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Background: Na+,K(+)-ATPase activity was evaluated in relation to membrane composition and molecular organization in erythrocyte membranes from obese patients by the amphyphylic molecule 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan). Its possible relationship with fat distribution and hyperinsulinaemia was also investigated., Design: Subjects were 10 obese men (OM), 12 women with subcutaneous obesity (FSO), 10 women with abdominal obesity (FAO) and 41 healthy lean subjects, 26 women (FC) and 15 men (MC). An oral glucose tolerance test was administered to all subjects to evaluate insulin secretion and glucose tolerance., Results: Na+,K(+)-ATPase activity was increased in all obese patients. Values were higher in FSO and FAO than in FC (with FAO greater than FSO) and in OM than in MC. The erythrocyte membrane cholesterol-to-phospholipid ratio was increased in obese patients and was significantly different in FSO patients compared with FC. The erythrocyte membrane protein-to-phospholipid ratio was also increased in all obese subjects, reaching statistical significance only in FSO vs. FC. The liquid crystalline phase, as tested by Laurdan generalized polarization (GP), was decreased in obese patients, indicating the presence of greater molecular environmental order; all patients groups showed lower GP values than control subjects, but only FAO reached statistical significance compared with FC. There was no evident correlation between membrane Na+,K(+)-ATPase activity and insulin levels, nor did membrane composition and properties show any evident relationship with insulin levels., Conclusion: Both increased Na+,K(+)-ATPase activity and altered fluidity and lipid composition were observed in the erythrocyte membrane of all obese patients. These findings are in line with previous observations by our group and indicate that the changes in Na+,K(+)-ATPase activity observed in obese patients could be related to changes in plasma membrane organization and composition.

Na+/K(+)- and Ca(2+)-ATPase are the major ATP-dependent membrane-bound enzymes that regulate the cation transmembrane gradient which is altered both in red blood cell (RBC) senescence and in RBCs of diabetic patients. In an attempt to clarify the possible connection between diabetes mellitus and ageing, we investigated the relationship between RBC ATP content, Na+/K(+)-ATPase, Ca(2+)-ATPase activities and ageing in healthy, insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) subjects. A significant correlation was found (r = -0.82; P < 0.001) between RBC ATP content and subject's age only in the control group. A significant reduction in Na+/K(+)-ATPase activity was observed in the older group (C2) of control subjects, in comparison with the younger (C1) one. In both IDDM and NIDDM subjects, the enzymatic activity was significantly decreased when compared with health subjects of similar age (P < 0.001). A significant negative correlation was found between age and enzymatic activity in healthy subjects (r = -0.60; P < 0.001). No difference was observed in the RBC membrane Ca(2+)-ATPase activity between younger (C1) and older (C2) healthy subjects. Ca(2+)-ATPase activity was significantly increased both in IDDM patients compared with C1 (P < 0.001) and in NIDDM patients compared with C2 (P < 0.001). The present data indicate that ageing causes a reduction in the erythrocyte ATP content in both healthy and diabetic subjects. In diabetic patients Na+/K(+)-ATPase activity decreases independently of age.

Plasma membrane lipid dynamics and cellular morphology were evaluated in endothelial cells obtained from umbilical cords of five women affected by insulin-dependent diabetes mellitus (IDDM) and six healthy pregnant women of similar age and gestational age. Endothelial cells were prepared by an adaptation of the method of Jaffe et al. Membrane fluidity was studied by means of the steady-state fluorescence anisotropy (r) of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), a fluorescent probe specifically anchoring at the membrane surface. Fluid phase endocytosis was evaluated by the measurement of the changes in fluorescence intensity of TMA-DPH at various times, owing to the internalization of the fluorescent marker in endocytic vesicles. The morphological and morphometric studies were performed by means of transmission electron microscopy (TEM). Endothelial cells obtained from IDDM women showed: (a) increased fluidity of the superficial region of the plasma membrane; (b) a more active fluid phase endocytosis compared with cells from healthy women; (c) increase in mitochondrial area, Weibel-Palade bodies and rough reticulum with wide cisternae. No statistically significant correlation was found between metabolic control and membrane fluidity and endocytosis. All the observed modifications suggest the presence of endothelial cell activation with membrane reshaping during IDDM. These alterations might play a central role in the pathophysiology of atherosclerosis and microangiopathy associated with diabetes mellitus.