Proton Fall or Bicarbonate Rise: GLYCOLYTIC RATE IN MOUSE ASTROCYTES IS PAVED BY INTRACELLULAR ALKALINIZATION

Shefeeq M Theparambil,Tobias Weber,Jana Schmälzle,Ivàn Ruminot,Joachim W Deitmer

doi:10.1074/jbc.m116.730143

Abstract

Glycolysis is the primary step for major energy production in the cell. There is strong evidence suggesting that glucose consumption and rate of glycolysis are highly modulated by cytosolic pH/[H(+)], but those can also be stimulated by an increase in the intracellular [HCO3 (-)]. Because proton and bicarbonate shift concomitantly, it remained unclear whether enhanced glucose consumption and glycolytic rate were mediated by the changes in intracellular [H(+)] or [HCO3 (-)]. We have asked whether glucose metabolism is enhanced by either a fall in intracellular [H(+)] or a rise in intracellular [HCO3 (-)], or by both, in mammalian astrocytes. We have recorded intracellular glucose in mouse astrocytes using a FRET-based nanosensor, while imposing different intracellular [H(+)] and [CO2]/[HCO3 (-)]. Glucose consumption and glycolytic rate were augmented by a fall in intracellular [H(+)], irrespective of a concomitant rise or fall in intracellular [HCO3 (-)]. Transport of HCO3 (-) into and out of astrocytes by the electrogenic sodium bicarbonate cotransporter (NBCe1) played a crucial role in causing changes in intracellular pH and [HCO3 (-)], but was not obligatory for the pH-dependent changes in glucose metabolism. Our results clearly show that it is the cytosolic pH that modulates glucose metabolism in cortical astrocytes, and possibly also in other cell types.

Highlights

The high energy requirement of the mammalian brain is primarily fueled by the degradation of blood-derived glucose
We have approached the question whether glucose consumption and glycolysis are stimulated by a fall in [Hϩi], a rise in bicarbonate, or by both, by measuring glucose in cultured astrocytes and in organotypic slices of mouse hippocampus in culture after imposing different pH and bicarbonate changes in the cells
The present study addresses a fundamental question in cellular glucose metabolism, i.e. whether pH-dependent stimulation of glycolysis reported in different cell types, including astrocytes, is mediated by a fall in [Hϩ]i or a rise in [HCO3Ϫ]i

Summary

Results

We have approached the question whether glucose consumption and glycolysis are stimulated by a fall in [Hϩi], a rise in bicarbonate, or by both, by measuring glucose in cultured astrocytes and in organotypic slices of mouse hippocampus in culture after imposing different pH and bicarbonate changes in the cells. Removing glucose and lactate resulted in a large and reversible decrease of the glucose signal by 18.9 Ϯ 0.3% with a rate of 3.7 Ϯ 0.26%/min (Fig. 1, I and J) These experiments suggest that a rise in glucose consumption is not necessarily coupled to the HCO3Ϫ transport via NBCe1, but are in line with a pH dependence of glucose metabolism. When external Kϩ was raised from 3 to 15 mM in the normal solution (5% CO2/26 mM HCO3Ϫ, pH 7.4), a sharp drop of the glucose signal of 13.8 Ϯ 0.47% was monitored in WT cells, but a small increase of glucose by 3.4 Ϯ 0.54% in NBCe1-KO cells was seen (Fig. 5, D and E) We used this depolarization protocol with 15 mM Kϩ to modulate the hypercapnic acidosis-associated Hϩi and HCO3Ϫ shifts in astrocytes. Our results agree with those of Ref. 10, and in addi-

85 HCO3- 26 mM 10 mM

Discussion

Experimental Procedures