sugar did not demonstrate any significant change during the study. Replacement of human C-peptide in IDDM patients leads to a redistribution in skin microvascular blood flow levels comparable to levels in healthy subjects by increasing the nutritive CBV relative to subpapillary arteriovenous shunt flow.

The effect of human proinsulin C-peptide on erythrocyte deformability in patients with Type I diabetes mellitus

Aims/hypothesis. In recent years, evidence has arisen that proinsulin C-peptide exerts biological effects especially on microcirculation, e. g. C-peptide has been shown to increase skin microcirculation in patients with Type I (insulin-dependent) diabetes mellitus and to activate endothelial nitric oxide synthase. This study aimed to investigate the influence of proinsulin C-peptide on erythrocyte deformability which was assessed by means of laser diffractoscopy. Methods. Blood samples from healthy control subjects (n = 10) and Type I diabetic patients (n = 15) completely deficient of C-peptide were analysed at shear stresses ranging from 0.3 to 30 Pa. Results. Erythrocyte deformability was lower in the group of Type I diabetic patients than in the control subjects. Preincubation of the diabetic blood samples with various concentrations of human proinsulin C-peptide for 8 h restored the deformability of erythrocytes, almost reaching the values of control samples. In contrast, proinsulin C-peptide did not modify the erythrocyte deformability of control subjects. Conclusion/interpretation. We conclude that proinsulin C-peptide is able to ameliorate the impaired deformability of erythrocytes in Type I diabetic patients and we hypothesise that this effect is mediated by restoration of Na+-K+-ATPase activity, which is known to be attenuated in diabetic patients. [Diabetologia (1999) 42: 465–471]

The influence of local capsaicin treatment on small nerve fibre function and neurovascular control in symptomatic diabetic neuropathy

Abstract Topical treatment wit capsaicin cream has been shown to be successful in the treatment of different symptomatic nerve disorders like diabetic neuropathy. Conflicting data exist on the effect of capsaicin on nerve function and neurovascular control especially in patients with diabetic neuropathy. The aim of this pilot study was to investigate the impact of topical capsaicin application on small nerve fibre function and neurovascular control. Capsaicin cream was applied to the feet of 13 patients with symptomatc diabetic neuropathy over a period of 8 weeks. Before and during the treatment period, we investigated the total symptoms score, the vibration, thermal (heat and cold) and pain perception thresholds, and the neurovascular responses to heat and acetylcholine stimuli. In addition, the serum plasma levels of substance P, a neurotransmitter of nociceptor C-fibres, were measured. A significant improvement in total symptoms score was observed during topical capsaicin treament (18.3±3.2 vs. 14.3±3.3; p<0.05). An improvement in the heat perception threshold was also found (12.7±0.4°C vs. 11.4±0.7°C; p<0.05), while other sensory nerve fibre functions remained unchanged. No significant change in neurovascular control was observed, neither after mild thermal injury nor after stimulation with acetylcholine. Serum substance P levels increased after initiation of topical capsaicin treatment (72.9±5.8 pg/ml vs. 81.7±5.0 pg/ml; p<0.05), but returned to baseline levels during further treatment (77.4±8.3 pg/ml; n. s.). In conclusion, topical treatment with capsaicin cream over a period of 8 weeks in patients with symptomatic diabetic neuropathy is effective without adverse effects on nerve fibre function or neurovascular control.

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 Ca2+-dependent and involves activation of red blood cell Na+, K+-ATPase.

Clinical and Laboratory Evaluation of Specific Chemiluminescence Assays for Intact and Total Proinsulin

Abstract Measurement of proinsulin is an important tool in the assessment of pancreatic β 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 β cell deterioration. The intact proinsulin CLIA has virtually no crossreactivity 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.

The influence of isolated small nerve fibre dysfunction on microvascular control in patients with diabetes mellitus.

Aim The aim of the study was to investigate the influence of isolated small nerve fibre dysfunction on microvascular skin blood flow and transcutaneous oxygen tension in patients with diabetes mellitus.

The physiological impact of proinsulin C-peptide

Abstract The proinsulin C-peptide fulfills an important function in the biosynthesis of insulin by facilitating the formation of the correct secondary and tertiary structure of the hormone. C-peptide and insulin are released in equimolar amounts to the circulation but C-peptide has generally been considered to be biologically inert. However, recent studies indicate that C-peptide administration to type 1 diabetes patients is accompanied by improved renal function, amelioration of autonomic dysfunction, stimulated tissue glucose utilization and augmented skin and muscle blood flow. The cellular mechanisms underlying these effects may be related to C-peptides capacity to stimulate both Na + , K + -ATPase and endothelial nitric oxide synthase (eNOS). The possibility that C-peptide may be beneficial in the long term treatment of patients with type 1 diabetes should be considered.

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.

Role of C-Peptide in the Regulation of Microvascular Blood Flow

During the recent years, the role of C-peptide, released from the pancreatic beta cell, in regulating microvascular blood flow, has received increasing attention. In type 1 diabetic patients, intravenous application of C-peptide in physiological concentrations was shown to increase microvascular blood flow, and to improve microvascular endothelial function and the endothelial release of NO. C-peptide was shown to impact microvascular blood flow by several interactive pathways, like stimulating Na+K+ATPase or the endothelial release of NO. There is increasing evidence, that in patients with declining beta cell function, the lack of C-peptide secretion might play a putative role in the development of microvascular blood flow abnormalities, which go beyond the effects of declining insulin secretion or increased blood glucose levels.

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) β. Several studies have shown that specific inhibition of the PKC isoform β 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 β- 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 β-specific concentration) and 400 nM (PKC β- 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 β activation. Ruboxistaurin is able to suppress this monocyte adhesion even in a PKC β-specific concentration. Further studies should evaluate these potential effects of ruboxistaurin in vivo.