Negative energy balance (NEB) during the perinatal period in cattle increases the incidence of mammary gland and uterine infectious diseases, such as mastitis, uteritis, retained fetal membranes (RFM), and endometritis. These diseases are associated with polymorphonuclear neutrophils (PMNs) dysfunction induced by the dramatic changes in steroid hormone levels. Studies have found that changes in estrogen (17β-estradiol, E2), a steroid hormone, may be responsible for the reduced immune response of PMNs before and after parturition (1). E2 in periparturient cows is known to increase in the 2 weeks immediately before parturition, rising from a basal level of 20 pg/mL to a peak of 450 pg/mL, then rapidly declining to the basal level (2). Many studies have confirmed that abnormal E2 levels may lead to perinatal diseases. For example, the level of E2 in cows that suffered from RFM was higher than that of normal cows 6 days before parturition (3, 4). Cows suffering from subclinical mastitis showed low circulating E2 levels (5). These results suggest that normal levels of E2 could relieve immunosuppression in periparturient cattle.

Previous studies have shown that ATP in PMNs is mainly produced by glucose metabolism, and elevated ATP levels are conducive to normal PMNs function during parturition (6). Regarding glucose metabolism in PMNs, glucose is first phosphorylated by hexokinase (HK) to produce glucose 6-phosphate (G6P), after which G6P is used for ATP and NADPH production via glycolysis and the pentose phosphate pathway (PPP) and glycogen synthesis (7). Hypoglycemia after parturition affects the maintenance of optimal intracellular glycogen levels and PMNs function, especially in cows suffering from uterine or mammary disease as described above. E2 has been shown to promote glycogen synthesis in various tissues and cells by regulating glycogen synthase kinase-3β (GSK-3β), for example, in the uterus (8), astrocytes (9), and neurons (10). However, to the best of our knowledge, there is no research on E2 regulation of glycogen synthesis in cattle PMNs. Reports have shown that glycolysis is the major pathway for ATP generation in PMNs, while the PPP is involved in NADPH generation (11). Both these pathways, together with the glycogen synthesis pathway, play important roles in glucose metabolism (12). As one of the most important regulatory enzymes in glycolysis, phosphofructosekinase-1 (PFK1) catalyzes the conversion of fructose-6-phosphate to fructose-1,6-diphosphate in response to cellular energy requirements, while glucose-6-phosphate dehydrogenase (G6PDH), the key enzyme in the PPP, fuels NADPH to produce superoxide. Numerous studies have shown that E2 plays an important regulatory role in glycolysis, the PPP, and other pathways of glucose metabolism in various types of cells, including MCF-7 breast cancer (13), uterine (14), and HeLa cervical cancer cells (15). Unfortunately, the specific mechanism of E2 action on glucose metabolism in PMNs is still unclear. It is, thus, worthwhile to improve our understanding of the role of glucose deficiency and E2 level on the immune potential of PMNs.

It is well-known that low extracellular glucose levels result in a deficiency in glucose uptake and utilization, and the glucose transport system of PMNs is responsible for the uptake of circulating glucose (16). To date, two glucose transporter families have been identified, including the main GLUT superfamily (GLUTs) and the sodium-glucose co-transporter SGLT family (SGLTs) (17). As the members of the GLUTs, the expression of GLUT1 and GLUT4 varies with PMNs biological conditions with glucose mainly transported across cell membranes by GLUT1 under physiological conditions (18). Meanwhile, SGLTs transport glucose through a secondary active transport mechanism, which promotes glucose uptake by using the sodium concentration gradient established by the Na⁺/K⁺-ATPase pump (19). As a member of SGLTs, the SGLT1 is mainly expressed in the kidney, heart and trachea. SGLT1 expression and its relationship to GLUT1/4 in cattle PMNs under changed glucose environments is completely unknown.

AMP-activated protein kinase (AMPK) senses available energy in cells by binding directly to ATP. Activated AMPK increases the translocation of glucose transporters and promotes ATP preservation and production. Once ATP production pathways such as glycolysis, fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) are activated, pathways involving ATP consumption, such as protein synthesis, fatty acid synthesis, gluconeogenesis and glycogen synthesis pathways, are reduced. However, reports on the role of AMPK in glucose metabolism are contradictory. Some studies have shown that activated AMPK phosphorylates key proteins of multiple pathways such as glycolysis, leading to enhanced catabolism and reduced anabolism (20, 21). Other studies have shown that AMPK activation is associated with glycogen accumulation rather than glycogen consumption (22, 23). Therefore, this study aimed to clarify the role of AMPK in the glucose metabolism of cattle PMNs.

The Bcl-2 superfamily both promotes and inhibits apoptosis, and the balance pro-apoptotic and anti-apoptotic proteins, such as Bax and Bcl-2, respectively, is critical for determining the survival time of mature PMNs. Previous study have found that the spontaneous apoptosis of human PMNs in the absence of sufficient nutrients could be inhibited by increased glucose in vitro (24). As is well-known, autophagy can supplement anabolic substrates and energy under low energy conditions by degrading internal cellular components (25) and autophagy markers including ATG5, Beclin1, LC3 and p62 play key roles in autophagy initiation in various cells (26). Studies have shown that E2 can promote autophagy, delay senescence (27) and inhibit apoptosis (28). Although increasing evidence shows that E2 can regulate cellular glucose metabolism, there are few studies on the mechanism of E2 regulation of PMNs autophagy.

The effects of E2 on the immune potential of cattle PMNs and its association with glucose levels have not been studied. Here, we investigated the effect and mechanism of E2 on glucose metabolism through regulation glucose uptake and utilization, to determine whether E2 enhances PMNs immune potential by activating autophagy under low-glucose conditions. This study provided valuable new perspectives on how E2 controls the immune potential of PMNs in cattle suffering from perinatal NEB.

We found that E2 promotes glycogen storage by promoting glycogen synthesis and maintains ATP homeostasis by enhancing glucose uptake and regulating autophagy in the context of changing extracellular glucose levels. These results suggested that E2 exerts a significant effect on both glucose uptake and utilization and, in particular, plays an important role in sustaining cell viability and promoting the glycogen storage and ATP content in situations of glucose deficiency in cattle PMNs in vitro. Therefore, E2 may be a key factor in maintaining the viability and enhancing the immune potential of PMNs for fulfilling immune function in periparturient cattle suffering from NEB.

Vazquez-Anon et al. (29) observed that hypoglycemia in periparturient cattle affects chemotaxis, phagocytosis, and killing capacity in PMNs due to reduced glycogen storage. Similarly, hyperglycemia during calving also impairs PMNs function and increases the risk of postpartum infection (30). Our results showed that PMNs viability was significantly affected by both low and high glucose, and the higher glucose condition significantly increased the PMNs glycogen content. Galvao et al. (31) found that treatment with glucose narrowed the difference in PMNs viability between cows suffering from uteritis and healthy cows, suggesting that hypoglycemia during the perinatal period is highly correlated with PMNs dysfunction and leads to disease susceptibility. It is well known that the E2 concentrations in perinatal cattle typically change from basal to peak and back to basal again. Our findings showed that different E2 concentrations significantly enhanced the viability of PMNs in low or high glucose environments in vitro, and we speculate that E2 exerts an active action on maintaining PMNs viability by regulating cellular glucose metabolism.

To further understand the E2 regulatory mechanism on PMNs glucose metabolism, we first evaluated the effect of E2 on PMNs glycogen synthesis. As the main energy source in PMNs, the glycogen content mostly depends on extracellular glucose uptake, and glucose deficiency leads to glycogen reduction in PMNs (31). Although previous studies have confirmed that E2 can promote utero glycogen synthesis in rats and rabbits (8, 32), the effect of E2 on glycogen synthesis in PMNs has been less studied. Our study showed that E2 promotes glycogen synthesis in cattle PMNs under different glucose concentrations in vitro, therefore, may benefit PMNs function in periparturient cattle suffering from NEB. ERα is the specific receptor for E2 and is a substrate of GSK-3β. E2 inhibits GSK-3β activity depending on the detachment of ERα from the ERα/GSK-3β complex (33). Reports have shown that E2 activates uterine epithelial cell proliferation by inhibiting GSK-3β-induced PI3K pathway activation (34). Our results showed that glycogen content depends on increased glucose concentration, and that E2 enhanced glycogen synthesis and inhibited GSK-3β activity in PMNs. Recent studies have shown that in mouse PMNs, the glycogen cycle can be used to produce energy at inflammatory sites where nutrients are limited (35). This suggested that moderate and well-timed amounts of E2 may contribute to the maintenance of the immune potential of PMNs in periparturient cattle by increasing the glycogen storage.

In terms of the glucose absorbed from the environment in PMNs, one part is stored in the form of glycogen while the other is competitively utilized between the PFK1-mediated glycolysis pathway and the G6PDH-mediated PPP. In mouse PMNs incubated with 25 mM glucose, there was a 50% dose-dependent reduction in G6PDH and oxygen production (36), and G6PDH deficiency in PMNs from diabetic mice resulted in reduced germicidal capacity and peroxide production (37). Therefore, we examined the activities of PFK1 and G6PDH in cattle PMNs and found that G6PDH activity decreased in response to increased glucose concentration. Addition of E2 to low- or normal-glucose medium attenuated G6PDH activity while E2 addition to high-glucose medium enhanced G6PDH activity, indicating that the impact of E2 on the PPP is involved in the extracellular glucose level. Newsholme et al. (38) found that the addition of glucose increased enzyme activity in glycolysis and ATP production in PMNs. Similarly, this study found that increased extracellular glucose enhanced PFK1 activity and ATP production. However, E2 significantly inhibited PFK1 activity and increased ATP content under low- or normal-glucose conditions, and enhanced PFK1 activity, and decreased ATP content under high-glucose conditions. These results raise an interesting question: E2 has been found to promote glycogen synthesis by inhibition of PFK1, G6PDH, and GSK-3β activity under low- or normal-glucose conditions, which ought to reduce the ATP content, however, our results showed an increase in ATP content. To answer this question, we next evaluated HK activity, glucose transporter expression, and autophagy levels in cattle PMNs.

Evidence has suggested that glucose uptake by cells is the result of functional coupling between GLUTs and HK (39). GLUT1 is expressed and up-regulated in a glucose-rich environment and mediates extracellular glucose uptake in mouse (40) and human (41) PMNs, and GLUT1, but not GLUT4, is regarded as the key link between numerous factors regulating glucose uptake in PMNs. However, we have not seen the report regarding the E2-mediated GLUT1 in PMNs, although E2 could up-regulate GLUT1 expression in breast cancer cells (42). Our study demonstrated that GLUT1, GLUT4, and SGLT1 protein are expressed in cattle PMNs. Low- and high-glucose promoted the expression of GLUT1 and SGLT1 and inhibited GLUT4 expression. We observed that the enhanced glucose uptake depends mainly on GLUT1 and SGLT1 in low-glucose environments, and which only relies on SGLT1 in the high-glucose environment. E2 enhanced the expression of GLUT1/4 and SGLT1 at low glucose levels, and inhibited GLUT4 expression at normal and high glucose levels, whereas E2 promoted SGLT1 expression at low- and normal-glucose levels and inhibited SGLT1 at high glucose levels. This demonstrates that E2 enhanced glucose uptake by promoting the expression of SGLT1 and GLUT1/4 in low glucose environments and preventing excessive glucose uptake by inhibiting SGLT1 and GLUT1/4 in high glucose environments. Thus, our results obviously defined that the promoting or inhibiting glucose uptake by E2 depends on glucose transporters function relating with the extracellular glucose level. Meanwhile, we found that HK activity was positively correlated with the level of environmental glucose and that HK activity was promoted by E2 under low glucose conditions while remaining unchanged under normal glucose conditions and being inhibited under high glucose conditions. This further provided the direct evidence for revealing glycogen increase in the perspective of intracellular glucose metabolism. Clearly, E2 enhanced the PMNs glycogen storage by upregulating glucose uptake, which lies in HK activity and the level of GLUT1/4 and SGLT1 expression in different glucose environments. Considering the process of glycogen synthesis, by its nature, needs to consume a large amount of ATP, here raises an interesting question of where the increased ATP come from when the activity of PFK1, G6PDH, and GSK-3β are inhibited by E2 in low- or normal-glucose environments in this study. Previous studies showed that activated AMPK enhance the plasma membrane localization of GLUT1 and GLUT4 in skeletal muscle (43) and leads to SGLT1 upregulation and glucose uptake promotion in cardiomyocytes (44). These finding remind us to evaluate AMPK activation may help to uncover the underlying relevance of E2 to cellular ATP content.

We observed that activated AMPK is synchronized with the increased glucose uptake, E2 promoted AMPK phosphorylation in the low- and normal-glucose environment, but inhibited AMPK activation under high-glucose conditions. This indicates that E2 can regulate ATP levels. Some studies have found that inhibiting glycolysis-dependent ATP production could activate AMPK (45) and activation of AMPK could increase the ATP level in ovarian cancer cells (46). Correspondingly, our results showed that E2 increased the ATP content by inhibiting PFK1 and activating AMPK at low and normal glucose levels, and yet E2 reduced the ATP content by enhancing PFK1 activity and inhibiting AMPK at high glucose levels. Therefore, it is reasonable to speculate that the change in ATP level resulting from addition of E2 at different glucose levels may depend on AMPK activity, and the increased ATP may be derived from an AMPK-dependent pathway, such as the autophagy pathway. We, therefore, next investigated autophagy-related proteins to answer this question.

Activation of autophagy at low glucose levels helps to maintain cellular energy homeostasis, whereas several identified signaling pathways also activate autophagy at high glucose levels (47). Ma et al. found that high glucose (20 mM) induced podocyte autophagy and damage by enhancing the expression of LC3 and Beclin1 proteins (48). Similarly, our study showed that autophagy was promoted in PMNs under low- and high-glucose conditions. The evidence showed that E2 could protect the vascular endothelium by promoting autophagy (49), but E2 protects cardiomyocytes from LPS damage by inhibiting autophagy (50). The controversial results may lies in the different conditions or cells. Our results suggested that E2 enhanced autophagy in low- or normal-glucose conditions and inhibited autophagy under high glucose conditions by regulating AMPK activity in PMNs in vitro. It may indicate that E2 promotes autophagy during energy deficiency or prevent damage to the cells by inhibiting autophagy in nutrient-rich environments. Therefore, the glucose environment is likely to be an essential prerequisite for E2-regulated autophagy for cattle PMNs. As known that glycolysis is the main pathway of ATP production in PMNs, whereas in the case of limited glucose supply, PMNs also can obtain the required energy through fatty acid oxidation (36). In human PMNs, autophagy provides sufficient free fatty acids through the decomposition of lipid droplets and supports the FAO-OXPHOS pathway to produce more ATP (51). This verifies our results from another aspect, that is, E2 may promote lipophagy to maintain a steady-state ATP content in cattle PMNs under deficient glucose conditions.

Both apoptosis and autophagy are necessary for PMNs survival, and the ATP content is lower in apoptotic PMNs (52), while E2 can protect mouse pancreatic β cells from apoptosis through the ERα mechanism (53). To further understand how E2 protects PMNs from spontaneous apoptosis, we analyzed the expression of autophagy- and apoptosis-related proteins Bcl-2 and Bax and the apoptosis rate. The results showed that low glucose promoted apoptosis and high glucose inhibited apoptosis. This indicated that autophagy is not sufficient to counteract spontaneous apoptosis under low-glucose conditions. The lower apoptosis rate in high-glucose, not in low-glucose conditions, could be attributed to enough ATP production resulting from increased autophagy and glycolysis being sufficient to inhibit spontaneous apoptosis. Moreover, our study also found that E2 inhibited apoptosis accompanied by inhibition of GSK-3β activity in PMNs under different glucose conditions, suggesting that E2-induced GSK-3β inactivation is the initial point of regulating glucose metabolism and the root cause of triggering or inhibiting lipophagy.

In conclusion, we demonstrated that E2 promotes glycogen synthesis and increases the ATP content of cattle PMNs by enhancing the activity of HK, expression of GLUT1/4 and SGLT1, and the level of autophagy, as well as by inhibiting the activity of PFK1, G6PDH and GSK-3β under conditions of glucose restriction. This finding suggested that the molecular mechanisms by which E2 controls cellular energy levels is essential for protecting the cells from apoptosis and reinforcing the immune potential of PMNs. More research is required to further elucidate the mechanism by which E2 regulates glucose metabolism. This study provided a meaningful understanding of the effects of E2 on PMNs function in periparturient cattle suffering from NEB.

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding authors.

The animal study was reviewed and approved by Inner Mongolia Nationalities University Animal Care and Use Committee (no. SCXK-2020-0002).

XW and YZ performed the experiments and wrote the manuscript. YL and MT collected and analyzed the data. QD and JM revised the manuscript. LD revised the manuscript and supervised the entire project. All authors read and approved the final manuscript.

This work was supported by National Natural Science Foundation of China (31760752 and 31260626). Natural Science Foundation of Inner Mongolia Autonomous Region (2018LH03009 and 2019LH03020). Young scientific and technological talents in Inner Mongolia (Inner Mongolia, China, Grant No. NJYT-20-B30).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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