Depletion of mitochondrial inorganic polyphosphate (polyP) in mammalian cells causes metabolic shift from oxidative phosphorylation to glycolysis

Abstract

Inorganic polyphosphate (polyP) is a linear polymer composed of up to a few hundred orthophosphates linked together by high-energy phosphoanhydride bonds, identical with those found in ATP. In mammalian mitochondria, polyP has been implicated in multiple processes, including energy metabolism, ion channels function, and the regulation of calcium signaling. However, the specific mechanisms of all these effects of polyP within the organelle remain poorly understood. The central goal of this study was to investigate how mitochondrial polyP participates in the regulation of the mammalian cellular energy metabolism. To accomplish this, we created HEK293 cells depleted of mitochondrial polyP, through the stable expression of the polyP hydrolyzing enzyme (scPPX). We found that these cells have significantly reduced rates of oxidative phosphorylation (OXPHOS), while their rates of glycolysis were elevated. Consistent with this, metabolomics assays confirmed increased levels of metabolites involved in glycolysis in these cells, compared with the wild-type samples. At the same time, key respiratory parameters of the isolated mitochondria were unchanged, suggesting that respiratory chain activity is not affected by the lack of mitochondrial polyP. However, we detected that mitochondria from cells that lack mitochondrial polyP are more fragmented when compared with those from wild-type cells. Based on these results, we propose that mitochondrial polyP plays an important role as a regulator of the metabolic switch between OXPHOS and glycolysis.

Keywords: glycolysis; inorganic polyphosphates; mitochondrial bioenergetics; oxidative phosphorylation; polyP.

Figure 1.. Mitochondrial polyP depletion decreases OXPHOS in mammalian cells.

Wt and MitoPPX HEK 293 cells were cultivated and the oxygen consumption rate (OCR) was assayed using the Seahorse technology. Each experiment was conducted in biological triplicates. (A) A representative graph showing decreased OXPHOS in MitoPPX cells compared with Wt cells after treatment with 0.5 μM FCCP. (B) Quantification of the differences in basal respiration, spare respiratory capacity, proton leak, and ATP production between Wt and MitoPPX cells. (C) Response of Wt and MitoPPX cells to the treatment with 0.5 μM FCCP and 0.5 μM rot/AA. In this case, oligomycin was not added to the samples to avoid the inhibition of the ATP synthase. Data in the graphs is shown as average ± SEM.

Figure 2.. MitoPPX cells show increased glycolysis compared with Wt cells.

(A) Analysis of the glycolysis rate in Wt and MitoPPX HEK 293 cells using the Glycolytic Rate Assay Kit and Seahorse technology. Each experiment was conducted in biological triplicates and a representative graph is shown. (B) Quantification of the data shown in A. (C) Quantification of cellular ATP levels in Wt and MitoPPX cells. Data in the graphs is shown as average ± SEM.

Figure 3.. Metabolomic comparison of Wt and MitoPPX cells.

Pathway analysis illustrating the primary differences between Wt and MitoPPX cells. The analysis was arranged by scores from pathway enrichment (y axis) and topology analysis (x axis). The size and the color of each circle is based on P-values and pathway impact values, respectively (Xia and Wishart 2011).

Figure 4.. Isolated mitochondria from MitoPPX cells show Wt activity.

(A) OCR measurements of mitochondria isolated from either Wt or MItoPPX cells. (B) Analysis of OCR and the respiratory control rate of Wt and MitoPPX cells during the respirometry experiment. (C) Steady-state levels of different components of the respiratory chain in from Wt and MitoPPX HEK 293 cells, using Western blotting. Immunoblot was conducted using a commercially available cocktail antibody, containing all the components of the ETC. Bands not labeled are unspecific. Actin was used as loading control. Data in the graphs are shown as average ± SEM.

Figure 5.. MitoPPX cells reveal increased mitochondrial fragmentation.

(A) Analysis of mitochondrial length in Wt and MitoPPX cells. Data was normalized with the total number of mitochondria counted in each picture. (B) Significant pictures of Wt and MitoPPX HEK 293 cells showing shorter mitochondria in MitoPPX cells, compared with Wt samples. Shorter mitochondria are a classical feature of increased mitochondrial fission. Mitochondria were labeled using TMRM. (C) Steady state levels of Drp1, Parkin and Bax in cell lysates of Wt and MitoPPX HEK 293 cells. Actin was used as loading control. Data in the graphs are shown as average of six pictures ± SEM.