Glycolytic metabolism and activation of Na+ pumping contribute to extracellular acidification in the central clock of the suprachiasmatic nucleus: Differential glucose sensitivity and utilization between oxidative and non-oxidative glycolytic pathways

Abstract

Background: The central clock of the suprachiasmatic nucleus (SCN) controls the metabolism of glucose and is sensitive to glucose shortage. However, it is only beginning to be understood how metabolic signals such as glucose availability regulate the SCN physiology. We previously showed that the ATP-sensitive K⁺ channel plays a glucose-sensing role in regulating SCN neuronal firing at times of glucose shortage. Nevertheless, it is unknown whether the energy-demanding Na⁺/K⁺-ATPase (NKA) is also sensitive to glucose availability. Furthermore, we recently showed that the metabolically active SCN constantly extrudes H⁺ to acidify extracellular pH (pHe). This study investigated whether the standing acidification is associated with Na⁺ pumping activity, energy metabolism, and glucose utilization, and whether glycolysis- and mitochondria-fueled NKAs may be differentially sensitive to glucose shortage.

Methods: Double-barreled pH-selective microelectrodes were used to determine the pHe in the SCN in hypothalamic slices.

Results: NKA inhibition with K⁺-free (0-K⁺) solution rapidly and reversibly alkalinized the pHe, an effect abolished by ouabain. Mitochondrial inhibition with cyanide acidified the pHe but did not inhibit 0-K⁺-induced alkalinization. Glycolytic inhibition with iodoacetate alkalinized the pHe, completely blocked cyanide-induced acidification, and nearly completely blocked 0-K⁺-induced alkalinization. The results indicate that glycolytic metabolism and activation of Na⁺ pumping contribute to the standing acidification. Glucoprivation also alkalinized the pHe, nearly completely eliminated cyanide-induced acidification, but only partially reduced 0-K⁺-induced alkalinization. In contrast, hypoglycemia preferentially and partially blocked cyanide-induced acidification. The result indicates sensitivity to glucose shortage for the mitochondria-associated oxidative glycolytic pathway.

Conclusion: Glycolytic metabolism and activation of glycolysis-fueled NKA Na⁺ pumping activity contribute to the standing acidification in the SCN. Furthermore, the oxidative and non-oxidative glycolytic pathways differ in their glucose sensitivity and utilization, with the oxidative glycolytic pathway susceptible to glucose shortage, and the non-oxidative glycolytic pathway able to maintain Na⁺ pumping even in glucoprivation.

Keywords: Glycolysis; Metabolism; Na(+)/K(+)-ATPase; Suprachiasmatic nucleus; pH.

Fig. 1

Na⁺/K⁺-ATPase (NKA) activity contributes to the standing extracellular acidification. (A) Effect of 5 mM ouabain on the extracellular pH (pHe) from a representative experiment. (B) Left: A representative result to show the effect of K⁺-free solution on the pHe in control and in 5 mM ouabain. Right: Statistics comparing the average amplitude of 0-K⁺-induced alkalinization in control and in 5 mM ouabain. ∗∗∗p < 0.001. (C) Statistics showing a similar amplitude of 0-K⁺-induced alkalinization recorded between day (ZT 4–11) and night (ZT 13–21).

Fig. 2

Cyanide effects on the resting extracellular pH (pHe) and 0-K⁺-induced alkalinization. (A) A representative result showing the pHe response to K⁺-free solution in control (a), in 1 mM NaCN to block mitochondrial respiration (b), and after washout of NaCN (c). Note the cyanide-induced large acidification following an initial small alkalinization (marked by arrow) (left). Right: Statistics showing a similar magnitude of cyanide-induced acidification between day (ZT 4–11) and night (ZT 13–20). (B) Superimposition of the 0-K⁺ responses (a, b, c; from a) indicating a reversible increase in the magnitude of 0-K⁺-induced alkalinization by cyanide (left). Note a moderate increase of t_1/2 (marked by arrows) by cyanide. Right: Statistics showing two different cyanide effects, a lack of effect (top) and an enhancing effect (bottom), on the magnitude of 0-K⁺-induced alkalinization recorded from two subgroups. ∗p < 0.05. (C) Superimposition of the 0-K⁺ responses (control, cyanide, and washout) from another experiment to indicate a marked increase in t_1/2 by cyanide (marked by arrows) (left). Right: Statistics showing the average t_1/2 value of the 0-K⁺-induced alkalinization in control and in cyanide. Note a more than twofold increase in the t_1/2 value by cyanide. ∗∗p < 0.01.

Fig. 3

Iodoacetate (IAA) effects on the resting extracellular pH (pHe), 0-K⁺-induced alkalinization, and cyanide-induced acidification. (A) A representative result showing the pHe response to glycolytic inhibition with 10 mM iodoacetate (left). Note the transient pHe responses to the wash-in and wash-out of the iodoacetate solution. Right: Statistics showing a similar magnitude of iodoacetate-induced alkalinization between day (ZT 4–11) and night (ZT 13–20). (B) A representative experiment to show a marked irreversible inhibition of 0-K⁺ response after 30 min exposure to 10 mM iodoacetate (left). Right: Statistics showing the average amplitude of 0-K⁺-induced alkalinization in control and after 30 min exposure of iodoacetate. ∗∗∗p < 0.001. (C) A representative experiment to show a complete block by iodoacetate of the cyanide response (left). Right: Statistics showing the average amplitude of cyanide-induced acidification in control and after 30 min exposure of iodoacetate. ∗∗p < 0.01.

Fig. 4

Glucoprivation effects on the resting extracellular pH (pHe) and 0-K⁺-induced alkalinization. (A) A representative result showing the pHe response to the removal of external glucose (left). Right: Statistics showing a similar magnitude in the alkaline shift induced by glucose-free solution for ∼30 min between day (ZT 7–11) and night (ZT 12–16). (B) A representative experiment to show the inhibition of 0-K⁺-induced alkalinization by glucose-free solution for ∼45 min (left). Right: Statistics showing the average amplitude of 0-K⁺-induced alkalinization in control and after 30–60 min withdrawal of glucose. ∗∗∗p < 0.001. (C) Statistics showing the effect of prolonged (6–9 h) glucoprivation on the standing acidification (left) and the magnitude of 0-K⁺-induced alkalinization (right). ∗∗∗p < 0.001.

Fig. 5

Glucoprivation effects on cyanide-induced acidification. (A) A representative experiment to show the effect of glucoprivation on the cyanide-induced extracellular pH (pHe) transient (left). Note the complete block of the cyanide response by glucose withdrawal for ∼30 min. Right, Statistics showing the average amplitude of cyanide-induced acidification in control and after 20–60 min withdrawal of glucose. ∗∗p < 0.01. (B) A representative result to demonstrate the differential effects of glucoprivation on cyanide-induced acidification and 0-K⁺-induced alkalinization in the same slice. The number marks the time in min before (control), during (0-Glc), and after (washout) glucose withdrawal. Note the complete inhibition and full recovery of cyanide-induced acid shifts in response to glucose withdrawal and readmission of glucose, respectively. Similar results were from two other experiments.

Fig. 6

Hypoglycemia (0.5 mM glucose) preferentially inhibits mitochondria-associated glycolysis. (A) Left: A representative experiment to show a small pHe response to the lowering of external glucose to 0.5 mM for 30 min. Right: Statistics showing the average amplitude of alkalinization induced by hypoglycemic and glucose-free solution (for 30 min). (B) A representative experiment to show the 0-K⁺-induced alkalinization in control and in 0.5 mM glucose solution for ∼45 min (left). Right: Statistics showing a similar amplitude of 0-K⁺-induced alkalinization in control and after 30–60 min application of 0.5 mM glucose solution. (C) Left: Effect of 0.5 mM glucose on the cyanide-induced pHe transient from a representative experiment. Right: Statistics showing a marked reduction in the cyanide-induced acidification by 0.5 mM glucose for ∼1 h. (D) A continuous recording trace (the same slice as in (C)) showing a full recovery of the cyanide response after return to control solution. ∗p < 0.05.