The cortex and hippocampus of 3-month-old mice APP/PS1 mice and wild type mice were assessed by immunostaining and WB analysis. Lactate contents in the cortex and hippocampus of APP/PS1 mice are 5.1 ± 1.28 mmol/g and 5.29 ± 1.76 mmol/g, which are lower than those of wild type mice in the cortex (14.8 ± 1.13 mmol/g) and hippocampus (15.91 ± 1.03 mmol/g) (*P < 0.05, n = 5; Fig. 1A). As arrowheads pointed, positive staining of GPR81 can be found in the cortex and hippocampus of APP/PS1 mice and wild type mice (Fig. 1B). In statistic, OD values of GPR81 in cortex and hippocampus of APP/PS1 are 21196 ± 2187 and 18503 ± 1895, which are lower than those of wild type mice (32741 ± 3012 in cortex and 27332.3 ± 2198 in hippocampus) (*P < 0.05, n = 5; Fig. 1C).
BACE1 and Aβ are increased in the cortex and hippocampus of 3-month-old APP/PS1 mice
BACE1 is positively labeled in the cortex and hippocampus of APP/PS1 mice and wild type mice (Fig. 3A). In the cortex and hippocampus of APP/PS1 mice, OD values of BACE1 are 38036 ± 1299 and 21917 ± 2229, which are higher than those of wild type mice, 21896 ± 1595 in cortex and 15333 ± 1052 in hippocampus (*P < 0.05, n = 5; Fig. 3B). Through WB analysis, there are positive protein bands of BACE1 in cortex and hippocampus of APP/PS1 (Fig. 3C). Relative expressions of BACE1 in cortex and hippocampus of APP/PS1 mice are 1.2 ± 0.05 and 0.59 ± 0.02. In cortex and hippocampus of wild type mice, relative quantities of BACE1 are 0.67 ± 0.02 and 0.19 ± 0.04 respectively. Relative levels of BACE1 in cortex and hippocampus of APP/PS1 mice are higher than those of wild type mice (*P < 0.05, n = 5; Fig. 3c). Based on WB analysis, there are positive bands of Aβ in the cortex and hippocampus of APP/PS1 mice (Fig. 3D). Aβ levels in cortex and hippocampus of APP/PS1 mice are 0.93 ± 0.05 and 0.19 ± 0.02, which are much higher than those of wild type mice, 0.3 ± 0.02 in cortex and 0.02 ± 0.01 in hippocampus (*P < 0.05, n = 5; Fig. 3D).
Lactate and its downstream PI3K/Akt/CREB signaling pathway is correlated with the reduction of DNMT1, and the increase of BACE1 and Aβ in 3-month-old APP/PS1 mice
Through correlative analysis, it is found that lactate content is positively correlated with expressions of p-PI3K, p-AKT and p-CREB. Relative levels of p-PI3K, p-AKT and p-CREB were investigated by ELISA analysis. Specifically, the relevant formulas of lactate and p-PI3K, lactate and p-AKT, lactate and p-CREB are Y = 0.02753*X − 0.001257, 0.02092*X + 0.05233 and Y = 0.04024*X + 0.3387 (**P < 0.01, n = 10; Fig. 4A). Importantly, p-CREB is positively correlative with DNMT1, and negatively correlative with BACE1. The formulas are Y = 7812*X + 9443 and Y = -36807*X + 54857 respectively (*P < 0.05, n = 10; Fig. 4B). Based on the results of immunostaining and WB, DNMT1 is negatively correlative with BACE1 and Aβ. The relevant formulas are Y = -2.275*X + 53287, Y =−0.0001197*X + 2.252 and Y = -8.903e-005*X + 1.493 (*P < 0.01, n = 10; Fig. 4C,D). These results signify that lactate deficit is correlative with DNMT1 reduction, contributing to BACE1 increase and Aβ production.
Lactate activates neuronal GPR81-PI3K/Akt/CREB signaling pathway in vitro Cultured neurons were divided into 5 groups and given different administrations. Neurons from 5 groups were given 0 mM, 5 mM, 10 mM, 15 mM lactate and 10 mM LDH inhibitor (sodium oxamate) for 24 hours, and named as Control, 5mM LAC, 10mM LAC, 15mM LAC, and Inhibitor. Besides, 10 mM LDH inhibitor (sodium oxamate) was given to reduce lactate level in cultured neurons. Then, expressions of GPR81, p-PI3K, p-Akt, p-CREB were assessed to identify the effect of lactate addition and lactate removal on GPR81-PI3K/Akt-CREB signaling pathway.
The cells were double stained by GPR81 (red) and Neu N (green) (Fig. 5). In statistic, OD values of GPR81 (red) are 46525 ± 1983, 55061 ± 1823, 61165 ± 2330, 52608 ± 1390 and 36800 ± 1711 in Control, 5 mM, 10 mM, 15mM and Inhibitor groups respectively. OD values of Neu N (green) are 35717 ± 3019, 35334 ± 1558, 33931 ± 1758, 34275 ± 1693 and 35161 ± 593 in each group. There is an increase of GPR81 expression in 10 mM group, and a reduction in GPR81 expression in the group of LDH Inhibitor (*P < 0.05, n = 5). No differences of Neu N expressions can be found in each group (Fig. 5). WB was used to detect relative expressions of p-PI3K, p-Akt, p-CREB in Control, 5mM, 10mM, 15mM and Inhibitor groups (Fig. 6A). In each group, relative levels of p-PI3K are 0.15 ± 0.02, 0.16 ± 0.01, 0.94 ± 0.02, 0.24 ± 0.04, 0.08 ± 0.04, relative quantities of p-Akt are 0.62 ± 0.04, 0.85 ± 0.01, 1.62 ± 0.05, 0.95 ± 0.01, 0.75 ± 0.03 and relative expressions of p-CREB are 0.27 ± 0.02, 0.24 ± 0.02, 0.65 ± 0.04, 0.31 ± 0.05, 0.12 ± 0.03 respectively. Statistically, Compared with Control group, 10 mM lactate increases the expressions of p-PI3K, p-Akt and p-CREB, while LDH Inhibitor decreases relative levels of p-Akt and p-CREB (**P < 0 .01, n = 5; *P < 0.05, n = 5; Fig. 6B).
These results reveal that lactate can effectively activate neuronal GPR81-PI3K/Akt/CREB signaling pathway in vitro. Otherwise, lactate removal, the addition of LDH inhibitor, leads to the suppression of GPR81-PI3K/Akt/CREB pathway. Thus, lactate is significant for the activation of GPR81-PI3K/Akt/CREB signaling pathway (Fig. 5,6).