Data_Sheet_1_Estrogen Regulates Glucose Metabolism in Cattle Neutrophils Through Autophagy.ZIP

Hypoglycemia resulting from a negative energy balance (NEB) in periparturient cattle is the major reason for a reduced glycogen content in polymorphonuclear neutrophils (PMNs). The lack of glycogen induces PMNs dysfunction and is responsible for the high incidence of perinatal diseases. The perinatal period is accompanied by dramatic changes in sex hormones levels of which estrogen (17β-estradiol, E2) has been shown to be closely associated with PMNs function. However, the precise regulatory mechanism of E2 on glucose metabolism in cattle PMNs has not been elucidated. Cattle PMNs were cultured in RPMI 1640 with 2.5 (LG), 5.5 (NG) and 25 (HG) mM glucose and E2 at 20 (EL), 200 (EM) and 450 (EH) pg/mL. We found that E2 maintained PMNs viability in different glucose conditions, and promoted glycogen synthesis by inhibiting PFK1, G6PDH and GSK-3β activity in LG while enhancing PFK1 and G6PDH activity and inhibiting GSK-3β activity in HG. E2 increased the ATP content in LG but decreased it in HG. This indicated that the E2-induced increase/decrease of ATP content may be independent of glycolysis and the pentose phosphate pathway (PPP). Further analysis showed that E2 promoted the activity of hexokinase (HK) and GLUT1, GLUT4 and SGLT1 expression in LG, while inhibiting GLUT1, GLUT4 and SGLT1 expression in HG. Finally, we found that E2 increased LC3, ATG5 and Beclin1 expression, inhibited p62 expression, promoting AMPK-dependent autophagy in LG, but with the opposite effect in HG. Moreover, E2 increased the Bcl-2/Bax ratio and decreased the apoptosis rate of PMNs in LG but had the opposite effect in HG. These results showed that E2 could promote AMPK-dependent autophagy and inhibit apoptosis in response to glucose-deficient environments. This study elucidated the detailed mechanism by which E2 promotes glycogen storage through enhancing glucose uptake and retarding glycolysis and the PPP in LG. Autophagy is essential for providing ATP to maintain the survival and immune potential of PMNs. These results provided significant evidence for further understanding the effects of E2 on PMNs immune potential during the hypoglycemia accompanying perinatal NEB in cattle.

围产期奶牛因负能量平衡（NEB）引起的低血糖症是导致多形核中性粒细胞（PMNs）中糖原含量降低的主要原因。糖原的缺乏导致PMNs功能异常，并是围产期疾病高发率的重要原因。围产期伴随着性激素水平的剧烈变化，其中雌激素（17β-雌二醇，E2）与PMNs功能的紧密相关性已被证实。然而，E2在奶牛PMNs中调节葡萄糖代谢的确切机制尚未阐明。在RPMI 1640培养基中，以2.5（LG）、5.5（NG）和25（HG）毫摩尔葡萄糖和E2在20（EL）、200（EM）和450（EH）皮克/毫升的浓度下培养奶牛PMNs。我们发现E2在不同葡萄糖条件下维持了PMNs的存活率，并通过抑制LG中的PFK1、G6PDH和GSK-3β活性，同时增强HG中的PFK1和G6PDH活性并抑制GSK-3β活性来促进糖原合成。E2在LG中增加了ATP含量，但在HG中降低了ATP含量。这表明E2诱导的ATP含量增加/减少可能独立于糖酵解和磷酸戊糖途径（PPP）。进一步分析显示，E2在LG中促进了己糖激酶（HK）和GLUT1、GLUT4和SGLT1的表达，而在HG中抑制了GLUT1、GLUT4和SGLT1的表达。最后，我们发现E2在LG中增加了LC3、ATG5和Beclin1的表达，抑制了p62的表达，促进了AMPK依赖性自噬，但在HG中产生了相反的效果。此外，E2在LG中增加了Bcl-2/Bax比率并降低了PMNs的凋亡率，但在HG中产生了相反的效果。这些结果表明，E2能够促进AMPK依赖性自噬并抑制凋亡，以应对葡萄糖缺乏的环境。本研究阐明了E2通过增强LG中的葡萄糖摄取和减缓糖酵解及PPP来促进糖原储存的详细机制。自噬对于为维持PMNs的存活和免疫潜能提供ATP至关重要。这些结果为深入了解E2在奶牛围产期NEB伴随的低血糖症中对PMNs免疫潜能的影响提供了重要的证据。