Study Overview
The overall design of this study is illustrated in Figure 1. Six different experimental techniques were used to characterize the response to acute ATP injection.
Metabolomics
Overview at 30 minutes
A total of 202 (50%) of 401 metabolites measured were significantly changed by ATP injection in 30 minutes (Table S1-S5, Figure S1-S12). The rank order of metabolites that were most changed is illustrated in Figure 2A. These metabolites belonged to 37 different biochemical pathways (Figure 2BC, Table S3) and showed FDRs <0.05, VIP ≥ 0.9, and t-test p values < 0.05 (Table S2). There was a generalized decrease in plasma amino acids. Methionine levels in particular were strongly decreased, dropping by 43% (Table S2). All amino acids—both essential and non-essential–were affected (Figure S3). The average decrease across the 19 amino acids measured was -5.0 ± 2.6 (Z-score ± SD; Table S2). Fifteen ceramides and a class of phospholipids enriched in lysosomal and exosomal membranes, bis(monoacylglycero)phosphates (BMP), were also decreased (Table S3, Figure S6). Phosphatidylinositol (PI) lipids were decreased. The polar head groups of the major phospholipids were increased. This included choline, phosphorylcholine, ethanolamine and myoinositol. Myoinositol, which is a phospholipid head group derived by PI lipid activation to inositol phosphates for calcium signaling and subsequent processing by phosphatases, was sharply increased by ATP injection. Other head groups like phosphorylcholine and ethanolamine were also increased. Phosphatidylethanolamine (PE) lipids were decreased, while their precursors phosphatidylserine (PS) lipids were increased. Several arachidonate-derived eicosanoids, including 5-HETE and 13S-HODE, and the endocannabinoid anandamide, were also increased (Figure S4,Table S2). Dopamine was strongly increased 30 minutes after ATP injection with a Z-score of +5.5 (Figure 2A, Table S2). Other striking effects of acute ATP injection included an increase in lactate, glycerol-3-phosphate, and pyrimidines like orotic acid, thymidine, and thymine. As expected, purine metabolites were strongly increased (Figure S2, Figure 2A-C, Figure S9). These included xanthosine, allantoin, inosine, hypoxanthine, xanthine, and uric acid. Allantoin is disproportionately increased compared to uric acid in the mouse because mice have an intact uricase gene, while primates do not [55]. Adenosine triphosphate (ATP) was rapidly metabolized and was not detectable in any of the plasma samples (Table S1; detection limit = 100 nM in heparinized plasma, data not shown).
The top 2 of 10 k-NN clusters at 30 minutes after ATP injection were responsible for 79% of the metabolic impact and contained 134 metabolites with VIP scores ≥ 1.0, from 36 different metabolic pathways. Seventy-one (71) metabolites were increased with a mean Z-score of +3.2 ± 1.8 (mean ± SD; cluster #2, Table S5, Figure S4) and belonged to a superpathway that included purines but was comprised of 26 different biochemical pathways. Sixty-three (63) metabolites were decreased with a mean Z-score of -3.4 ± 2.0, including 19 of 19 measured amino acids, (cluster #1, Table S6, Figure S5) and belonged to a second superpathway comprised of an overlapping set of 26 different biochemical pathways (Tables S2-S5).
Overview at 4 hours
A total of 54 (13%) of 401 metabolites measured were changed at 4 hours after ATP injection (Figure 2D-F, Tables S1, S6-S9, Figure S1-S12). After 4 hours of recovery, one of 8 animals had recovered sufficiently to be indistinguishable from controls by multivariate analysis, while most others had not recovered completely. This is seen graphically by the overlap of sample points and the 95% confidence limits shown in the 2D PLSDA plots (Figure 2G; blue and pink circles), and in the dendrograms that show a clean statistical separation of ATP- and saline-treated animals at 30 minutes, but an intermixed response after 4 hours (Figure 2H). During subacute recovery measured 4 hours after eATP injection, phospholipids and sphingolipids, were decreased. Several bile acids like glycocholic and taurocholic acid remained decreased. Increased turnover of phospholipids was evidenced by an increase in the phospholipid head groups phosphorylcholine, ethanolamine, and myoinositol. Several markers of cellular oxidation were increased 4 hours after ATP injection. These included increased oxidized glutathione (GS-SG) and cystine (CysS-SCys), and increased markers of carnosine metabolism such as 1-methylhistidine and histidine. Changes in nitrogen metabolism were marked by increases in agmatine, homoarginine, and urea (Figure S4).
The top 3 of 10 k-NN clusters at 4 hours after ATP injection were responsible for 88% of the metabolic impact and contained 45 metabolites with VIP scores ≥ 1.5 that belonged to 27 different metabolic pathways (Table S9-S11). Thirty-two (32) metabolites in the top k-NN cluster were increased with a mean Z-score of +2.2 ± 0.7 (mean ± SD; cluster #3, Table S10) and belonged to a superpathway comprised of 19 different biochemical pathways. The second two superpathways contained 13 metabolites that were decreased with a Z-score range of -2.0 to -2.7 and were made up of 7 biochemical pathways (clusters #7 and #8, Table S9, S11).
A heat map of the 30 most increased or decreased metabolites is shown in Figure 2I. The proportional effects of purinergic signaling on all the biochemical pathways measured at 30 minutes and 4 hours after injection are illustrated in the Cytoscape map in Figure S1. Quantitative changes in purines, amino acid, methylation, sulfur, polyamine, and nitrogen metabolism are illustrated in Figure S9. Principal components analysis showed that metabolomics explained 81.2% and 74.9% of the phenotypic variance in animals at 30 minutes and 4 hours after ATP injection, respectively (Figure S10).
eATP and the microbiome
Eleven products of microbiome metabolism increased 30 minutes after eATP injection (Figure S7). These included an increase in the cysteine precursor O-acetylserine, the leucine precursor isopropylmalic acid, the carnitine metabolite trimethylamine-oxide (TMAO), the histamine metabolite imidazoleacetic acid, two phenylketones from microbial tyrosine metabolism, 4-hydroxyphenyllactic acid and 4-hydroxyphenylpyruvic acid, and the aryl hydrocarbon receptor-binding immunomodulatory molecule and tryptophan metabolite indoxyl-3-sulfate [56]. The mean Z-score for increased microbiome metabolites in the plasma was +3.5 ± 1.5 (Table S2). Only two microbiome metabolites, butyrylcarnitine (Z = -5.7) and vitamin K2 (menaquinone; Z = -1.2) were decreased 30 minutes after ATP injection. Unsubstituted purine, thought to be a marker of purine-rich food intake [57], was decreased (Z = -5.2) 30 min after ATP. The significance of this is not yet understood. No microbiome metabolites were abnormal 4 hours after ATP injection, although glycocholic, taurocholic, and taurodeoxycholic bile acids remained low (Z = -1.5 ± 0.2; Table S6).
eATP and plasma vitamin concentrations
A broad range of vitamins were acutely changed in the plasma 30 minutes after ATP injection (Figure S8). Thiamine (B1), niacin (B3), pyridoxic acid (B6), and choline were increased by a mean Z-score of +3.4 ± 1.6 (Table S2). In contrast, the plasma intermediates and effectors of 1-carbon metabolism were decreased. These included serine, glycine, and trimethyl-glycine (betaine), with a mean Z-score of -4.4 ± 1.8 (Table S2). Vitamin D3 (cholecalciferol) was also decreased (Z-score = -1.5), although active 1,25-dihydroxy Vitamin D3 was unchanged (Table S2). Thiamine (B1), niacin (B3), and pyridoxic acid (B6) remained increased in the plasma 4 hours after eATP injection, with a mean Z-score of +1.8 ± 1.1 (Table S6, Figure S8). Other vitamins and cofactors that were increased at 4 hours included 5-methyl tetrahydrofolic acid (mTHF), dimethylglycine, flavin adenine dinucleotide (FAD; B2), and L-carnitine with a mean Z-score of +1.7 ± 0.9. No vitamins were decreased 4 hours after ATP injection (Table S6 and S9).
Breathomics
Overview
Accurate measurements of exhaled gases requires normalization for minute volumes using the rate of CO2 production [48]. We found that ATP injection stimulated the release of volatile organic molecules ranging from 1 to 5 carbons in length in the first 10 minutes (Figure 3A-H). These included the three different 1-carbon species: carbon monoxide (CO), methanol, and methane. One 2-carbon, sulfur-containing volatile was increased by ATP injection: dimethylsulfide. The remaining volatiles that were produced by acute hyperpurinergia included acetaldehyde (C2), acetone (C3), butyraldehyde (C4), and isoprene (C5; Figure 3A-H).
Chemokines and Cytokines
Cytokines were measured at baseline, 30-minutes and 4-hours after ATP or saline injection to permit comparison of metabolomic and cytokine data at these time points. The chemokine CXCL1, also known as KC and GROa, was increased 2.8 times compared to saline injections (526 ± 118 pg/ml vs 188 ± 75; p < 0.0002). CXCL1 binds the G-protein coupled receptor CXCR2 and facilitates the arrest of rolling neutrophils and monocytes at sites of inflammation [58]. The anti-inflammatory interleukin, IL10 was also increased (95 ± 56 vs 36 ± 12; p < 0.04) (Figure 3I-K). ATP was known to stimulate IL10 secretion from microglial cells in culture [59], but had not been studied in animals. By 4 hours, each of these had returned to baseline levels. IL6 trended toward being increased at 30-minutes but animal-to-animal variability in the saline controls limited a stronger statistical conclusion without a larger sample size (Figure 3 K). IL1b, TNFa, IFNg, and IL12p40 were measured but unchanged at 30 minutes and 4 hours after IP ATP injection (data not shown).
eATP Effects on Corticosterone Release
Previous studies have shown that adrenal corticoid synthesis and release are directly stimulated by purinergic signaling at the adrenal cortex, independent of ACTH [60]. We found that plasma corticosterone peaked 30 minutes after injection of ATP, then trended below baseline levels by 4 hours (Figure 3L). This pattern of response was consistent with acute stimulation of corticosterone release, followed by feedback inhibition of hypothalamic corticotropin releasing hormone (CRH) and ACTH. CRH and ACTH levels were not measured in this study.
eATP Effects on Body Temperature
We tested several nucleotides for their hypometabolic effects at the high dose of 0.5 µmol/g IP in both males and females (Figure 4AB, Figure S10). All adenine-containing purines (adenosine, AMP, ADP, and ATP) produced a decrease in rectal temperature with a nadir that was reached 30-60 minutes after injection and recovery by 120 minutes. This effect lasted longer when the dose was administered intravenously instead of IP (Figure 4C). The behavioral changes caused by ATP also lasted longer when given IV (Figure 4D). ADP was most potent at these high doses of 0.5 µmol/g IP in both males and females (Figure 4AB). We next evaluated the gender-specific potency of each purine under non-saturating doses of 0 to 0.20 µmol/g measured at 15 minutes to reflect the initial phase of the metabolic response. Under these conditions we found that females were about 70% more sensitive to the hypothermic effects of ATP, i.e., had a more rapid decrease in temperature (Figure 4E, Table 1), while males were more than twice (108%) as sensitive to ADP (Figure 4F, Table 1). AMP and adenosine were equally potent in both males and females (Figure 4GH). We also examined the metabolic effects of several other purines and pyrimidines and cyclic nucleotides at equimolar doses of 0.5 µmol/g IP compared to saline and ATP (Figure S11). In males, only cAMP showed a hypometabolic effect similar to ATP. In females, both cAMP and GTP showed some activity, but both were less potent than ATP.
eATP Effects on Behavior
The behavioral effects of ATP injection were stereotyped and dose dependent. The purinergic behavioral response scale scored the change in 6 behavioral characteristics: open field avoidance, decreased exploratory behavior or locomotor activity, rapid shallow breathing or panting, shivering or rigors, piloerection, and imbalance or ataxia. The onset of behavioral changes after a high dose (0.5 µmol/g) of ATP IP occurred within 1- 2 minutes, peaked at 30 minutes, and resolved by 60 minutes of IP injection (Figure 4I). Normal movements and self-grooming behavior gradually reappeared after 45 - 60 minutes, but the abnormal behavioral features were prolonged after an intravenous (IV) dose of ATP (Figure 4D). When non-saturating, low doses of ATP were given IP (0.025 - 0.20 moles/g; Figure 4J), and outcomes were measured in the linear initial phase 15 minutes after injection, significant gender differences were observed in the response to extracellular ATP (eATP injection). Males were 37% ± 3% more sensitive to the behavioral effects produced by eATP than females (male behavioral response slope b = 37.1 ± 1; female b = 27.4 ± 0.7; p<000.1; Figure 4J, Table 1).
eATP Effects on Whole Body Metabolism
The effects of eATP injection on whole body metabolism and oxygen consumption were quantified in Comprehensive Lab Animal Monitoring System (CLAMS cages, Figure 4K-N) using indirect calorimetry. By 26 minutes after a dose of 0.5 µmol/g ATP, whole body oxygen consumption (VO2) dropped by 74% ± 6% (5,303 to 1,382 ml/kg/hr, p<0.0001; Figure 4K) and the rate of CO2 production (VCO2) dropped by 76% ± 18% (4323 to 1034 ml/kg/hr, p<0.0001; Figure 4L). The respiratory exchange ratio (RER = VO2/VCO2) shifted from 0.84 ± 0.08, reflecting a balanced usage of fat and carbohydrate to nearly complete dependence on fatty acids with an RER = 0.70 ± 0.062, p<0.006; Figure 4M, Table 1). Locomotor activity declined in both saline and ATP injected animals when placed in the wire-bottomed CLAMS cages for analysis, but the ATP-injected animals were nearly motionless between 26-52 minutes (Figure 4N, Table 1).
eATP Activates a Latent Metabolic Memory Response in the MIA Model of ASD
Acute temperature response
In the MIA model, pregnant female mice are exposed to a simulated viral infection by injection with the toll-like receptor 3 (TLR3) agonist poly(IC). This produces offspring with neurodevelopmental abnormalities associated with both autism spectrum disorders [61] and schizophrenia [62]. We administered ATP or saline to adult MIA offspring of poly(IC)-treated females and wild-type control offspring from saline-treated dams. We used a lower dose of 0.05 moles/g in females compared to 0.2 µmol/g in males because of the increased sensitivity of females to the hypothermic effects of ATP. All animals were 8-9 months of age. This is the human biological age equivalent of 35-38 years of age (see Materials and Methods). When the male MIA animals were given 0.2 µmol/g ATP, they had a 3.6 ± 0.3C mean decrease in temperature (Figure 5A). MIA females had a 2.5 ± 0.3C mean reduction in temperature following the 0.05 µmol/g dose of ATP (Figure 5B). Control males and females showed a similar short-term hypothermic response to ATP injection (Figure 5AB, red squares).
Subacute temperature response
We next recorded the body temperatures in MIA animals over 5 days after a single injection of 0.2 moles/g ATP (Figure 5CD). Although both MIA and control groups had a similar acute response to ATP injection in the first hour, their subacute response over the next 5 days differed. ATP injection produced a significant rise in basal body temperature for days 1-3 after injection only in the MIA mice (gold triangles; 0.7 ± 0.1˚ in males, p < 0.0001; 0.6 ± 0.1˚ in females, p< 0.003), and not the unprimed wild-type controls, or the MIA animals treated with saline (Figure 5CD, purple vs gold triangles). The core body temperature of the MIA mice then returned to baseline by 5 days after ATP injection.
Month-long temperature response in MIA mice after poly(IC) challenge
We next followed the basal body temperature in 8-month old MIA males and controls for 28 days after a postnatal dose of poly(IC) or saline (Figure 5E). This experiment unmasked a triphasic temperature response to poly(IC) in both the MIA and control animals: 1) An initial increase in temperature on day 1 after poly(IC) (red and gold squares), 2) a decrease in temperature on days 2-4 to below the pre-challenge baseline, 3) a return to baseline in control animals by 5 days (red squares), or a rebound increase of 0.8C that was sustained between 6 to 14 days (36.3 ± 0.5 vs 35.5 ± 0.2; p <0.0001) in the MIA animals challenged with poly(IC), with a gradual return to baseline only after 28 days (gold squares). Poly(IC) injection in both the MIA and control mice produced a similar magnitude of hypothermia on days 2 to 4. Note that the MIA animals with ASD-like behaviors maintained a 0.5˚C lower body temperature than saline-treated control animals before the challenge even at 8 months of age (Figure 5E, purple vs black circles to the left of the y-axis; Day 0 control temperature = 36.3 ± 0.3˚C vs poly(IC) = 35.8 ± 0.4; 0.5˚C difference, p <0.03).