Effect of insulin on human erythrocyte membrane fluidity in diabetes mellitus

Abstract

Summary. The effect of insulin in vitro on the fluidity of the human erythrocyte membrane in Type I (insulin-dependent) diabetic patients and healthy control subjects was investigated using a fluorescence technique. It was found that the addition of 10 -9 mol/1 porcine insulin significantly increased fluorescent probe lateral mobility in the membrane lipid layer but did not appear to produce any conformational changes of membrane proteins.Key words: Insulin, erythrocyte membrane, lateral mobility,Interest in hormone interactions with cell membranes has increased considerably during recent years. One hypothesis postulates that changes of membrane lipid structure and microviscosity may play the role of a hormonal information transducer [1].The existence of insulin receptors in erythrocyte membranes has been observed recently [2,3] although their physiological significance requires elucidation [4]. Altered rheological properties of erythrocytes in diabetes have been reported [5][6][7][8][9]. These alterations are related to chariged erythrocyte membrane viscosity and were confirmed by decreased rotational mobility of a fluorescent probe [10] and decreased lateral mobility of pyrene in erythrocyte membranes from insulin-dependent diabetics. Because changes in membrane lipid fluidity are considered to be responsible for information transfer through the membrane, studies were undertaken to investigate the effect of insulin in vitro on the fluidity of erythrocyte membranes in diabetes. Materials and MethodsThe method used to investigate the lateral diffusion of phospholipid molecules in the membrane influenced by insulin was an optical method using the fluorescent label pyrene, which is incorporated deep in the hydrophobic membrane region [11,12]. A measure of the lateral mobility of the probe is the formation rate of excimers during the collision of an excited pyrene molecule and a molecule in the ground state. The excimer fluorescence maximum (470 nm) is well separated from the monomer emission band (396 nm) on the fluorescence spectrum and the intensity ratio I470/I396 of these two peaks is a measure of the lateral mobility of the probe in the membrane (Fig. 1). The wavelength of fluorescence excitation was 339 nm.Blood was taken from 13 Type I (insulin-dependent) diabetic patients all of whom had retinopathy and neuropathy. The mean age of patients was 39.5 + 2.6 years, age of onset of diabetes 24.2 + 2.0 years and insulin dosage 0.88 + 0.12 IU/kg body weight.Blood was taken into 3% sodium citrate after an overnight fast and before the morning insulin and centrifuged at 3000rev/min for 10 rain. After removing plasma and the leucocyte layer, erythrocytes were washed three times with phosphate-buffered saline (PBS: 9 g/1 NaC1 in 10 mmol/1 phosphate buffer, pH 7.4).Erythrocyte membranes were obtained by the method of hypotonic haemolysis [13] and suspended in PBS to give a protein content of 100gg/ml. Pyrene was dissolved in methanol (2.10-3mol/1) and added to the membrane samples at a final probe concentration of 10 ...

Correction by insulin added in vitro of abnormal membrane fluidity of the erythrocytes from Type 1 (insulin-dependent) diabetic patients

“…Plasma membrane of hepatocytes from streptozocin-diabetic rats have been shown to have low fluorescence polarization values, suggesting an increase in membrane fluidity with the DPH probe [21]. Bryszewska and Leyko [9] demonstrated increased membrane viscosity by using a different technique, measuring the lateral mobility of a lipophilic probe (pyrene). Bryszewska et al [22] found a higher membrane cholesterol/phospholipid ratio.…”

“…Modifications in the erythrocyte membrane could be the cause of these theological abnormalities. Biochemical abnormalities have been described [5][6][7] in the membrane, and change in its viscosity have been demonstrated by the fluorescence polarization technique [7][8][9] or electron spin resonance [10]. However, these results have been challenged [11].…”

Non-enzymatic glycosylation and the chronic complications of diabetes: an overview

“…It is clear that various physical properties of the erythrocyte membrane, such as deformability [24], doublet formation [25] and microviscosity [26] may be altered in poorly controlled diabetes. There is, however, no evidence which directly links nonenzymatic glycosylation with these abnormalities, and other factors such as pH, changes in the lipid composition of the membrane [27], lipid-protein interactions [28] or a direct insulin effect [29] may be important.…”

Long-Term Pharmacologic Doses of Vitamin E Only Moderately Affect the Erythrocytes of Patients with Type 1 Diabetes Mellitus

“…The relative increase of polyunsaturated acids (PUFA; mainly 20:4 and/or 22:6) found by the three groups mentioned seems in contradiction to others (11,12). In T1DM, the unifying mechanism behind all of these changes has been postulated to be the deficiency in insulin because it has been observed that insulin treatment, which is known to improve membrane fluidity (4,5) and blood viscosity (13), also increases conversion of dietary (n-6) fatty acids (mainly linoleic acid, 18:2) to PUFA (mainly arachidonic acid, 20:4) (14).…”

“…The increased blood viscosity seen in diabetes (1) and more so in patients with established complications (2) has been ascribed to a decrease in erythrocyte deformability (3) and to changes in erythrocyte membrane fluidity (4,5). The extent to which these changes are due solely to alterations in the lipid composition of the erythrocyte membrane is still controversial.…”