Erythrocyte membrane integrity and protein activity in high-fat diet fed male Wistar rats

High-fat diet (HFD) has been reported to induce systemic inflammation and oxidative stress that may affect the structural integrity of erythrocytes and alter their ability to undergo deformation. This study was therefore designed to investigate the effect of HFD feeding on erythrocyte membrane integrity and protein activity in male Wistar rats. Ten animals (100–120 g) were grouped equally and exposed to either standard diet or HFD (25% fat) for 42 days. Thereafter, retro-orbital sinus blood was collected under anesthesia (thiopental), and aliquots were analyzed for erythrocyte sedimentation rate (ESR), osmotic fragility, and mean corpuscular fragility. Erythrocyte ghost membranes were also isolated from blood sample aliquots and analyzed for total protein concentration, malondialdehyde (MDA), Na+K+-ATPase activity, Ca2+Mg2+-ATPase activity, and intercellular adhesion molecule (ICAM)-4 level. Osmotic fragility and mean corpuscular fragility were significantly increased (P < 0.05) in the HFD-fed group compared to control. ESR (mm/h) (64.60 ± 2.34 vs. 21.20 ± 1.53), membrane MDA (µMol) (3.66 ± 0.86 vs. 0.43 ± 0.08), and ICAM-4 (ng/ml) (1.68 ± 0.23 vs. 0.49 ± 0.16) levels were also increased (P < 0.05) in the group 2 (HFD) compared to group 1 (standard diet). Compared to standard diet group, erythrocyte membrane total protein concentration (10.46 ± 0.96 vs 6.00 ± 0.38 g/dl) and Na+K+ATPase activity (1.37 ± 0.22 vs 0.22 ± 0.03 × 107 µmol pi/mg protein/h) was reduced (P < 0.05) in the HFD group, while Ca2+Mg2+-ATPase exhibited a 27.9% increase in activity. This study suggests that HFD may compromise the structural and functional integrity of erythrocytes by activating systemic inflammation, erythrocyte membrane, and protein oxidation as well as dysregulated membrane ATPase activity required to maintain erythrocyte deformability in male Wistar rat.


Introduction
Healthy diets are vital to nutrition and may be described as a necessity for good health and long life. Constant intake of unbalanced or skewed dietary regimens have been known to impact the body negatively resulting in the development of a variety of conditions such as hyperlipidemia, hyperinsulinemia, hematological alterations, and aging etc. [1][2][3]. High-fat diets (HFDs) have been associated with a number of disease conditions such as obesity, metabolic syndrome, vascular impairment, diabetes mellitus, cardiovascular disease, and cancer [4,5]. HFD intake has also been associated with the modifications of cell membrane lipids and proteins resulting in cellular dysfunction [6][7][8].
In erythrocytes, cellular dysfunction has been reported following diet-induced obesity and has been suggested to serve as a mediator of atherosclerosis [6]. High-fat diet feeding in rats and fish has also been shown to result in alterations in the phospholipid composition and cholesterol content of the erythrocyte membrane, which in turn may affect erythrocyte osmotic fragility [9] and erythrocyte deformability [10]. More so, alterations in erythrocyte fragility are associated with lipid peroxidation and changes in membrane fluidity, membrane lipid content, and protein conformation [7,8] which define deformability of the cell.
The ability of erythrocytes to change shape under stress without rupturing (erythrocyte deformability) is dependent on the membrane properties and structural interactions between the components of the membrane [11,12]. The integrity of the erythrocyte membrane and function is maintained by an intertwined relationship existing among the trans-membrane protein-rich layer, phospholipid bilayer, and a two-dimensional cytoskeleton having spectrin, actin, and protein 4.1 as principal components [11]. These structural components and interactions provide the erythrocyte with the flexibility, durability, and tensile strength to undergo large deformations in microcirculatory blood flow [13] and facilitate cell-signaling events [14]. Deformability of the erythrocyte is a response to fluid force which is regulated by the activities of ion pumps-Na-K-ATPase and Ca-Mg-ATPase [11][12][13]. Regulation of membrane fluidity is closely related to maintenance of intracellular Ca 2+ within normal limits, optimal intracellular concentrations of cations, and provision of energy for sodium-calcium ion exchange as well as for cotransport of other substances. The regulation of erythrocyte volume and water balance by Ca-Mg-ATPase and Na-K-ATPase also helps to maintain the surface area-to-volume ratio and cytoplasmic rheology that defines deformability [11].
Though HFD-induced lipid peroxidation and alteration in cytoskeletal protein and phospholipids have been reported in erythrocytes [6,[8][9][10], however, effects of HFD on the activity of membrane pumps that are essential for erythrocyte morphology and deformability have not been reported. Dysfunction of these pumps has been reported to affect cell morphological alterations and rigidity which may lead to osmotic fragility, phagocytosis, lysis fragmentation, and reduced lifespan. This study was therefore designed to investigate the effects of HFD feeding on erythrocyte integrity (osmotic fragility, sedimentation rate), membrane lipid peroxidation (malondialdehyde), pump activities (Ca 2+ Mg 2+ ATPase and Na + /K + ATPase activities), and cytoskeletal protein (intercellular adhesion molecule-4 (ICAM-4) level in male Wistar rats.

Animals and groupings
Ten male Wistar rats (110 ± 10 g; 8 weeks old; n = 5 per group) were housed in well-aerated plastic cages, maintained on alternating day and night cycles and allowed free access to food and water ad libitum. Animals received humane care, and all experimental protocols were in accordance with the guidelines of the University of Ibadan Animal Care and use in Research Ethical Committee (ACUREC-UI). The animals were divided into two equal groups and maintained on either standard diet or HFD for 42 days, respectively.

Tissue collection and osmotic fragility test
At the end of respective dietary exposure periods, retroorbital blood samples were collected from animals in each group under thiopentone (50 mg/kg) anesthesia into ethylenediaminetetraacetic acid (EDTA), heparinized and citrate buffer coated bottles. Heparinized blood samples were analyzed for erythrocyte osmotic fragility test within 1 h of collection as described by Turgeon [16]. Briefly, whole blood (0.02 ml) was added to 5 ml of varying concentrations (0%, 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%) of buffered sodium chloride (NaCl) (pH = 7.4). The mixture was allowed to stand for 30 min at room temperature and centrifuged at 1500 rpm for 10 min. The absorbance of the supernatant obtained was thereafter taken at 540 nm. Percentage hemolysis was expressed with reference to hemolysis in distilled water. The concentration of the buffered NaCl at which 50% of erythrocytes that lyzed was taken as the mean corpuscular fragility (MCF) and the ability of the diet to stabilize or destabilize the membrane was calculated using the following equation [17]: Citrated blood samples were evaluated for erythrocyte sedimentation rate (ESR) using the Westergren tube method [18,19]. Briefly, the citrated blood samples were aspirated into the Westergren tube up to mark 0 and placed in the rack at room temperature undisturbed. The erythrocyte sedimentation was determined within an hour as the rate at which cells sediment in millimeters in the tube with a column of serum at the top of the tube.

Isolation and analysis of erythrocyte ghost membranes
Red blood cell ghost membrane was prepared from blood sample collected in EDTA-lined sample bottles as described by Niggli et al. [20] which is based on the principle of hypotonic lysing developed by Dodge et al. [21] involving repeated cold centrifugation at low ionic strength. Erythrocytes were washed thrice in 0.1 M Tris-HCl buffer (pH 7.4), lysed in Tris-HCl buffer (15 nM, pH 7.4) for 1 h at 4 °C, and then vortexed. The RBC hemolysates were centrifuged at 18,000 rpm (4 °C) for 30 min to precipitate the erythrocyte membrane. The membrane pellet was then washed four times over centrifugation (1800 rpm, 4 °C, and 15 min) and then suspended in 0.1 M Tris-HCl buffer. The erythrocyte ghost membrane isolated was analyzed for total protein concentration [22], malondialdehyde (MDA) level [23], Na + -K + -ATPase [24], Ca 2+ -Mg 2+ ATPase [25] activity, and intercellular adhesion molecule (ICAM)-4 level [26], respectively.

Statistical analysis
The results obtained are expressed as mean ± SEM, and statistical differences between groups were determined using the two-tailed unpaired Student T-test and Mann Whitney post-hoc test with significance taken at P < 0.05.

Proximate analysis of standard and HFD formulations
The analysis of the diet showed that the formulated standard diet had a percent crude fat composition of 3.71% while that of the HFD was 25.72% (Table 1).
Values are mean ± SEM; n = 5. * P < 0.05 significantly different compared to standard diet. Std Diet, standard diet; HFD, high-fat diet

Effect of HFD on erythrocyte membrane protein concentration and ATPase activity
Total erythrocyte membrane protein (g/dl) was significantly (P < 0.05) reduced in the HFD-treated group (6.00 ± 0.38) compared to standard diet group (10.46 ± 0.96). Evaluation of ATPase activity showed significant reduction in Na + K + -ATPase (× 10 7 µmol pi/ mg protein/h) activity in the HFD-fed group (0. 22

Discussion
Erythrocytes primarily function to transport and distribute oxygen to body cells and return carbon dioxide back to the lungs from the cells [27]. In achieving this, erythrocytes undergo membrane deformations in order to be able to pass through the microcirculation and deliver oxygen as well as needed nutrients to the cells [28]. Erythrocyte deformability has also been observed to influence oxygen delivery, acid-base balance functions, and the survival rate of erythrocyte in circulating plasma [11]. It also aids the exit of erythrocytes from the bone marrow, reduces bulk viscosity in larger vessels, and prevents phagocytic clearance of erythrocytes by macrophages [29]. Disruption in erythrocyte deformability contributes to alterations in microcirculatory blood flow that play a pivotal role in multiple organ failure and death arising from impairment in tissue oxygenation [30,31].   Table 3 Membrane total protein concentration, Na + -K + ATPase, and Ca ++ -Mg ++ ATPase activity in standard-and HFD-fed rats Values are mean ± SEM; n = 5 ** P < 0.01 significantly different compared to standard diet The deformability of the erythrocyte and its functions has been reported to be influenced by its plasma membrane compositional elements and their interactions [9,32]. Dietary conditions, especially high-fat diet, has been reported to affect the cholesterol content, fatty acid, and protein matrix of erythrocyte membrane resulting in alterations of its membrane cholesterol-phospholipid ratio [33,34], increased erythrocyte phagocytic susceptibility and lipid peroxidation [6], and reductions in erythrocyte membrane p55 and band 4.2 skeletal proteins [8] and alter erythrocyte shape and deformability index [35]. In this study, increased osmotic fragility and mean corpuscular fragility were observed in the HFD group. This suggests increased susceptibility of erythrocytes to undergo hemolysis in this treatment group, which is in accordance with the reports of Kalmath et al. [7] who in an in vitro study showed that HFD induces hypo-osmotic fragility in rat erythrocytes.
Several studies have indicated that high-fat consumption causes overproduction of circulating free fatty acids and systemic inflammation, which can result in cellular and molecular damage via oxidative stress [10,33,36]. This study also shows an increase in erythrocyte membrane lipid peroxidation and sedimentation rate suggesting not only increased systemic inflammatory conditions in the HFD group but also increased oxidative damage to erythrocyte membrane lipids and hence compromised cholesterol-phospholipids ratio and structural integrity of the cell. Oxidative stress has been implicated in the alteration of membrane integrity and fragility of erythrocytes resulting in a dysfunctional propensity for them to flow into microcirculatory vascular beds, adhere to endothelial monolayers, and hemolyze [37]. Furthermore, ICAM-4, an erythrocyte-specific intercellular adhesion molecule that binds to αVβ3 integrins on endothelial cells [26], was increased in the HFD group. This suggests increased susceptibility of these cells to bind to macrophages in the spleen and undergo hemolysis. High-fat diet feeding has been reported to increase circulating chemokine level, promote its binding to erythrocyte Duffy antigen receptor for chemokine (DARC) [6,38], and trigger macrophage attack of erythrocyte by increasing externalization of phosphatidylserine in erythrocyte membrane [6]. Hence, upregulation of cell adhesion molecule (ICAM-4) as observed in this study suggests an activation of pro-inflammatory responses in erythrocytes [5,39] which can increase its susceptibility to be phagocytized by macrophages.
Gas exchange, the primary function of erythrocytes, is a passive diffusional process that presents with no direct metabolic demand but requires a rheologically competent cell [40]. The disco-like shape of erythrocytes allows it to deform, fold, and squeeze against the endothelial walls of capillaries, thus exposing maximal surface area and offering minimal diffusional distances for rapid oxygen and carbon dioxide exchanges across the capillary walls.
For maximum efficiency, the optimal rheology of erythrocytes is maintained by its ability to regulate cell volume below the maximum spherical volume that can be accommodated by the membrane area of the cell [14]. The volume control in mature erythrocytes is depended on the presence of active and passive membrane transporter on their membranes. The ability of the erythrocytes to regulate its volume in association with its net cation content is also important to its function and lifespan. A large number of membrane proteins including Na + -K + ATPase and Ca 2+ -Mg 2+ ATPase are involved in cation homeostasis and water content regulation [11,14]. Na + -K + ATPase catalyzes the energy-dependent transport of Na + and K + across membrane thus regulating cell volume and ionic uptake [11], and its activity can be modulated by membrane lipid content [41]. This study shows a reduction in the total protein content of the membrane in the HFD treatment group possibly as a result of protein oxidation [42], arising from high-fat diet-induced oxidative stress [6,36]. This reduction in membrane protein level may also account for the reduction in Na + -K + ATPase activity observed in this study, an observation that has been reported to decrease erythrocyte deformability and predispose to rheological induced cardiovascular disease conditions [43].
The membrane-bound Ca 2+ ATPase has been reported to maintain intracellular calcium level within narrow range compared to extracellular free Ca 2+ and regulate erythrocyte membrane fluidity [11], while Mg 2+ ATPase regulates the membrane aminophospholipid translocase activity which can affect Ca 2+ ATPase [44,45]. The results from this study show a slight increase in the activity of Ca 2+ Mg 2+ ATPase in the HFD treatment group, which suggest likely increases in erythrocyte intracellular Ca 2+ levels in this group. Increases in erythrocyte intracellular calcium levels have been associated with increased osmotic fragility [46] and hence may also contribute to the increased erythrocyte osmotic fragility observed in the HFD treatment group.
This study suggests that prolonged high-fat dietary intake may compromise the rheology and integrity of erythrocytes through increased systemic inflammation, cellular oxidative stress, membrane protein oxidation, and decreased activity of membrane proteins necessary for the maintenance of erythrocyte volume and stability in male Wistar rats.