Mitochondrial lactate dehydrogenase is involved in oxidative-energy metabolism in human astrocytoma cells (CCF-STTG1)

Abstract

Lactate has long been regarded as an end product of anaerobic energy production and its fate in cerebral metabolism has not been precisely delineated. In this report, we demonstrate, for the first time, the ability of a human astrocytic cell line (CCF-STTG1) to consume lactate and to generate ATP via oxidative phosphorylation. (13)C-NMR and HPLC analyses aided in the identification of tricarboxylic acid (TCA) cyle metabolites and ATP in the astrocytic mitochondria incubated with lactate. Oxamate, an inhibitor of lactate dehydrogenase (LDH), abolished mitochondrial lactate consumption. Electrophoretic and fluorescence microscopic analyses helped localize LDH in the mitochondria. Taken together, this study implicates lactate as an important contributor to ATP metabolism in the brain, a finding that may significantly change our notion of how this important organ manipulates its energy budget.

Figure 1. Lactate utilization by an astrocytic cell line.

CCF-STTG1 cultures were supplemented with 2.5 mM lactate in serum free media. Lactate measurements were performed using HPLC (Reezex organic acid column) at various time intervals. Viable cell counts were performed using the Trypan Blue Exclusion Assay. • Corresponds to viable cell number of the astrocytic cells grown in serum free α-MEM+2.5 mM lactate. ○ Corresponds to viable cell number of astrocytic cells grown in serum free α-MEM. ▪ Corresponds to relative amount of lactate levels in the spent fluid (lactate cultures). n = 3, mean±SD

Figure 2. Lactate consumption by mitochondria derived from this astrocytic cell line.

I) Mitochondria were incubated with 5mM lactate and 0.1mM NAD⁺. The time dependent consumption of lactate by the mitochondria was measured using HPLC. II) The purity of the mitochondrial and cytoplasmic fractions were confirmed using VDAC and F-actin respectively. III) Mitochondrial lactate consumption was confirmed using NMR spectroscopy. Data were acquired after 15min and 60min respectively. n = 3, mean±SD

Figure 3. Lactate as a source of mitochondrial energy in an astrocytic cell line.

Mitochondria were incubated with 5 mM lactate or 5 mM citrate, 0.1 mM NAD⁺, and 0.1 mM ADP for varying time intervals. Nucleotide levels were measured by HPLC using a C₁₈ reverse phase column. I) ATP/ADP ratio in mitochondria. Open bar □ = ADP, and closed bar ▪ = ATP II) NAD⁺/NADH ratio in mitochondria incubated with lactate. Open bar □ = NADH, and closed bar ▪ = NAD⁺. Peaks were confirmed by utilizing known standards and by spiking the samples. n = 3, mean±SD

Figure 4. Oxidative metabolism of lactate in astrocytic mitochondria.

Mitochondria isolated from CCF-STTG1 cells were incubated in 10 mM ¹³C₃–lactate, 0.1 mM NAD⁺ and 1 µM NaN₃ for varying time intervals. Accumulation of TCA cycle intermediates were measured via I) HPLC and II) NMR spectroscopy. HPLC fractions were also confirmed by enzymatic assays (citrate, succinate, and fumarate).

Figure 5. Lactate promotes aerobic respiration in astrocytic mitochondria.

I) Mitochondria from an astrocytic cell line were isolated and oxygen consumption was measured over a 5 min period, utilizing an oxygen electrode (Orion®). Mitochondria were incubated with 5 mM substrate, 0.5 mM NAD⁺, and 0.5 mM ADP. A reaction buffer blank was also analyzed to ensure proper instrument calibration. II) In gel activity of cytochrome C oxidase. A) CCF-STTG1 cells incubated with 2.5 mM lactate. B) CCF-STTG1 cells incubated with 2.5mM glucose. C) CCF-STTG1 cells; incubated with 2.5 mM citrate.

Figure 6. Mitochondrial lactate metabolism.

Mitochondria from CCF-STTG1 cells were isolated and incubated with 5 mM lactate, and 0.1 mM NAD⁺ for varying time intervals within the presence or in absence of 10 mM oxamate. Relative lactate levels were measured by HPLC. • = mitochondria in the absence of 10mM oxamate. ▴ = mitochondria in the presence of 10mM oxamate. n = 3; SD

Figure 7. BN PAGE analyses of LDH in an astrocytic cell line.

I) Mitochondrial fraction. II) Soluble fraction. A) Coomassie stain for LDH from porcine heart (Sigma). B) Coomassie stain for LDH from porcine muscle (Sigma). C) In gel detection of LDH activity with 0 mM NAD⁺. D) In gel detection of LDH activity with 0.1 mM NAD⁺. E) In gel detection of LDH activity with 0.5 mM NAD⁺. F) In gel detection of LDH activity with 0.5 mM NAD⁺ and 2 mM AgNO₃.

Figure 8. 2D immunoblot analysis of LDH.

Activity bands were excised from BN PAGE experiment and ran on a 10% SDS-PAGE. A) Upper band (mitochondrial fraction). B) Lower band (mitochondrial fraction). C) Band from soluble fraction

Figure 9. Localization of LDH in the mitochondria.

Mitochondria were isolated and separated into A) Mitoplast and B) Outer membrane and inner membrane space fractions. I) Immunoblot for LDH1 in the mitochondrial fractions. II) Immunoblot for Cyt C and SDH to determine purity of mitochondrial fractions. Std corresponds to LDH from porcine heart (Sigma).

Figure 10. Lactate dehydrogenase localization in an astrocytic cell line.

A) Hoechst stain for the nucleus. B) Rhodamine B stain utilized for mitochondrial localization. C) FITC tagged secondary for anti-LDH. D) Merged image of Hoechst, Rhodamine B, and FITC. Note: Yellow spots are indicative of LDH associated with the mitochondria.

Figure 11. The versatile role of a mitochondrial LDH in an astrocytic cell line.

Lactate may be obtained either directly via uptake or by conversion of glucose through the glycolytic pathways.