No abstract

Circulating leukocyte-platelet heterophilic aggregates produce procoagulant, oxidative and mitogenic substances, and can cause microembolism in capillaries as well as acute arterial thrombosis. Our aim was to determine if there was any difference in the number of circulating heterophilic aggregates between diabetic patients and controls, if the formation of aggregates correlated with the actual HgbA1c level, duration of diabetes and postprandial rise in serum glucose level, with different vascular complications and whether decreasing postprandial serum glucose had any effect on heterophilic aggregate formation. The number of circulating heterophilic aggregates was measured in 90 diabetic patients (Type 1, 29; Type 2, 61) and in 23 control subjects by a flow-cytometric assay, and the result was given as percentage of the respective leukocyte subsets. There was no significant difference in lymphocyte-platelet and neutrophil-platelet aggregate number in patients and controls; however, there was a significant difference in the percentage of monocyte-platelet aggregates between the diabetic and control group (Type 1, 43.0 +/- 17.8; Type 2, 34.9 +/- 12.5; control, 24.6 +/- 8.2; P < 0.01 and P < 0.5, respectively). Patients with proliferative retinopathy and nephropathy showed the highest number of monocyte-platelet aggregates. No significant correlation was, however, found with HgbA1c. In Type 2 diabetes a non-significant, but remarkable, tendency between elevation of postprandial serum glucose levels and platelet-monocyte aggregate formation was observed and acarbose seemed to be effective in decreasing both. This study provides further support that heterophilic aggregates might have role in the pathogenesis of diabetic vascular complications.

Isothermal titration calorimetry (ITC) has an increasing significance in enzyme kinetic studies owing to its general applicability and sensitivity. In the present work, we aimed at developing a simple ITC-based screening procedure for the measurement of human salivary a-amylase (HSA) activity. Reaction of two substrates was studied with three independent methods (ITC, HPLC and spectrophotometry). ITC experiments were made using free and chromophore-containing maltooligomers of different length as substrates. Detailed studies revealed that maltoheptaose or longer oligomers could model properly starch and the presence of aromatic chromophore group did not affect the K M values considerably. It is the first time, when ITC was used to investigate of HSA-catalysed hydrolysis of different substrates (2-chloro-4-nitrophenyl-4-O-a-Dgalactopyranosyl-maltoside, maltoheptaose and starch) in the presence of acarbose inhibitor. All measured IC 50 values are in micromolar range (0.9, 18.6 and 29.0 mM, respectively) and increased in parallel with the degree of polymerisation of substrates.

Glycogen phosphorylase enzymes (GP) catalyse reversible reactions; the glucose transfer from glycogen to inorganic phosphate (Pi, phosphorolysis) or the reverse glucose transfer from glucose-1-phosphate (G-1-P) to glycogen (synthesis). Rabbit muscle GPb (rmGPb) was used as a model enzyme to study the reversible enzyme reaction. To follow both directions of this reversible reaction, we have developed a novel isothermal titration calorimetry (ITC) method for the determination of the direct reaction rate. The preference of forward or reverse reaction was ensured by the 0.1 or 10 concentration ratios of G-1-P/Pi, respectively. Substrate specificity was studied using different maltooligosaccharides and glycogen. Based on the KM values, glycogen and 2-chloro-4-nitrophenyl maltoheptaoside (CNP-G7) were found to be analogous substrates, which allowed to optimize the method by taking advantage of the CNP chromophore being detectable in HPLC. In case of CNP-G7, substrate inhibition was observed and characterised by Ki of 23± 7 mM. Inhibition of human GP is a promising strategy for the treatment of diabetes.Our ITC measurements have confirmed that caffeine and glucopyranosylidene-spirothiohydantoin (GTH), as known GPb inhibitors, inhibit the rmGPb-catalysed reversible reaction in both directions. Ki values obtained in the direction of synthesis (1.92±0.14 mM for caffeine and 11.5± 2.0 μM for GTH) have been shown to be in good agreement with the Ki values obtained in the direction of phosphorolysis (4.05±0.26 mM for caffeine and 13.8±1.6 μM for GTH). The higher difference between the inhibition constants of caffeine was explained by the non-competitive mechanism. The described ITC method using the developed experimental design and reaction conditions is suitable for activity measurements of different phosphorylase enzymes on various substrates and is applicable for inhibition studies as well.

Fenugreek is a common herb possessing several bioactive components including diosgenin. Here, dietary fenugreek seed flour and diosgenin were evaluated on a model of endothelium-dependent vasorelaxation by abdominal aortas isolated from rats receiving high-fat, high-sugar diet (HFHSD). 60 male Wistar rats were randomized into six groups: (i) negative control getting conventional rat feed regimen; (ii) positive control receiving HFHSD; (iii) a test group fed 2 g/kg bw/day fenugreek seed flour (containing 10 mg/kg bw/day diosgenin) + HFHSD; (iv) three test groups fed 1, 10 and 50 mg/kg bw/day diosgenin + HFHSD. Alimentary treatments were carried out for six weeks. The abdominal aortas were isolated, and 2 mm wide rings were sectioned off and mounted at a resting tension of 10 mN in organ baths containing Krebs solution (36 °C) exposed to 95% O2 and 5% CO2. After 60-min incubation, a norepinephrine concentration-response (E/c) curve was generated to determine their half-maximal effective concentration (EC50) value. After 60-min wash-out, a pre-contraction with norepinephrine EC50 was made, followed by an acetylcholine E/c curve. Plasma glutathione levels, glutathione-handling enzyme activities and blood antioxidant capacities were also determined. HFHSD significantly decreased the dilatory response to acetylcholine and increased plasma glutathione levels and these effects were significantly reversed by fenugreek seed flour, 10 and 50 mg/kg bw/day diosgenin. Both fenugreek and diosgenin treatments prevent HFHSD-induced endothelial dysfunction and redox changes. As fenugreek treatment was more effective at lower acetylcholine concentrations than diosgenin treatments, components of fenugreek other than diosgenin may contribute to the beneficial effects of dietary fenugreek seed flour.

In developed, developing and low-income countries alike, type 2 diabetes mellitus (T2DM) is one of the most common chronic diseases, the severity of which is substantially a consequence of multiple organ complications that occur due to long-term progression of the disease before diagnosis and treatment. Despite enormous investment into the characterization of the disease, its long-term management remains problematic, with those afflicted enduring significant degradation in quality-of-life. Current research efforts into the etiology and pathogenesis of T2DM, are focused on defining aberrations in cellular physiology that result in development of insulin resistance and strategies for increasing insulin sensitivity, along with downstream effects on T2DM pathogenesis. Ongoing use of plant-derived naturally occurring materials to delay the onset of the disease or alleviate symptoms is viewed by clinicians as particularly desirable due to well-established efficacy and minimal toxicity of such preparations, along with generally lower per-patient costs, in comparison to many modern pharmaceuticals. A particularly attractive candidate in this respect, is fenugreek, a plant that has been used as a flavouring in human diet through recorded history. The present study assessed the insulin-sensitizing effect of fenugreek seeds in a cohort of human volunteers, and tested a hypothesis that melanin-concentrating hormone (MCH) acts as a critical determinant of this effect. A test of the hypothesis was undertaken using a hyperinsulinemic euglycemic glucose clamp approach to assess insulin sensitivity in response to oral administration of a fenugreek seed preparation to healthy subjects. Outcomes of these evaluations demonstrated significant improvement in glucose tolerance, especially in patients with impaired glucose responses. Outcome data further suggested that fenugreek seed intake-mediated improvement in insulin sensitivity correlated with reduction in MCH levels.

Introduction. Mannose-binding lectin (MBL) activates complement system and has been suggested to play a role in vascular complications in diabetics. Carotid intima-media thickness (cIMT) detects subclinical atherosclerosis. We evaluated the association of MBL and IMT in type 2 diabetic (T2DM) patients. Methods. Serum MBL levels and cIMT were measured in a total of 103 diabetics and in 98 age-matched healthy controls. Results. There was no significant difference in MBL level in T2DM versus controls. As expected, IMT was significantly higher in T2DM patients than in controls (P = 0.001). In T2DM, the lowest cIMT was seen in patients with normal MBL level (500–1000) while cIMT continuously increased with both high MBL and absolute MBL deficiency states. This was especially significant in high MBL versus normal MBL T2DM patients (P = 0.002). According to multiple regression analysis the main predictors of IMT in T2DM are age (P < 0.003), ApoA level (P = 0.023), and the MBL (P = 0.036). Conclusions. Our results suggest a dual role of MBL as a risk factor for cIMT in T2DM. MBL may also be used as a marker of macrovascular disease, as both low and high levels indicate the susceptibility for atherosclerosis in T2DM.

The versatile biological activity of gallotannins has been investigated for a long time, including their use as α‐amylase inhibitors for the treatment of diabetes and its complications. The effectiveness of gallotannins on a wide range of enzymes refers to promiscuity. We proved that gallotannins are non‐specific promiscuous α‐amylase inhibitors, which exert their effect through their aggregates. A gallotannin of Aleppo oak origin fulfilled all the criteria for aggregators; significant changes could be observed in the IC50 values in the presence of Triton™ X‐100 detergent (from 2.3 to 110 μg/ml) and after enzyme–inhibitor preincubation (from 2.3 to 0.65 μg/ml). Increasing the enzyme concentration also led to the moderation of the inhibition by gallotannin. In addition, we observed that gallotannin molecules are those, which are involved in aggregation, and discrete protein molecules are adsorbed to the aggregates. This was revealed by the increasing particle size of gallotannin, which became three orders of magnitude higher after 150 min, whereas the size of α‐amylase remained unchanged. Consequently, gallotannins should be used as anti‐diabetic drugs only if the necessity of higher dose due to their promiscuity is taken into account. Aggregation propensity should not be ignored in case of in vivo applications.