Diphosphosinositol Polyphosphates and Energy Metabolism: Assay for ATP/ADP Ratio

Abstract

Several inositol compounds undergo rapid cycles of phosphorylation and dephosphorylation. These cycles are dependent on ATP and energy metabolism. Therefore, interfering with the cellular energy metabolism can change the concentration of rapidly turning over inositols. Many pharmacological inhibitors, apart from their intended action, also affect the energy metabolism of the cells and lower ATP. This can unspecifically influence rapidly turning over inositol phosphates. Thus, the ATP concentration should be checked when reduced inositol phosphates are observed after application of pharmacological inhibitors. A luminescence-based assay for the measurement of ATP and ADP is described. ATP is measured luminometrically using firefly luciferase. Detection of ADP is performed in a two-step enzymatic procedure: (1) The sample ATP is degraded to AMP and (2) ADP is phosphorylated to ATP, which can then be measured luminometrically. This method gives a better signal-to-noise ratio than other methods that do not degrade the sample ATP, but convert ADP directly to ATP and then measure the sum of ATP plus ADP.

New Horizons in Cellular Regulation by Inositol Polyphosphates: Insights from the Pancreaticβ-Cell

“…Disturbances of ATP and energy production in pancreatic b-cells is an established phenomenon in diabetic models (Ma et al, 2012), and as we have stated in the introduction, the initiation of glucose-induced insulin secretion is dependent on changes in the ATP: ADP ratio. 5-PP-InsP 5 levels seem to be exquisitely sensitive to reductions in cellular ATP levels, even more so than the lipids (Nagel et al, 2010). Thus, there could be a direct coupling of disrupted energy metabolism to a reduced activity of IP6K1.…”

“…Shears, also noting the kinetic properties of IP6Ks with regard to ATP, has (Voglmaier et al, 1996;Saiardi et al, 2000), they are potentially sensitive to cellular ATP levels. Thus, reductions in cellular ATP:ADP ratio can lead to significant reductions in PP-InsP 5 (Nagel et al, 2010). The IP6Ks are also sensitive to inhibition by oxidation resulting from a sensitive cysteine in the catalytic site (Onnebo and Saiardi, 2009).…”

Inositol Pyrophosphates: Energetic, Omnipresent and Versatile Signalling Molecules

“…Lowering the cellular ATP concentration by treatment with sodium azide, oligomycin, or certain kinase inhibitors has been shown to significantly reduce the levels of PP-IPs. 54,80 Interestingly, at a low ATP/ADP ratio, IP6Ks can switch to being ADP phosphotransferases, transferring the 1-phosphate from IP 6 to ADP to generate I(2,3,4,5,6)P 5 , 81 depleting cytosolic IP 6 and perhaps further lowering IP 7 synthesis. It is possible that the dual enzymatic activity of IP6Ks allows them to function as cellular adenylate energy sensors, converting IP 6 to IP 7 or IP 5 under high or low energy conditions respectively, so that these products may transduce information on the cellular energy status to regulate different metabolic and signalling pathways.…”

Inositol pyrophosphates: Why so many phosphates?

“…As a consequence, physiologically-relevant alterations in [ATP] (1–5 mM; (Soboll et al, 1978)) may impact kinase activity and hence modify the cellular levels of 5-InsP 7 (Nagel et al, 2010). That is, the prevailing levels of 5-InsP 7 may reflect the degree of the cell’s bioenergetic well-being.…”

“…That is, the prevailing levels of 5-InsP 7 may reflect the degree of the cell’s bioenergetic well-being. In pancreatic β-cells this may be a significant event in organismal metabolic homeostasis: increased transport of serum glucose into β-cells stimulates the rate of glycolytic ATP production, elevating levels of 5-InsP 7 , which promotes exocytic insulin secretion (Barker and Berggren, 2013; Illies et al, 2007; Nagel et al, 2010). It is tempting to rationalize the significance of the multiple phosphates of 5-InsP 7 as their being fundamental to the polyphosphate’s “energetic” properties that closely ties its synthesis to cellular ATP availability, aided by the enzymology of IP6Ks (i.e., their low ATP affinity).…”