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Hyperglycemia and Red Blood Cells: too sweet to survive

       Sustained untreated hyperglycemia is associated with complications at molecular, cellular, and organ levels in the body that ultimately lead to comorbidities including cardiovascular-related pathologies, neuropathies, nephropathies, blindness, limb amputations, etc. Mature RBCs are unique in their structure and function; being without cellular organelles including nucleus and mitochondria, they are highly sensitive and responsive to the molecular changes in their microenvironment in general and elevated glucose in particular. They lack the ability to synthesize new proteins, replenish its enzyme-based antioxidant machinery, and replace any cellular components in the event of oxidative damage. Although they are dependent on glycolytic processing of glucose for their energy requirements, sustained exposure to hyperglycemia significantly impacts their structure as well as function and leads to early aging of the circulating RBCs with shortened lifespan.


         Loss of deformability due to hyperglycemia prohibits them to reversibly change their shape and squeeze through the microvasculature, a hallmark of RBC functionality for nutrient and gaseous exchanges. This mini-review of literature signifies the effect of hyperglycemia on RBCs in terms of eryptosis, lipid peroxidation in the cell membrane to compromise membrane integrity which significantly alters its deformity and coaguability, and adherence to endothelial surface leading loss of functionality and life-span.


Keywords: Clinical; Diabetes; Erythrocyte, glycolysis, hyperglycemia; RBCs


Introduction

        The prevalence of diabetes is steadily increasing worldwide that can be apprised from the recently released updates from World Health Organization (WHO) which states that the number of people suffering from diabetes has quadrupled to an estimated 422 million adults in the world since the publication of the first report by WHO in 1980 [ 1]. The complications and comorbidities associated with diabetes include macroangiopathies, e.g., coronary artery disease, peripheral arterial disease, and cerebrovascular stroke, as well as microangiopathies, e.g., nephropathy, neuropathy, and retinopathy. Majority of these complications evolve from intricate cellular and molecular mechanisms which are altered either directly or indirectly, partially or fully in response to sustained exposure to uncontrolled hyperglycemia emanating from multitude of factors with metabolic disorders in the center [ 2]. The situation worsens further with advancing age [ 3].


       Given that different cell types diverge in handling of glucose, the undesired consequences of hyperglycemia and metabolic pathways involved therein incur a cell-relevant series of pathological changes at cellular level that lead to organ dysfunction [ 4]. The malfunctioning metabolic pathways primarily result in diabetes-induced overproduction and accumulation of reactive oxygen species (ROS), elevated oxidative stress, hyperosmolarity, and formation of irreversible glycation products, i.e., advanced glycation end products (AGEs). These metabolic changes are inter-related and serve as a trigger for the each other. A continuous higher glucose exposure increases ROS production in the cells via glycation reaction and electron transport chain in the mitochondria while AGEs along with insulin and angiotensin-II trigger ROS production via membrane-bound NADH activation [ 5]. The failure of the cells to eliminate the undesired end products of glucose metabolism negatively impacts various signaling pathways in the cells (i.e., protein kinases) and leads to altered gene expression at molecular levels and toxicity of β cells and pancreatic toxicity at organ level [ 6]. Either insufficient insulin production from pancreatic β cells or inefficient use of the presenting insulin causes abnormal glucose utilization and hyperglycemia, which are the mainstay abnormalities in DM. With insulin as pivotal to enhance cell sensitivity to glucose uptake in most cases [ 7], these effects are more obvious in the case where presence of insulin is not mandatory for glucose uptake into the cells and, hence, are more sensitive to hyperglycemia [ 8]. These include vascular endothelial cells, neuronal cells, pancreatic β cells, renal cells, retinal cells, cardiac cells, brain cells and red blood cells (RBCs). In the wake of their insulin-independent glucose uptake, these cells are inherently predisposed to equilibrate with high glucose concentrations in their microenvironment [ 9].


        Unlike other body cells, mature RBCs lack many organelles including the nucleus, mitochondria, and ribosomes, rendering them incapable to proliferate and unique in handling of glucose uptake its anaerobic metabolic pathways for its energy requirements. Our current review of literature focuses on the effect of hyperglycemia in terms of changes in membrane lipid peroxidation and membrane fluidity and integrity that leads to altered functionality and life span. The clinical scenarios wherein uncontrolled hyperglycemia impacts the RBCs in patients with DM and gestational diabetes has also been discussed.


The red blood cells

       RBCs constitute a vast population of blood tissue cells which is maintained critically (4.5-5 × 106 cells/μL) via production and destruction of cells to ensure that oxygen supply to the body tissues is optimally maintained as part of the homeostasis. RBCs are biconcave dumbbell shaped, 7-8 μm in diameter, and with an average life span of 100-120 days at the end of which they are sequestered from the peripheral circulation and processed by macrophages in the splenic and hepatic sinusoids. An interplay between the membrane-expressed phosphatidylserine and CD47 during RBCs aging act as pro- and anti-apoptotic signals respectively to help in their recognition by the macrophages for erythrophagocytosis [10]. Approximately 1012 new erythrocytes are produced per day through an intricate and highly regulated process with a central role for erythropoietin to act as a stimulant of the blood cell precursors [11, 12]. They originate from hematopoietic stem cell-derived progenitors in the bone marrow that undergo a series of sequential divisions and differentiations and lose their nucleus to form mature RBCs besides producing granulocytes, monocytes, platelets, and both B and T lymphocytes. Besides containing hemoglobin for respiratory gas transport, RBCs possess high-level flexibility of their membranes that permit the cells to deform and squeeze through as small as 2-3 μm diameter microvasculature [13]. Nevertheless, a sustained uncontrolled hyperglycemia causes membrane lipid peroxidation and osmotic fragility in RBCs [14]. As a result of these changes, there is an irreversible membranous cross-linking, inactivation of enzymes, and loss of cellular elasticity due to stiffness of cell membranes of RBCs which result in increased tendency of their aggregation and difficulty in flowing through the microvasculature [15]. Besides losing membrane flexibility and becoming stiff, the aging RBCs also exhaust their energy-producing (ATP) capability that contributes to their elimination by the reticuloendothelial system [16]. Hence, rigidity of RBCs' cell membrane is an important indicator of their health and physiological functioning.


RBCs and glucose metabolism

         One of the fundamental requirements of every cell in the human body is to ensure adequate supply of energy in the form of ATP to support its functionality, converting glucose into pyruvate via glycolysis which then enters Krebs cycle to release ATP in the process (Fig. 1). As glycolysis is a cytoplasmic pathway and does not require mitochondria or oxygen, the process remains an exclusive source of energy and, hence, is indispensable for survival of the mature RBCs [17]. Therefore, enzymopathies that hamper glycolytic pathway may cause sustained reduction in the available energy that plunges at times lower than the requirements of mature RBCs to perpetuate their routine functions, thus resulting in significant lessening of their life span [18]. There is ample evidence in literature that persistent exposure to hyperglycemia leads to shortening of the RBCs' life span [19-21]. Using a modified carbon monoxide breath test, a recent study has reported a whopping 16.9% reduction in RBCs lifespan in DM-II patients [21].An outline of glucose metabolism to produce energy (ATP) in a typical human body cell. Pyruvate is then converted to acetyl-CoA by the mitochondrial enzyme pyruvate dehydrogenase (PDH), making this pathway and the following pathways unavailable in RBCs, which do not have mitochondria. Acetyl-CoA enters Krebs cycle and is burned to CO2 producing electron-rich carriers NADH and FADH2. These molecules then run through electron transport chain (ETC) in the inner mitochondrial membrane and produce ATP by oxidative phosphorylation. ETC is considered as the major source of ATP and requires both O2 and mitochondria.


         Glucose enters the cells by facilitated diffusion through glucose transporter 1 (GLUT1), one of the 13 members of the glucose transporting protein family spanning the erythrocyte cell membrane [22]. Encoded by SLC2A1 gene, GLUT1 is expressed in species-specific manner. It is exclusively expressed in species such as humans and primates which are deficient in synthesizing ascorbic acid from glucose [23]. Besides species-specific expression, differential expression of GLUT1 has been observed in ascorbic acid-synthesis competent mammals such as mice during neonatal and post-neonatal stages of development and switches between GLUT1 and GLUT4. This is unlike human erythrocytes which consistently express GLUT1 as only irrespective of the stage of development [24].


          From amongst all the cell lineages in the human body, RBCs have the highest propensity of GLUT1 expression with more than 200,000 molecules/cell and account for nearly 10% of the protein mass in cells [25]. Many of these GLUT1 transporters are inactive and are considered as masked and inaccessible to glucose for its transport; however, it can be activated by treatment with cytochalasin B [26]. The significance of such high-level presence of GLUT1 is to allow the blood to carry glucose and free access to glucose between plasma and the cell itself and enable accumulation of glucose across the erythrocyte cell membrane [25]. Kinetic studies using 14C-labeled D-glucose have confirmed that permeation of glucose across the RBC membrane is five orders of magnitude higher than what is expected if it were by simple diffusion [27]. GLUT1 shows high affinity for glucose and facilitates glucose uptake by the cells in insulin-independent fashion. Once inside the cell, glucose gets converted to pyruvate and then to lactate by lactate dehydrogenase which is important to recycling NAD+ for glycolysis to continue (Fig. 2). Besides glucose, GLUT1 also allows transport of other sugars including mannose, galactose, glucosamine, and reduced ascorbic acid [28]. A structural change in GLUT1, especially in the outer domain of the transporter, has been reported in diabetic patients that causes altered glucose transport across the erythrocyte cell membrane [29].An overview on glucose uptake and metabolism in RBCs.


Hyperglycemic exposure and RBCs

        RBCs are incessantly exposed to oxidative, chemical, ionic, and mechanical stressors during their life span and therefore need a meticulous homeostatic mechanism to survive in the circulation. Higher glucose levels than the physiological limits in the plasma play havoc with RBCs, and the effect of an uncontrolled elevated glucose exposure is manifested in different ways on the RBCs encompassing changes in cell membrane flexibility to the intracellular metabolism which will be discussed at length below.


Hyperglycemia and eryptosis

         Lacking the nucleus as well as the mitochondria, RBCs undergo programmed cell death by the process of eryptosis which is equivalent to apoptosis in their counterpart cells possessing both the nucleus and mitochondria [30]. It is stimulated by the influx of Ca+2 into the cells, osmotic shock, oxidative stress, energy depletion, and dysregulation of various kinases; all these are typical features of exposure to sustained hyperglycemia. Study on the erythrocytes from rodent model of streptozotocin-induced diabetes showed increased eryptotic RBCs in the diabetic animals in comparison with the control non-diabetic animals. At molecular levels, RBCs from the diabetic animals showed extensive caspase-3 immunoreactivity thus suggesting its role in programmed lysis of RBCs [31]. Prior studies have also reported the presence of Fas, FasL, Fas-associated death domain, caspase-8, and caspase-3 in mature circulating erythrocytes [32]. Although the exact mechanism underlying the enhanced age-related susceptibility of circulating RBCs to eryptosis remains less explored, depleted phospholipid translocase activity along with high-level phosphatidylserine externalization as compared to the young RBCs are considered as the two prime contributory factors in this regard. On the other hand, fetal RBCs are more resistant to eryptosis as compared to their adult and aged counterparts. However, their resistance to eryptosis lasts only until they get overwhelmed by oxidative stress during postnatal life [33]. Eryptosis may be inhibited by several xenobiotics and endogenous biomolecules including nitric oxide and erythropoietin and is characterized by erythrocyte shrinkage, blabbing with concomitant translocation of phosphatidylserine to the cell membrane [34]. A recent study has shown that increase in the burden of AGEs results in elevated oxidant stress in the RBCs due to chronic hyperglycemic exposure that renders the cells susceptible to eryptosis [35]. The study reports the pro-eryptotic properties of three of the commonly observed AGEs including carboxymethyllysine, carboxyethyllysine, and Arg-pyrimidine. Additionally, pro-eryptotic activity of methylglyoxal-modified albumin has also been reported via morphological and membranous changes in the cells during sustained hyperglycemia in diabetic patients. Elevated oxidative stress in the extracellular milieu promotes caspase-3 activation in the RBCs in diabetic patients that impairs the maintenance of cell shape and morphology thus contributing towards altered hemorheological properties of the cells that result in hemorheological disorders and shorten the life span of RBCs [36].


Hyperglycemia and RBC cell membrane

          The unique biconcave dumbbell-shaped morphology and deformability of the RBCs' cell membrane that allow the cell to increase its area-to-volume ratio are two of the mandatory features required for physical adaptation of the cells to pass through the narrow capillary beds and splenic sinusoids. RBCs that lose deformability due to whichever reason become rigid and less deformable which significantly promote their aging and elimination from the circulation.


          Empirically, the cell membrane is the only organized structural component of RBCs which is responsible for various characteristics of the cell and its biological function, i.e., antigenic, transport and exchange of materials across the membrane, and mechanical [37]. It possesses lipid bilayer structure which besides others, includes phospholipids and cholesterol, traversed by various proteins with different domains, i.e., hydrophobic transmembrane domains embedded in the membrane, extracellular domains, and intracellular cytoplasmic domains [38]. Moreover, the lipid bilayer is tethered by cytoskeletal proteins including spectrin, ankyrin, and band 3. While spectrin is a heterodimer composed of α and β subunits, it is connected to the membrane lipid bilayer by ankyrin that interacts with β spectrin and the integral membrane protein band 3 which is responsible for RBCs' morphology and deformability [39]. Regulated by membranous protein systems including Syk and Lyn [40, 41], band 3 also links some key biochemical enzymes to balance the use of glucose either in glycolysis for production of ATP or in the pentose phosphate pathway [42]. This is important since the presence of higher level glucose decreases the rate of the glycolysis and an accompanied increase in the activity of the pentose phosphate pathway thus producing NADPH to protect the cell against the possible oxidative stress [43]. Any mutation in the RBCs' membranous proteins is accompanied by altered morphology, decreased stability, and/or increased removal.


          When subjected to a high-glucose conditions, RBCs show influx of glucose via insulin-independent glucose transporter GLUT1, causing high intracellular glucose concentration. In vitro experiments have shown that increased intracellular glucose produces a hyperosmolar state to favor rapid inward movement of water thus causing a reversible increase in mean corpuscular volume (MCV) [44]. These data have clinical significance and emphasize hyperglycemia as one of the factors causing increase in MCV in diabetics. The persistently elevated glucose levels also result in the formation of AGEs in the erythrocytes. Human erythrocytes cultured in low (5 mM) and high glucose (30 mM) concentration for 5 days showed significantly higher levels of AGEs (N-(carboxyethyl)lysine and N-(carboxymethyl)lysine) in the cells [45]. The formation of AGEs as well as non-enzymatic glycosylation of membranous proteins irreversibly cross-links the cytoskeletal proteins to disrupt RBCs' elasticity and functionality and may ultimately cause cell death [46]. This mechanism is also manifested as one of the long-term complications of diabetes through its consequences in accelerated atherosclerosis, glomerular dysfunction, endothelial dysfunction, and altered extracellular matrix composition [47].


           Another important effect of hyperglycemia is through interference with the lipid constituents of the RBC membrane. The lipid components are vital for physiological hemostasis of RBCs. These lipid components in the membranes of fresh, untreated erythrocytes of diabetic patients undergo peroxidation which correlates well with the levels of hyperglycemia as well as glycosylated hemoglobin HbA1c levels [48]. The amount of glycated hemoglobin in the human RBCs changes with age and also with their exposure to higher levels of glucose in time-dependent manner [49]. Similar observations have been reported in the streptozotocin-treated rodent model of experimentally induced diabetes which showed significantly higher lipid peroxidation in the erythrocyte membrane of the diabetic animals as compared to the non-diabetic control group (50-84% higher vs control) [50]. Lipid composition analysis of the erythrocyte membrane shows that sphingomyelin is significantly reduced whereas the levels of phosphatidylethanolamine increase in the hyperglycemic rats. In vitro exposure of RBCs to physiological (5 mM) and higher (45 and 100 mM) glucose concentration showed that the cells exposed to higher glucose concentrations had higher level of lipid peroxidation besides concomitant reduction in RBCs glutathione-s-transferase and glutathione reductase enzyme activities [51].


          The process of lipid peroxidation includes a complex chain reaction encompassing the generation of lipid radicals, alteration in the double bonds, and their rearrangement in the molecular structure of the unsaturated lipids with eventual destruction of the lipid membrane [52]. The process may proceed non-enzymatically via free-radical chain reactions or require the presence of enzymes, i.e., lipoxygenases, for which polyunsaturated fatty acids and cholesterol are more sensitive substrates. These molecular reactions generate undesired end products with altered lipid membrane properties including their permeability and deformability. Furthermore, lipid peroxidation in the cell membrane in response to hyperglycemia is multifarious. Higher influx of glucose due to the hyperglycemic microenvironment enhances the rate of intracellular glycolysis and the resultant metabolite NADPH that promotes membrane lipid peroxidation in the presence of cytochrome P-450. This system is mainly found in the liver microsomes and the microsomal lipid peroxidation depends upon NADPH-cytochrome P-450 reductase [48]. Inside the erythrocytes, oxyhemoglobin may act like CYP-450 in the presence of NADPH in response to hyperglycemia [53, 54].


           An alternative mechanism of lipid peroxidation in response to hyperglycemia is triggered by the oxidative stress that stems from an imbalance between the rate generation of ROS and antioxidant activity of the cell [55]. Most of the ROS generated in the cells are neutralized by the antioxidant machinery of the cells that includes both non-enzyme systems, i.e., vitamin C, tocopherol, etc., and the enzyme-based systems, i.e., catalase, glutathione, thioredoxin and peroxiredoxin-2. Nevertheless, excessive generation of the ROS and oxygen radicals combined with diminished antioxidant activity inside the cell lead to enhanced oxidative stress in the cell [56]. The inability of RBCs to synthesize new proteins and failure to substitute the degrading enzyme machinery involved in detoxification besides incompetence to replace any cellular components damaged due to exposure to oxidative stress render the cells more susceptible to oxidative damage [57]. The ROS formed in excess of the antioxidant capacity of the cells can initiate peroxidation of the RBCs' cell membrane phospholipids and result in the accumulation of malonyldialdehyde (MDA), an end product of the membrane lipid peroxidation which is commonly used as indicator of lipid peroxidation activity [58]. MDA causes membrane rigidity, cellular dehydration, and diminished RBC deformability which significantly reduces RBCs' survival [59].


          The cumulative effect of changes in the lipid and fatty acid profile of the RBC cell membrane itself, the associated changes in the cytoskeletal structure supporting the membrane and the membrane-bound receptors, and the enzyme machinery embedded in the cell membrane lead to serious consequences in terms of functionality and life span of the cells [60]. In terms of functionality, the cell membrane loses its fluidity due to lipid peroxidation which renders them hypercoagulable and adherent to endothelial cells [61]. Additionally, the elevated levels of oxidative stress perpetuate the aging process in RBCs [56]. Insulin treatment of the animals significantly alleviates the effects of hyperglycemia in terms of lipid peroxidation [50] besides reducing the intracellular oxidative stress [62]. On the same note, ferulic acid treatment significantly reduces glycation of hemoglobin, lipid peroxidation, and Na+/K+-ATPase activity in the erythrocytes exposed to higher glucose concentration [63]. Hyperglycemia in diabetes mellitus also causes a reduction in the activity of Na+/K+-ATPase [64]. Insulin causes induction of this enzyme to activate the Na+/K+ pump to allow electrolyte balance and membrane potential while insulin resistance during diabetes diminishes the activity of Na+/K+-ATPase activity. Moreover, factors associated with hyperglycemia such as oxidative stress and glycation of proteins also decrease the activity of this enzyme to cause erythrocyte morphological dysfunction [65]. 

Clinical implications of hyperglycemic effects on RBCs. Diabetes mellitus

          As discussed earlier, RBCs possess a spectrin-based cytoskeleton wherein a phospholipid bilayer membrane is supported by an underlying spectrin-actin cytoskeletal complex [66]. The plasma membrane is anchored to the spectrin network mainly by protein ankyrin and the transmembrane protein bands 3, 4.1, and 4.2. This provides the cells with elasticity and mechanical stability to endure the shear stress during its traversing through the microvascular structures. The integrity and structure of the membrane and cytoskeleton are vital for proper RBC functioning and to maintain their deformability [67]. In uncontrolled diabetes mellitus, persistent exposure to hyperglycemia not only tends to increase the red cell count [68] but also incurs damage to the cytoskeleton of the RBCs' membrane besides inducing a low-grade chronic inflammatory response which contributes to the development of complications.


          Both hyperglycemia and low-grade inflammation are linked with ultra-structural and functional changes in RBCs in the diabetic patients [69, 70]. Due to increase in glucose concentration, the RBC deformability is reduced with a concomitantly increased tendency to aggregate and deviate from the normal shape of RBCs as compared to the healthy subjects [69]. RBCs in diabetic patients appear swollen and display echinocytes' morphology where the cells show altered membrane characteristics and display evenly spaced projections on the surface in comparison with the age-matched healthy controls [71]. These morphological changes significantly affect their deformability as well. As diabetic microangiopathy and accelerated atherosclerosis cause narrowing of the capillary diameter due to the thickening of the basal membrane and accumulation of several metabolites, reduction in deformability of RBCs may further complicate the situation. Hence, appropriate flexibility and deformability of RBCs is required in diabetics to alleviate blood flow disturbances and occlusion which otherwise may lead to poor tissue perfusion. At the RBCs' level, oxidative stress, ROS, AGEs, non-enzymatic glycosylation of membranous proteins, lipid peroxidation, and Na+/K+ ATPase pump malfunction are some of the important factors which cause alteration of membrane proteins and lipid composition, reduction in elasticity, increase in fragility, reduction in enzyme activities, dysfunction of RBCs, and ultimately leading to reduced life span of RBCs.


Gestational diabetes

          Gestational diabetes (GD) impacts at least 2-4% pregnancies and has clinical implications reaching from altered placental characteristics to macrosomia due to maternal hyperglycemia and hyperinsulinemia [72, 73]. It has been reported that exposure to GD also causes platelet hyperactivity with little change in total platelet number [74]. On the contrary, the number of RBCs increases with concurrent changes in the mean corpuscular volume (MCV) in GD patients as compared to the control pregnant women without GD. Additionally, GD alters the fatty acid profile of the RBCs' cell membrane and causes perturbations in the cell membrane [75]. There is a significant drop in arachidonic acid and docosahexaenoic acid contents in the RBCs' cell membrane in GD patients in comparison with the control non-GD patients. Similar observations have also been reported for ethanolamine phosphoglycerides. Interestingly, such depletion was observed more in the outer leaflet of the cell membrane where the receptor and enzyme activity is more pronounced and, therefore, has been attributed to insulin resistance in GD patients [76]. Such changes have been assigned more to the disease process itself rather than showing any ethnic or racial differences [76].


           A recently published review of literature has reported a distinct fatty acid profile in pregnant women with GD [77]. Women with GD have more saturated and less polyunsaturated fatty acids in their RBC cell membrane as compared to normoglycemic women. A direct comparison of RBC cell membrane contents of healthy pregnant women with those from pregnant women with GD for sialic acid (SA), membrane fluidity, and Na+/K+ adenosine triphosphatase (ATPase) activity showed SA contents and membrane fluidity were significantly increased in pregnant women with GD [78]. On the contrary, Na+/K+-ATPase activity was significantly reduced in pregnant women with GD. These observations may be the cause of altered blood viscosity and placental perfusion in pregnant women with GD as compared with the normal pregnancy. Various studies have also reported these changes in RBCs in combination with glycosylation of hemoglobin (Hb) which significantly influences its oxygen binding capacity and affinity. The glycosylation status of membrane proteins and Hb during gestation has clinical implications in terms of predicting the pregnancies at risk as well as malformation of infants in the mothers with GD.


            In conclusion, hyperglycemia is one of the most important manifestations of diabetes mellitus and it forms the basis of most diabetic complications at cellular, tissue, and organ levels. Similar to any other cell in the human body, hyperglycemia significantly affects RBCs in terms of their morphology and functionality. Persistent exposure to hyperglycemia causes molecular changes in the cell membrane which becomes more fragile and loses its flexibility that influence the deformability of RBCs and cells become hypercoagulable and adherent. These molecular and cellular changes significantly contribute to reduced blood flow in the microvasculature thus leading to tissue hypoxia.



Acknowledgements

We thank SRC for supporting this student research project.

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Conflicts of interest

The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

This article does not contain any studies with human participants or animals performed by any of the authors.

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