diphenylhexatriene and Diabetes-Mellitus--Type-1

The involvement of platelets in the pathogenesis of diabetic vascular complications is supported by several studies. Type 1 diabetic (T1D) platelets show increased adhesiveness and aggregation related to a modification of nitric oxide synthase activity. Moreover, different cell types from diabetic patients showed a decreased membrane Na(+) /K(+) -ATPase activity, which might be involved in diabetic complications. The aim of this study was to investigate whether T1D at onset is able to induce alterations of platelet physicochemical and functional properties and whether these changes are affected by hyperglycaemia.. The study was performed on 50 young subjects: 30 patients (1-14 years) affected by T1D and 20 age- and gender-matched healthy subjects. We analyzed platelet membrane fluidity by fluorescent anisotropy of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene and 1,6-diphenyl-1,3,5-hexatriene, Na(+) /K(+) -ATPase, nitric oxide, and peroxynitrite production.. In T1D subjects, we observed an increased fluidity in the plasma membrane outer part and greater rigidity in the internal part compared with that in controls. Na(+) /K(+) -ATPase activity and nitric oxide levels were significantly reduced, while peroxynitrite production was increased compared with that in controls. Moreover, correlations found between the above parameters were correlated with fasting glycaemia and haemoglobin A(1c).. T1D patients exhibit structural and functional modifications of platelet membrane properties and alterations of nitric oxide metabolism due to diabetes per se and not to chronic hyperglycaemia, insulin therapy, or ageing. These results support the hypothesis that oxidative attack could be an important early event in the pathogenesis of diabetic complications.

Topics: Adolescent; Blood Platelets; Cardiovascular Diseases; Child; Child, Preschool; Diabetes Mellitus, Type 1; Diphenylhexatriene; Female; Fluorescence Polarization; Humans; Infant; Male; Membrane Fluidity; Nitric Oxide; Peroxynitrous Acid; Risk; Sodium-Potassium-Exchanging ATPase

The aim of the present study was to evaluate the action of plasma from insulin-dependent diabetic (IDDM) pregnant women on nitric oxide synthase (NOS) activity in cultured human umbilical vein endothelial cells (HUVECs). We also studied the effect of the plasma on cytosolic calcium and on Na+/K+-adenosine triphosphatase (ATPase) activity. Dynamic fluorescence studies of membrane fluidity were contemporarily performed to detect a direct effect of plasma on the endothelial cell membrane. We observed a significant increase in NOS activity, intracellular calcium, and Na+/K+-ATPase activity in cultured HUVECs exposed to IDDM plasma. Our dynamic fluorescence study showed a different microenvironmental organization of the cellular membrane after incubation with plasma from IDDM pregnant women, with a marked decrease in microheterogeneity as evaluated in terms of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) lifetime distribution width. The present investigation suggests that plasma from IDDM pregnant women can cause a generalized disturbance in the function of endothelial cells cultured from healthy subjects. Such a modification might play a central role in the pathogenesis of the vascular complications of the disease.

Topics: Adult; Blood Physiological Phenomena; Calcium; Cell Membrane; Cells, Cultured; Cytosol; Diabetes Mellitus, Type 1; Diphenylhexatriene; Endothelium, Vascular; Female; Fluorescent Dyes; Fluorometry; Humans; Membrane Fluidity; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Pregnancy; Pregnancy in Diabetics; Sodium-Potassium-Exchanging ATPase; Umbilical Veins

The aim of the present work was to analyze the effect of LDL obtained from type 1 diabetic patients in good metabolic control on human umbilical vein endothelial cells (HUVECs) after a short incubation period to detect possible atherogenic modifications of endothelial properties. Cultured HUVECs were incubated for 3 h with culture medium alone (control HUVEC), with native LDL from 12 healthy men (control LDL), or with native LDL from 12 type 1 diabetic men (type 1 LDL) (100 pg/ml). After the incubation, the following parameters were evaluated: nitric oxide synthase (NOS) activity, cytoplasmic Ca2+ levels, Na+-K+-ATPase activity, plasma membrane fluidity determined by means of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), and plasma membrane conjugated diene (CD) content. The same experiments were repeated after bradykinin stimulation or in the presence of the antioxidant butylated hydroxytoluene (BHT), and nitric oxide (NO) production in intact HUVECs was also evaluated. HUVECs incubated with control LDL in comparison with control HUVECs showed a decreased fluidity of the membrane surface evaluated by TMA-DPH and a higher CD content. These alterations were prevented by the presence of BHT. HUVECs incubated with type 1 LDL in comparison with both control HUVECs and cells incubated with control LDL showed 1) increased NOS and Na+-K+-ATPase activity, cytoplasmic Ca2+ levels, and CD content, and 2) decreased fluidity of the membrane surface evaluated by TMA-DPH. These modifications were blunted--but not abolished--by the presence of BHT. After bradykinin stimulation either in the absence or in the presence of BHT, both cytoplasmic Ca2+ levels and NO production were increased in control HUVECs and in HUVECs incubated with control LDL, while a reduced response was observed in HUVECs incubated with type 1 LDL. The alterations observed in the endothelial function after the cell-LDL interaction might play a central role in the atherogenic process in diabetes.

Topics: Adult; Calcium; Cell Membrane; Cholesterol; Cholesterol, HDL; Cholesterol, LDL; Diabetes Mellitus, Type 1; Diphenylhexatriene; Endothelium, Vascular; Fluorescent Dyes; Humans; Lipoproteins, LDL; Male; Membrane Fluidity; Nitric Oxide Synthase; Phospholipids; Reference Values; Sodium-Potassium-Exchanging ATPase; Triglycerides; Umbilical Veins

An abnormality of the physical properties of the cell membrane may underlie the defect that unites the clinical and biochemical abnormalities found in subjects with diabetic nephropathy. The cell membrane is linked both structurally and functionally with the cytoskeleton. The fluorescence anisotropy, a measure of membrane fluidity, was studied at baseline and after modulation of cytoskeletal proteins by thiol group alkylation with N-ethylmaleimide (NEM). 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to assess anisotropy in the deep hydrophobic regions of the lipid bilayer and trimethylammonium-diphenylhexatriene (TMA-DPH) was used to assess the superficial, relatively hydrophilic regions. We compared 17 subjects with insulin-dependent diabetes mellitus (IDDM) and nephropathy with 17 control subjects with IDDM and 24 non-diabetic control subjects. Median TMA-DPH anisotropy (0.271 (0.239-0.332) vs 0.269 (0.258-0.281) vs 0.275 (0.246-0.287)) and DPH anisotropy (0.221 (0.193-0.261) vs 0.227 (0.197-0.253) vs 0.226 (0.193-0.245)) were similar in erythrocytes from the three groups. However after alkylation of protein thiol groups with NEM clear differences emerged. In the control subjects with and without IDDM there was a significant fall in TMA-DPH anisotropy compared to the subjects with diabetic nephropathy in whom the addition of NEM had no effect (deltaTMA-DPH anisotropy -0.005 (-0.020 - +0.006) vs -0.005 (-0.011 - +0.016) vs +0.002 (-0.010 - +0.008) p < 0.001). This finding was confirmed when the deep regions of the lipid bilayer were assessed using DPH (deltaDPH anisotropy -0.017 (-0.029 - -0.007.) vs -0.015 (-0.029 - +0.001) vs + 0.003 (-0.021 - +0.018) p < 0.001). We conclude that cytoskeletal modulation of the physical properties of the cell membrane lipids by proteins is abnormal in subjects with diabetic nephropathy. Such an abnormality could explain some of the clinical and metabolic abnormalities found in this condition.

Topics: Adult; Angiotensin-Converting Enzyme Inhibitors; Case-Control Studies; Creatinine; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Diphenylhexatriene; Erythrocyte Membrane; Ethylmaleimide; Female; Fluorescence Polarization; Fluorescent Dyes; Glycated Hemoglobin; Humans; Male; Membrane Fluidity; Middle Aged; Sulfhydryl Reagents; Triglycerides

The causes of the reduced activity of Na+/K+-adenosine triphosphatase (ATPase) in human diabetes are still the object of controversy. The aim of this work was to investigate the mechanisms of inhibition by means of the study of the Na+/K+-ATPase purified from human placenta. We purified Na+/K+-ATPase from term placentas of six healthy women and six age-matched women with insulin-dependent diabetes mellitus (IDDM) in good metabolic control. The enzymatic activity was reduced in both the microsomal fraction and the purified Na+/K+-ATPase obtained from diabetic women, whereas no difference was found in the number of active molecules determined by anthroyl ouabain binding. The Na+/K+-ATPase purified from women with IDDM did not show any modification in the ouabain affinity or changes in the physicochemical structure of the ouabain binding site investigated by dynamic fluorescence or alterations in lateral diffusion. The activation energy of the enzyme was increased, whereas the tryptophan accessibility of the enzyme was lower in women with IDDM. The fluidity of the lipid anulus of the enzyme was higher in women with IDDM than in control women, as suggested by fluorescence polarization of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene. The adenosine triphosphate-binding site, investigated by anisotropy decay studies of the fluorescent probe pyrene isothiocyanate, was modified in women with IDDM. It appears that the Na+/K+-ATPase of human placenta is altered in its disposition in IDDM.

Topics: Acrylamide; Acrylamides; Adult; Anthracenes; Binding Sites; Diabetes Mellitus, Type 1; Diffusion; Diphenylhexatriene; Enzyme Activation; Female; Fluorescence Polarization; Fluorescent Dyes; Humans; Isothiocyanates; Kinetics; Membrane Fluidity; Microsomes; Ouabain; Placenta; Pregnancy; Pregnancy in Diabetics; Sodium-Potassium-Exchanging ATPase; Spectrometry, Fluorescence; Tryptophan

A modified platelet response to aggregating stimuli is supposed to play a role in the pathogenesis of diabetic macroangiopathy. We studied the fluidity and microheterogeneity of the external surface of the platelet membrane and the activities of the plasma membrane Na+-K+-ATPase and Ca2+-ATPase in 21 men with type 1 diabetes and in 20 control subjects before and after in vitro thrombin addition. In the resting state, platelets from type 1 diabetic patients showed an increased fluidity and microheterogeneity of the platelet membrane, a higher Ca2+-ATPase activity, and a reduced Na+-K+-ATPase activity in comparison with platelets from healthy subjects. The fatty acid composition was also modified, with increased C 16:1 and decreased C 18:0 content. Control cells incubated with thrombin showed a modification of the membrane parameters opposite to the response observed in type 1 cells after the stimulation. The incubation of control platelets in the resting state with high concentrations of glucose modified the fluidity of the plasma membrane Na+-K+-ATPase and Ca2+-ATPase activities in an opposite way in comparison with the alterations observed in type 1 platelets. This study suggests that in type 1 diabetic patients, the platelet membrane responds to activation with a molecular remodeling different from the response of healthy subjects. The abnormal organization of the membrane might contribute to the altered platelet functions in type 1 diabetic patients, but acute exposure to high glucose levels does not seem able to modify the platelet membrane in the way observed in type 1 diabetes.

Topics: Adult; Blood Platelets; Calcium-Transporting ATPases; Cell Membrane; Diabetes Mellitus, Type 1; Diphenylhexatriene; Fatty Acids; Fluorescence Polarization; Fluorescent Dyes; Glucose; Humans; Male; Membrane Fluidity; Sodium-Potassium-Exchanging ATPase

The interaction between lipoproteins and the platelet membrane has been proved to cause a modification in cellular functions. We studied 12 men with insulin-dependent diabetes mellitus (IDDM), 14 men with noninsulin-dependent diabetes mellitus (NIDDM), and 26 age-matched healthy men on the same diet. We determined fluidity by measuring the fluorescence polarization (P) of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) both in platelet membranes and in lipoproteins isolated by ultracentrifugation in NaBr density gradient. The lipid composition of lipoproteins and of platelet membranes was determined by enzymatic methods. The fluidity of platelet membranes was significantly increased both in patients affected by NIDDM and in subjects with IDDM compared with normal subjects. Low-density lipoproteins (LDL) showed an increased fluidity only in NIDDM patients. A percent increase in the triglyceride content was observed in all lipoprotein fractions in diabetic subjects. Increased phospholipid content was found in the platelet membranes from IDDM and NIDDM patients. The change in LDL fluidity observed in NIDDM patients might determine altered interactions between the lipoprotein and cellular receptors. The role of lipoproteins in the modulation of the platelet membrane properties in diabetes mellitus deserves further studies.

Topics: Adult; Blood Platelets; Cell Membrane; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diphenylhexatriene; Fluorescence Polarization; Humans; Lipids; Lipoproteins; Male; Membrane Fluidity; Membrane Lipids

Sodium-lithium countertransport (SLC) activity at a standard physiological sodium concentration is raised in uncomplicated IDDM, for which the kinetic mechanism is a raised maximum velocity (Vmax). Diabetic patients with nephropathy do not have raised values for Vmax but a low Michaelis constant (km). Transporter activity could be influenced by its membrane lipid environment. This was assessed in 21 control subjects, 32 uncomplicated diabetic patients, 17 patients with diabetic nephropathy and 11 patients with non-diabetic nephropathy by measuring the fluorescence anisotropy of DPH and TMA-DPH to assess different membrane regions. Standard SLC was higher in all the patient groups compared to the control subjects: 0.307 +/- 0.020 mmol Li/h x 1 cells in uncomplicated IDDM; 0.300 +/- 0.032 in diabetic nephropathy patients and 0.276 +/- 0.019 in non-diabetic nephropathy patients vs 0.216 +/- 0.011 mmol Li/h x 1 cells in control subjects (p < 0.001, p < 0.05, p < 0.05, respectively). This was due to raised Vmax values in the uncomplicated group: 0.528 +/- 0.035 vs 0.385 +/- 0.022 mmol Li/h x 1 cells in control subjects (p = 0.001) and low values for km in the diabetic nephropathy group: 58 (27-170) vs 106 (81-161) mmol/l in control subjects (p < 0.001). Raised SLC in the non-diabetic nephropathy group was largely due to raised Vmax: 0.460 +/- 0.030 mmol Li/h x 1 cells; p = 0.053, with no difference in km: 99.5 (74-137).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Biological Transport; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Diphenylhexatriene; Erythrocyte Membrane; Female; Fluorescence Polarization; Fluorescent Dyes; Humans; Lithium; Male; Membrane Fluidity; Middle Aged; Sodium

Exposure of human erythrocytes to elevated temperatures induces a decrease in stability of the cell membrane. Thermally induced haemolysis of erythrocytes from patients with type 1 diabetes and from healthy control individuals was measured as a function of duration of exposure to heat between 48.0 and 54.0 degrees C. Results indicate that the thermosensitivity of erythrocytes from patients with type 1 diabetes is lower than for control individuals. Activation energies for lysis were similar for both control and 'diabetic' erythrocytes, being 298.3 and 287.7 kJ/mol, respectively. The steady-state fluorescence anisotropy measurement of TMA-DPH for each step of haemolysis was employed as a parameter characterizing membrane fluidity. We found that 'diabetic' erythrocyte membranes had significantly decreased fluidity. The relationship between fluidity and rate of haemolysis indicates that the rate-limiting step in the haemolysis reaction involves the rupturing of the membrane bilayer.

Topics: Diabetes Mellitus, Type 1; Diphenylhexatriene; Erythrocyte Membrane; Fluorescent Dyes; Hemolysis; Hot Temperature; Humans; In Vitro Techniques; Membrane Fluidity

The susceptibility to peroxidative stress of low density lipoprotein (LDL), induced by incubation with CuSO4, has been studied in eleven diabetic patients and eleven control subjects. Our results suggest that oxidized LDL (OX-LDL) of diabetic patients have a significant higher reactivity to 2,4,6-trinitrobenzene sulfonic acid (TNBS) than controls, that indicates a lower susceptibility of LDL to oxidative stress. Furthermore using the fluorescence polarization (Pf) of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its phosphatidylcholine derivative (DPH-PC) we have shown that peroxidation induces a decrease of fluidity in OX-LDL of controls and diabetic patients, both at the lipoprotein surface, where is localized DPH-PC and at the interface between lipoprotein surface and core which is probed by DPH.

Topics: Adult; Copper; Copper Sulfate; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diphenylhexatriene; Female; Fluorescence Polarization; Humans; Lipid Peroxidation; Lipids; Lipoproteins, LDL; Male; Middle Aged; Oxidation-Reduction; Phosphatidylcholines; Trinitrobenzenesulfonic Acid

The significance of the two most common hallmarks of the diabetic state, hyperglycaemia and hyperlipidaemia, was investigated in terms of disorders of cell membrane dynamics. In order to examine whether the alterations in cell membrane lipid bilayer dynamics are somehow related to protein chemical modifications in plasma low-(LDL) and high-density lipoproteins (HDL) and blood cell membranes, we compared 19 poorly controlled diabetic subjects with 19 age- and sex-matched controls. The extent of (non-enzymatic) glycation, lipid peroxidation and the cholesterol/phospholipid ratio were increased in plasma low density lipoproteins and high density lipoproteins from diabetic patients. The mean steady-state fluorescence polarization values in 1,6-diphenyl-1,3,5-hexatriene-labelled isolated erythrocyte membranes from diabetic subjects were significantly greater than from control subjects (0.186 +/- 0.008 vs 0.173 +/- 0.006, p < 0.001); the fluorescence polarization values in erythrocyte membranes from diabetic and control subjects positively correlated with the extent of membrane protein glycation, lipid peroxidation and the cholesterol content. The cholesterol to phospholipid molar ratios in low density lipoproteins and high-density lipoproteins from diabetic and control subjects correlated significantly with the fluorescence polarization values in erythrocyte membranes from these subjects. Furthermore, the extent of glycation of low density lipoproteins appears to be strongly correlated with the extent of lipoprotein lipid peroxidation (r = 0.789, p < 0.001). The atherosclerotic potential of plasma lipoproteins in diabetes mellitus was discussed in terms of membrane and plasma protein chemical modifications.

Topics: Adult; Apoproteins; Blood Glucose; Blood Proteins; Cholesterol; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diphenylhexatriene; Erythrocyte Membrane; Female; Fluorescence Polarization; Glycated Hemoglobin; Glycosylation; Humans; Lipid Peroxidation; Lipoproteins; Lipoproteins, HDL; Lipoproteins, LDL; Male; Membrane Fluidity; Membrane Proteins; Middle Aged; Phospholipids; Thiobarbituric Acid Reactive Substances

Fluorescence polarization of red blood cell (RBC) membranes evaluated using DPH was measured in patients with uncontrolled insulin-dependent diabetes mellitus and in controls. The effect of in vitro addition of pentoxifylline and propentofylline (10(-5) and 10(-4) M) was studied. The fluorescence polarization parameter (P) was lower in the diabetic patients (p = 0.20 +/- 0.03, n = 8) as compared to the controls (p = 0.28 +/- 0.02), n = 8), reflecting an increase in probe mobility in the hydrophobic environment in the depth of the double lipid layer. In vitro addition of pentoxifylline had no effect on the fluorescence parameter of RBC membranes from controls, whereas both concentrations of pentoxifylline studied (p = 0.24 +/- 0.03) significantly increased the fluorescence parameter of RBC membranes from diabetics. Propentofylline had no effect. These findings suggest that the active site responsible for improved membrane fluidity of RBC from diabetics is located on the methyl radical present in the pentoxifylline molecule but not in the propentofylline molecule.

Topics: Diabetes Mellitus, Type 1; Diphenylhexatriene; Erythrocyte Membrane; Fluorescence Polarization; Humans; Membrane Fluidity; Pentoxifylline; Theobromine; Xanthines

Diphenylhexatriene transverse distribution has been studied in normal and diabetic erythrocyte membrane ghosts using fluorescence polarization and fluorescence quenching methods. Acrylamide quenched the fluorescence of diphenylhexatriene according to a dynamic mechanism in agreement with Stern-Volmer equation. Nonlinear least-squares analysis based on quenching results has shown greater accessibility of fluorophore to quencher molecules in diabetic ghosts (37.2 +/- 3.2% in normal vs. 67.5 +/- 6.4% in diabetic membranes). Steady-state fluorescence anisotropy measurements evidenced the lowered membrane lipid fluidity in diabetics (anisotropy values: 0.166 +/- 0.011 in normal subjects vs. 0.193 +/- 0.018 in diabetics). A model mechanism is proposed which attributes the lowered capacity of lipid bilayer in diabetes to the increased ordering and more compact structure of membrane phospholipids. The implications of the results for the resolving of steady-state anisotropy data are discussed.

Topics: Diabetes Mellitus, Type 1; Diphenylhexatriene; Erythrocyte Membrane; Fluorescence Polarization; Humans; Kinetics; Polyenes; Reference Values