Metabolomics reveals critical adrenergic regulatory checkpoints in glycolysis and pentose-phosphate pathways in embryonic heart

Cardiac energy demands during early embryonic periods are sufficiently met through glycolysis, but as development proceeds, oxidative phosphorylation in mitochondria becomes increasingly Adrenergic hormones are known stimulate mammals and are essential for embryonic development, but relatively little is known about their effects on metabolism in the embryonic heart. Here, we show that embryos lacking adrenergic stimulation have approximately 10-fold less cardiac ATP compared to littermate controls. Despite this deficit in steady-state ATP, neither the rates of ATP formation or degradation were affected in adrenergic-deficient hearts, suggesting that ATP synthesis and hydrolysis mechanisms were fully operational. We thus hypothesized that adrenergic hormones stimulate metabolism of glucose to provide chemical substrates for oxidation in mitochondria. compared to controls (●), normalized to β-actin. The β-actin panel is re-used as a representative image of six replicated experiments probing for GAPDH, G-6-PDH and PDH. Numerical values below x-axes refer to the number ( n ) of samples analyzed. Student’s t-test was used to compare means between competent and deficient groups. *p < 0.05.


Abstract
Cardiac energy demands during early embryonic periods are sufficiently met through glycolysis, but as development proceeds, oxidative phosphorylation in mitochondria becomes increasingly vital. Adrenergic hormones are known to stimulate metabolism in adult mammals and are essential for embryonic development, but relatively little is known about their effects on metabolism in the embryonic heart. Here, we show that embryos lacking adrenergic stimulation have approximately 10-fold less cardiac ATP compared to littermate controls. Despite this deficit in steady-state ATP, neither the rates of ATP formation or degradation were affected in adrenergic-deficient hearts, suggesting that ATP synthesis and hydrolysis mechanisms were fully operational. We thus hypothesized that adrenergic hormones stimulate metabolism of glucose to provide chemical substrates for oxidation in mitochondria. To test this hypothesis, we employed a metabolomics-based approach using liquid chromatography/mass spectrometry (LC/MS). Our results showed glucose-1-phosphate and glucose-6-phosphate concentrations were not significantly altered, but several downstream metabolites in both glycolytic and pentosephosphate pathways were significantly lower compared to controls. Further, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) as key enzymes in those respective metabolic pathways whose activity was significantly (p < 0.05) and substantially (80% and 40%, respectively) lower in adrenergic-deficient hearts.
Addition of pyruvate and to a lesser extent, ribose, led to significant recovery of steady-state ATP concentrations. These results demonstrate that without adrenergic stimulation, glucose metabolism in the embryonic heart is severely impaired in multiple pathways, ultimately leading to insufficient metabolic substrate availability for successful transition to aerobic respiration needed for survival.
to promote oxidative phosphorylation of ADP to ATP in mitochondria. Early embryonic heart development is characterized by a glycolytic metabolism with carbohydrates as the primary energy source (1)(2)(3)(4). However, as energy demands increase, cardiac metabolism undergoes an "embryonic-shift" in metabolism from anaerobic to aerobic (5). The mechanisms underlying the embryonic-shift initiating mitochondrial metabolism during heart development are not well-understood. The adrenergic hormones, epinephrine (EPI) and norepinephrine (NE), are regulators of stress and the sympathetic nervous system in adult mammals. Adrenergic hormones, produced in the heart as early as embryonic day 8.5 (E8.5), are also active and essential during embryonic heart development (5)(6)(7). Targeted disruption of the essential dopamine βhydroxylase (Dbh) gene prevents NE and EPI biosynthesis, and leads to embryonic lethality due to heart failure in mice (6,8). Subsequent disruption of the phenylethanolamine nmethyltransferase (Pnmt) gene leads to loss of EPI, but does not impede prenatal development (9,10). Thus, NE is crucial for embryonic heart development and survival, but the regulatory roles and physiological targets of adrenergic stimulation during embryonic heart development remain unknown.
Adrenergic-deficient (Dbh -/-) embryos appear to develop and function normally through approximately embryonic day 9.5 (E9.5), but begin to show cardiac distress between E10.5-E12 (8). These adrenergic-deficient (Dbh -/-) embryos undergo steady deterioration that ultimately ends in death within 24-hours of initial symptoms. To gain insight into transcriptional changes between Dbh +/+ and Dbh -/-, a genomic-wide expression screen showed that the largest set of differentially expressed genes are involved in metabolism (11). Further evidence demonstrated that Dbh -/embryos are energy-depleted with >95% ATP/ADP reduction by E11.5 (12). Oxygen consumption rates (OCR), an indicator of mitochondrial respiration, are established in E10.5 hearts; however, Dbh -/hearts lag behind by ~24 hours later compared to age-matched littermate controls. These results established that NE and EPI influence embryonic heart energy metabolism; however, the mechanism(s) and metabolic targets regulated by adrenergic hormones remain unknown.
Aerobic metabolism in mitochondria becomes increasingly important as the heart transitions from embryonic to fetal stages. Several studies using genetic mutations to disrupt aerobic respiration and/or mitochondrial structure/function have resulted in embryonic lethality due to heart failure (5,(13)(14)(15)(16). Similarly, lack of adrenergic hormones leads to delayed transition to aerobic metabolism in mitochondria and is associated with embryonic lethality due to apparent heart failure. We thus hypothesized that adrenergic hormones facilitate the embryonic-shift in metabolism from anaerobic glycolysis towards aerobic oxidative phosphorylation in mitochondria. This study focuses primarily on carbohydrate metabolism since lipid metabolism is not fully established until late fetal and neonatal stages (17)(18)(19). We tested our hypothesis using metabolomics analysis and measurements of key enzymatic reaction rates to evaluate metabolic changes in response to adrenergic deficiency in embryonic mouse hearts. In addition, we were able to show that metabolic blockade could be overcome, in part, by addition of downstream metabolites, leading to significant recovery of steady-state ATP in adrenergicdeficient embryonic hearts.

Results
Influence of adrenergic hormones on embryonic heart metabolism -To determine if cardiac energy levels were impaired in adrenergic-deficient embryonic hearts, we first isolated E11.5 mouse hearts to measure steady-state ATP concentrations. Our results show a dramatic and significant 10fold decrease (p < 0.01) in steady-state ATP concentrations in adrenergic-deficient hearts compared with adrenergic-competent littermate controls ( Fig 1A). To determine whether adrenergic deficiency slows ATP production or increases ATP utilization/degradation as a potential cause of energy depletion, we measured rates of ATP synthesis and hydrolysis in adrenergic-competent and adrenergic-deficient embryonic hearts (Fig 1B-E). Embryonic heart lysates were incubated with pyruvate and malate prior to measurements to provide substrates for ATP synthesis. We observed no significant difference in ATP synthesis rates in adrenergiccompetent and adrenergic-deficient hearts, suggesting that ATP synthesis rates were not impaired when the hearts were provided with necessary substrates and conditions. Similarly, no significant differences were observed in ATP hydrolysis rates in adrenergic-competent and adrenergic-deficient hearts, suggesting that the observed depletion of steady-state ATP concentrations may not necessarily be the result of increased rates of ATP hydrolysis. These data suggest ATP synthesis and hydrolysis rates were not impaired in adrenergic-deficient embryonic hearts when supplied with sufficient substrates.
To test whether metabolic substrates were limited in adrenergic-deficient embryonic hearts, we utilized LC/MS to generate metabolomic profiles. The results are shown in the Figure 2 heat map depicting relative concentrations of 47 key metabolites involved in carbohydrate, amino acid, and nucleotide metabolism. Other metabolites in these pathways, such as Acetyl CoA, were also evaluated, but not included here because we did not obtain reliable measurements for them due to limiting concentrations in both adrenergiccompetent control and adrenergic-deficient hearts at this embryonic stage of development (E11.5). Initial analysis of the heat map data clearly reveals that many but not all of the measured metabolites were substantially decreased in adrenergicdeficient hearts relative to littermate controls (Fig.  2). Remarkably, adrenergic-deficient hearts show significant decreases in several metabolites from pentose phosphate pathway (PPP), glycolysis, and TCA cycle, suggesting that glucose metabolism was severely compromised (Fig 2). On the other hand, adrenergic-deficient hearts had comparable glucose-6-phosphate, glucose-1-phosphate, and glyceraldehyde-3-phosphate concentrations compared to adrenergic-competent controls, indicating that there was sufficient starting substrate (glucose). Nevertheless, there were ~10fold decreases in downstream glycolytic metabolites such as 1,3-diphosphoglycerate (p < 0.05) and phosphoenolpyruvate, indicating the "pay-off" phase of glycolysis was likely impaired. In contrast, lactate concentrations did not differ between adrenergic-deficient and adrenergiccompetent hearts suggesting that some glycolytic activity remains or that lactate levels may be stabilized from embryonic/maternal circulation.
Another pathway that was clearly abrogated in adrenergic-deficient hearts was the pentosephosphate pathway (PPP) as evidenced by the five-fold decreases in critically important intermediate metabolites including ribose-5phosphate (p < 0.05) and phosphoribosyl pyrophosphate (PRPP) as well as two-fold decreases in sedoheptulose-7-phosphate. Consistent with these results, we also found major decreases in cardiac concentrations of many nucleotides, which rely in large part on the PPP for crucial substrates for their biosynthesis.
Further, adrenergic-deficient hearts had significantly lower concentrations of several tricarboxylic acid (TCA) cycle and related intermediates, including citrate/isocitrate, aconitate, malate, glutamate, and aspartate, that likely contributed to impaired mitochondrial metabolism (Fig 2). In addition, adrenergicdeficient hearts had two-fold lower pantothenate concentrations (not significant) compared to adrenergic-competent controls suggesting that CoA biosynthesis, an essential precursor for acetyl CoA and succinyl CoA, may also be compromised (20). These results demonstrate that entry and phosphorylation of glucose is functional; however, further utilization to produce downstream metabolic intermediates for glycolysis/glucose oxidation and PPP may be compromised in adrenergic-deficient hearts.
In addition, several metabolites involved in oxidation/reduction were significantly decreased in adrenergic-deficient hearts (Fig 2). Notably, adrenergic-deficient hearts had a significant tenfold decrease in glutathione (p < 0.05), 1.5-fold decrease in glutathione disulfide (p < 0.05), and two-fold decrease in taurine (p < 0.05) suggesting that these hearts may be experiencing some oxidative stress due to limited antioxidants (21)(22)(23). In addition, NAD + and NADH concentrations were significantly decreased (5-fold, p < 0.05 and 500-fold, p < 0.05, respectively) in adrenergicdeficient hearts. NADP + and NADPH concentrations were also significantly decreased (10-fold, p < 0.05 and greater than 30-fold, respectively) in adrenergic-deficient hearts. These data thus suggest that there is increased oxidative and decreased reducing capacity in adrenergicdeficient hearts, indicating that metabolic pathways with redox enzymes, such as dehydrogenases, may have limited activities due to limitations in essential NAD + /NADH and NADP + /NADPH cofactors (24).
Based on these metabolomic data, the redox state of the embryonic heart appeared to be substantially less reduced in the absence of adrenergic stimulation. To quantify this, we calculated the ratios of NAD + /NADH, NADP+/NADPH, and GSSG/GSH from the LC/MS data. Remarkably, all three ratios were clearly elevated in adrenergic-deficient hearts compared to competent controls (Fig 3). It may be important to also note that in each of these examples, the oxidized forms (i.e., NAD+, NADP+, and GSSG) were also decreased in adrenergic-deficient hearts compared to controls (Fig. 2), and as seen from the ratio-metric displays in Fig. 3, the reduced forms were decreased even further, resulting in a shift to a more oxidized state that is less favorable for energy production. These results indicate that an adrenergic-deficient state leads to an overall shift towards increased oxidized and corresponding decreased reduced forms of critical enzymatic co-factors necessary for metabolic oxidation-reduction reactions.
Consistent with these findings, we found that the ratios of ATP/ADP, ADP/AMP, and ATP/AMP were all decreased in adrenergic-deficient E11.5 hearts relative to competent controls ( Fig. 4 A, B, and C, respectively). Further, the overall "Energy Charge" or "EC": (25,26) in E11.5 mouse hearts showed that adrenergic-competent control hearts had an EC of 0.90 ± 0.005, consistent with normal, healthy tissues. In contrast, the EC of adrenergic-deficient hearts was significantly lower (0.66 ± 0.091, p < 0.05; Fig  4D). It is important to note that the redox and energy ratios in Figs 3 and 4 are not based on absolute values, but instead are relative comparisons calculated from the metabolite pools measured by LC/MS. These results nevertheless indicate that adrenergic-deficient hearts had significantly diminished energy status by E11.5 compared to adrenergic-competent controls.
AMPK activation compromised in absence of adrenergic hormones -Significant decreases in ATP/AMP and EC in adrenergic-deficient hearts suggested potential involvement of AMPK (27)(28)(29). We measured protein expression of the catalytic subunit, α-AMPK, and its phosphorylated form, pAMPK (Thr172) in adrenergic-deficient embryonic trunks containing the heart to assess AMPK activity. Adrenergic-deficient embryos had significantly decreased α-AMPK protein concentrations compared to adrenergic-competent controls.
Interestingly, pAMPK protein concentrations were not significantly affected in adrenergic-deficient embryos (Fig 5A and B). pAMPK normalized to total α-AMPK also indicated that adrenergic-deficient embryos have slightly more activated AMPK compared to adrenergic-competent embryos ( Fig 5C, not significant). These data suggest that AMPK activation is intact in adrenergic-deficient embryos; however, this signaling is not sufficient for survival, possibly due to lower overall α-AMPK protein.
Glycolysis and the Pentose Phosphate Pathway are impaired in the absence of adrenergic stimulation -Using the metabolomic data, we examined relative amounts of reactants (substrates) and products for known metabolic reactions to identify other potential targets of adrenergic stimulation. Adrenergic-deficient hearts had comparable glyceraldehyde-3-phosphate concentrations, but decreased 1,3diphosphoglycerate concentrations compared to adrenergic-competent controls suggesting impairment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Fig 6A, see yellow highlights). To determine whether lack of adrenergic stimulation affected GAPDH activity, we measured GAPDH rate kinetics. Our results showed that adrenergic-deficient hearts had significantly decreased GAPDH activity (80% reduction, p < 0.05) compared to adrenergiccompetent controls (Fig 6B). GAPDH protein concentrations were similar in adrenergic-deficient and adrenergic-competent embryos, suggesting adrenergic control of GAPDH likely occurs through post-translational mechanisms (Fig 6, C and D). These results identify glycolysis and GAPDH as a metabolic pathway and checkpoint, respectively, impaired by adrenergic deficiency.
Adrenergic-deficient hearts also had significantly lower concentrations of ribose-5-phosphate and PRPP, but comparable levels of glucose-6phosphate in adrenergic-deficient hearts suggesting that glucose-6-phosphate dehydrogenase (G-6-PDH), a key rate-limiting enzymatic reaction in the PPP, may also be impaired ( Fig 7A, see yellow highlights). G-6-PDH activity was significantly lower (40% reduction, p < 0.05) in adrenergic-deficient hearts ( Fig  7B), however G-6-PDH protein concentrations did not differ compared to adrenergic-competent controls (Fig 7, C and D). Consequently, this result suggests G-6-PDH protein concentrations are not limiting, but rather that G-6-PDH activity may be lower due to lack of post-translational regulation in the absence of adrenergic stimulation. Many nucleotides, including GTP, UTP, CTP, and dATP, were also significantly decreased in adrenergic-deficient hearts suggesting that impaired G-6-PDH activity may also impact nucleotide biosynthesis. Thus, G-6-PDH was another major metabolic checkpoint affected by adrenergic deficiency.
Adrenergic deficiency changes phosphorylation status of GAPDH and G-6-PDH -Significant decreases in GAPDH and G-6-PDH activity, but similar protein concentrations in adrenergicdeficient embryos compared to adrenergiccompetent controls suggested that adrenergic hormones regulate GAPDH and G-6-PDH activity via post-translational mechanisms. To identify post-translational changes in adrenergic-deficient embryos, we immunoprecipitated phospho-serine and acetyl-lysine proteins, respectively, and phosphorylated /acetylated GAPDH and G-6-PDH were detected using western blot (Fig 8). When comparing the total amounts of GAPDH, phospho-GAPDH, and acetylated GAPDH for adrenergicdeficient and adrenergic-competent controls, no significant differences were observed (Fig. 8A). The same was true for G-6-PDH when similar analysis was performed (Fig. 8B). In contrast, the ratio of pGAPDH relative to total GAPDH protein was found to be significantly decreased in adrenergic-deficient embryos in comparison to adrenergic-competent controls whereas there was no change in the ratio of acetylated GAPDH to total GAPDH protein in these groups (Fig. 8C). There was a similar lack of significant change in the ratio of acetylated G-6-PDH to total G-6-PDH though a slight tendency towards increased acetylated G-6-PDH was apparent in the adrenergic-deficient group (Fig. 8D). Moreover, there was a significant increase in the ratio of phosphorylated G-6-PDH to total G-6-PDH protein in the adrenergic-deficient group relative to adrenergic-competent controls (p < 0.01; Fig.  8D). These results show that lack of adrenergic stimulation alters the phosphorylation status of GAPDH and G-6-PDH during embryonic development, thereby providing a potential mechanistic explanation for how adrenergic hormones may regulate the activity of these enzymes during embryonic development.

Lack of adrenergic stimulation does not affect pyruvate
dehydrogenase -TCA cycle intermediates, citrate/isocitrate, aconitate, and malate, were significantly decreased in adrenergicdeficient hearts implicating impaired mitochondrial metabolism (Fig 9A). We measured pyruvate dehydrogenase (PDH) activity to determine whether entry into the TCA cycle was impaired in adrenergic-deficient hearts. PDH activity did not differ in adrenergic-deficient and adrenergic-competent hearts (Fig 9B). Adrenergicdeficient hearts appeared to have slightly increased PDH activity (not significant) compared to controls, indicating that PDH clearly is not impaired by lack of adrenergic stimulation. PDH concentrations were also not affected between adrenergic-deficient and adrenergic-competent hearts (Fig 9, C and D). These results indicate that adrenergic deficiency had little influence on PDH activity, thereby suggesting that addition of pyruvate substrate may aid in rescuing the observed metabolic deficiency.
Pyruvate rescues adrenergic-deficient OCR and steady-state ATP -By E11.5, adrenergic-deficient embryonic hearts had significantly lower amounts of metabolites needed to produce ATP and NADH, indicating that at this developmental stage, these embryos may be metabolically starved relative to adrenergic-competent controls. We thus attempted to rescue cardiac energy levels by providing essential metabolites that were limiting, such as pyruvate (for TCA cycle) and ribose (for PPP) to embryonic hearts ex vivo. Pyruvate significantly rescued OCR (~50% increase, p < 0.05) and steady-state ATP concentrations (~40% increase, p < 0.001) compared to untreated adrenergic-deficient controls. Ribose had no effect on OCR in adrenergic-deficient hearts, but significantly rescued steady-state ATP concentrations (~25% increase, p < 0.05; Fig.  10A-C). Addition of pyruvate and ribose did not have an additive effect on ATP concentrations in adrenergic-deficient hearts ( Figure  10C) suggesting that energy deficiency is not further rescued with both pyruvate and ribose. These results support the hypothesis that adrenergicdeficient embryonic hearts are unable to produce sufficient chemical substrates (e.g., pyruvate and ribose) for glucose oxidation and nucleotide synthesis, respectively.

Discussion
The aim of this study was to identify metabolic targets of adrenergic stimulation necessary for the embryonic-shift from anaerobic to aerobic metabolism during a critical period of cardiac development. Recent evidence demonstrated that the electron transport chain is activated beginning at E11.5 via closing of the mitochondrial transition pore (30). Therefore, impaired flux from glycolysis to the TCA cycle would disrupt essential ATP production from the mitochondria that normally would occur during this critical transition period in cardiac development (5). Our metabolomics analysis revealed adrenergicdeficient embryonic hearts have a severely compromised glucose metabolism impeding ATP production. Wealso demonstrated that adrenergicdeficient hearts appear to harbor a more oxidized intracellular environment compared to adrenergiccompetent controls, and thus may have impairments in anaplerotic processes including nucleotide, amino acid, and other biosynthesis pathways. Our results also indicated an increased trend of AMPK activation via phosphorylation in adrenergic-deficient embryos, however energy status is not sufficiently restored likely due to decreased total α-AMPK.
Further, we have identified two key enzymes impaired as a consequence of adrenergic deficiency that impact two major avenues of ATP production: (i) GAPDH of glycolysis/aerobic respiration and (ii) G-6-PDH of PPP.

Redox shift during Embryonic Development
Normal developmental processes, such as differentiation and organ formation, requires gradual increases in cellular oxidation and redox signaling. However, excessive increases in oxidation can lead to abnormal development and embryonic lethality (31)(32)(33)(34). NADPH, GSH, and other antioxidants have essential roles preventing excessive oxidized levels during development (35)(36)(37). GSSG is formed as GSH peroxidase reduces reactive oxygen species to more stable species, H 2 O. NADPH produced from G-6-PDH is required to regenerate GSH from GSSG via GSH reductase thus maintaining low GSSG levels (33,38). Relative ratios of NAD(H) also serve as an indicator of the cellular redox and metabolic states (33,39). Our results show significant increases in NAD + /NADH and GSSG/GSH ratios as well as non-significant increases in NADP + /NADPH ratios suggesting that the redox state in adrenergic-deficient hearts is shifted to a more oxidized state with less reducing potential needed to drive metabolic reactions including glycolysis, PPP, and TCA cycle pathways..

Glyceraldehyde-3-phosphate
Dehydrogenase (GAPDH) GAPDH, an essential enzyme of glycolysis that converts glyceraldehyde-3-phosphate to 1,3diphosphoglycerate, was identified as an important target affected by the absence of adrenergic influence. Decreased GAPDH activity in adrenergic-deficient hearts was accompanied by decreased phosphorylation of GAPDH, and decreased 1,3-diphosphoglycerate and phosphoenolpyruvate concentrations implicating adrenergic regulation of GAPDH. One potential regulatory mechanism following adrenergic stimulation is phosphorylation of GAPDH to increase activity (40,41). Studies have shown several kinases regulated by adrenergic stimulation; including Akt/PKB, CaMKIIβ, PKC, and AMPK, phosphorylate GAPDH to enhance activity (42)(43)(44)(45). Thus, the observed decrease in the ration of phosphorylated to total GAPDH protein in adrenergic-deficient embryos is consistent with the decreased enzymatic activity observed for GAPDH in these embryos. Although activation of AMPK appears to be intact in adrenergic-deficient embryos, decreased α-AMPK protein concentrations may be preventing sufficient GAPDH phosphorylation and activation leading to disruption of the glycolysis pathway.. Evidence also suggests that GAPDH activity is highly sensitive to oxidative stress (37,(46)(47)(48)(49)(50), and hence, the limitations in important NAD + /NADH co-factors likely also contributed to the observed decreases in GAPDH activity in adrenergicdeficient hearts.
Interestingly, however, cardiac lactate concentrations were not significantly affected by adrenergic deficiency. Possible explanations for this finding could be minimal GAPDH activity is compensated by extra-cardiac (including maternal circulation) lactate or other pyruvate sources, such as alanine, glycine, and/or glycerol-3-phosphate (which was not diminished in concentration in adrenergic-deficient heartssee Fig. 2 heat map) help maintain lactate concentrations under anaerobic conditions (51)(52)(53). This would indicate that adrenergic hormones are necessary to shift the fate of glucose from anaerobic lactate formation to glucose oxidation and aerobic respiration during this phase of embryonic development ("Embryonic-Shift") (5). Another explanation is lactate produced from glucose feeds into the TCA cycle (54). Since the TCA cycle is compromised in adrenergic-deficient embryonic hearts, this may also contribute to the relatively unaffected lactate concentrations in these hearts compared with those found in adrenergic-competent controls.

Glucose-6-phosphate Dehydrogenase (G-6-PDH)
Our metabolomic results also identified G-6-PDH, the rate limiting enzyme in PPP that converts glucose-6-phosphate to 6-phosphogluconolactone, as another target of adrenergic stimulation. We show that adrenergic-deficient embryos have decreased G-6-PDH activity, increased ratios of phosphorylated to total G-6-PDH protein, and decreased ribose-5-phosphate, PRPP, and nucleotide concentrations indicating G-6-PDH and PPP are effectively regulated by adrenergic stimulation during embryonic development. Other studies have also shown adrenergic regulation of PPP in working rat hearts exposed to NE and other adrenergic agonists with increased G-6-PDH activity, PRPP concentrations, and adenine nucleotide biosynthesis (59)(60)(61). During development, G-6-PDH has been shown to be protective against oxidative stress resulting from placental circulation and nutrient/respiratory exchange (31,38). Additionally, phosphorylation of G-6-PDH has been shown to decrease activity leading to increased oxidative stress (62,63). The decreased G-6-PDH activity and increased NAD + /NADH and NADP + /NADPH indicates adrenergic-deficient embryos have shifted to a more oxidized status with consequently less reducing power that could contribute to oxidative stress. Therefore, our results suggest that adrenergic stimulation may regulate G-6-PDH by reducing phosphorylation status, which in turn leads to increased enzymatic activity thereby allowing for protection against oxidative stresses in the embryonic heart.
Further, observed deficits in ribose-5-phosphate concentrations in adrenergic-deficient hearts suggested that addition of d-ribose may also be an effective rescue strategy to bypass the G-6-PDH reaction and improve energy status. Consistent with this hypothesis, we showed that addition of ribose significantly improved ATP concentrations in adrenergic-deficient hearts, though it was interestingly neither additive or synergistic when applied in conjunction with pyruvate, possibly reflecting the overall increase in oxidized status under conditions when adrenergic hormones are not present.

Metabolic Rescue of Energy Metabolism
Our observations of comparable glucose-6phosphate, but decreased citrate/isocitrate and malate suggested that pyruvate oxidation and PDH activity may have been impaired in adrenergicdeficient hearts. Intriguingly, however, PDH activity was not affected in adrenergic-deficient hearts, indicating that not all metabolic dehydrogenase enzymes were equally affected by the absence of adrenergic stimulation possibly due to differing sensitivities to oxidation (55,56). For example, GAPDH and G-6-PDH have been shown to be sensitive to oxidative stress while oxidative environments have lesser effects on PDH (57,58).
Since PDH rates were similar to adrenergiccompetent controls, addition of the metabolic substrate pyruvate to adrenergic-deficient hearts should provide effective rescue that could facilitate restoration of cellular energy levels. Exvivo delivery of pyruvate to adrenergic-deficient embryonic hearts substantially improved OCR and steady-state ATP levels, demonstrating that apparent metabolic starvation could be overcome by addition of the appropriate substrate(s). We previously showed that adrenergic-deficient embryos have larger and more elongated mitochondria compared to controls, consistent with a "starvation" phenotype (12,64,65). Taken together, these results suggest that mitochondrial metabolism is capable of functioning in adrenergic-deficient embryos, but impaired GAPDH activity may limit the amount of pyruvate substrate entering mitochondria to fuel aerobic respiration.

Summary
Adrenergic-deficient embryos have severely compromised glucose metabolism, specifically involving glycolysis, PPP, and the TCA cycle ( Fig  10). We have shown that the absence of adrenergic hormones selectively affects GAPDH and G-6-PDH during embryonic cardiac development, suggesting that adrenergic stimulation is necessary to enable GAPDH and G-6-PDH to produce sufficient metabolic fuel and anaplerotic substrates, respectively, for successful transition from anaerobic to aerobic metabolism during a crucial phase of embryonic development.

Experimental Procedures
Mice -Female (2-6 months) and male (2-12 months) C57BL/6J mice mated in the present studies were housed in a 12 hour light/12 hour dark cycle and given ad libitum access to food and water. All procedures and handling of mice were conducted in accordance with and approved by the University of Central Florida Institutional Animal Care and Use Committee and the guidelines established by the National Institutes of Health (NIH) for vertebrate animal research (66,67). The Dbh mouse colony was kindly provided by the Palmiter laboratory (University of Washington, Seattle, WA) (8) and maintained as described previously (11,12,68). Timed pregnancies were determined by the presence of a vaginal plug at E0.5. Pregnancies were confirmed by highresolution ultrasound at E8.5 using a Visualsonics Vevo3100 instrument with an MX250 transducer.
Embryonic Tissue Collections -All embryos appearing healthy in color, size (crown-rump length = 6-7 mm), texture, morphology, heartbeat, and blood circulation during microscopic examination were used for this study. All unhealthy and dead embryos were discarded. No differences were seen upon gross examination of adrenergic-deficient (Dbh -/-) and adrenergiccompetent control (Dbh +/+ and Dbh +/-) embryonic hearts at time of isolation. Embryonic heads were isolated for genotyping. For LC-MS metabolomics analysis, enzyme activity assays, and immunoblotting E11.5 hearts or trunks containing the heart were flash-frozen in liquid nitrogen and stored at -80°C for further analysis.
Ex-vivo embryonic heart culture -E11.5 hearts were isolated under aseptic conditions and cultured overnight in Dulbecco's Modified Eagle's medium (DMEM) (no glucose, no glutamine, no phenol red) (ThermoFisher Scientific) containing 5% fetal bovine serum (Hyclone Laboratories) that was charcoal-stripped of catecholamines and steroid hormones. The medium was additionally supplemented with penicillin G (100,000U/l) and streptomycin (100mg/l). Cultures were incubated overnight prior to next day steady-state ATP measurements.
Reagents -Chemical reagents were purchased from Sigma-Aldrich, except where noted otherwise.
Steady-state ATP measurements -Isolated E11.5 embryonic hearts were flash-frozen in liquid nitrogen and stored at -80°C. Samples were homogenized with 6% trichloroacetic acid. Exvivo embryonic heart cultures were placed in DMEM media supplemented with pyruvate (11mM), ribose (6.6mM), or control (no supplement) following overnight incubation. Cultures were incubated for 2 h, then collected and sonicated in 50L media and 10L 6% trichloroacetic acid. All samples were centrifuged at 6000g for 5 min at 4°C. Supernatant was collected and neutralized with 1X Tris-acetate, as described previously (12,69). ATP measurements were performed with the ATPlite Bioluminescence Assay (Perkin Elmer) according to the manufacturer's protocol.
Standard curves were generated with known concentrations of ATP. Luminescence was measured with Envision Multilabel plate reader, and ATP measurements were normalized to total protein concentrations determined using ThermoScientific NanoDrop 8000 Spectrophotometer.
Liquid Chromatography-Mass Spectrometry -Isolated E11.5 embryonic hearts were flash-frozen in liquid nitrogen and stored at -80°C. Samples were prepared as previously described (73). Briefly, metabolites were serially extracted from tissue powder of each heart in 80% methanol (1 then 0.5 mL) and extracts were dried under nitrogen gas and stored at -80º C. Samples were suspended in 120 μL of 50% methanol and, for amino acid detection, were derivatized with 2% (v/v) triethylamine and benzyl chloroformate. Metabolites were separated by reverse phase HPLC (Shimadzu) and identified by single reaction monitoring on a triple-quadrupole mass spectrometer (Thermo QuantumUltra). Data were analyzed using a publicly available MzRockmachine learning tool kit (http://code.google.com/p/mzrock/), which automated metabolite identification based on retention time, whole molecule mass, collision energy, and fragment mass. Selected metabolites were also analyzed using XCalibur Qual Browser (Thermo Scientific). We attempted to measure pyruvate and acetyl CoA by LC/MS and other biochemical techniques; however, detection was limited in both adrenergic-competent as well as deficient controls, possibly due to rapid flux from pyruvate to acetyl CoA to citrate (74,75).
GAPDH, G-6-PDH, and PDH Activities -GAPDH Activity Assay kit (catalog number ab204732) was purchased from Abcam. G-6-PDH and PDH Activity Assay kits (catalog numbers MAK105 and MAK183) were purchased from Sigma-Aldrich. Standard curves were generated for these activity assays with known concentrations of NADH. Flash-frozen embryonic hearts were sonicated for 5 s in cold PBS, or buffer specified in manufacturers' protocols. For the GAPDH assay, extracts were incubated on ice for 10 min, centrifuged at 10,000g for 5 min at 4°C, and supernatant was collected. Absorbance was measured at 450nm in 25L extracts in a clear 96 well plate in 5 min intervals for 40 min, according to the manufacturer's protocol. For the G-6-PDH assay, extracts were centrifuged at 15,000g for 10-min at 4°C and the supernatant was collected. Absorbance was measured at 450nm in 25L extracts in a clear 96 well plate in 5 min intervals for 40 min, according to the manufacturer's protocol. For the PDH assay, extracts were incubated on ice for 10 min, centrifuged at 10,000g for 5 min at 4°C, and the supernatant was collected. Absorbance was measured at 450nm in 25L extracts in a clear 96 well plate in 2 min intervals for 40 min, according to the manufacturer's protocol. . This procedure was replicated by probing six individual blots for GAPDH, G-6-PDH, and β-actin, then stripping each blot with Restore Fluorescent Western Blot Stripping Buffer (ThermoFisher Sci) and reprobing for PDH. pAMPK and β-actin were also probed together, then stripped and re-probed for AMPK. Blots were visualized using a LICOR Odyssey Infrared imaging system. All blots were analyzed using ImageStudio software, and normalized to β-actin.
OCR Measurements -Ex-vivo E11.5 mouse hearts were isolated as mentioned above and incubated overnight in a Seahorse Biosciences XF24 Islet Capture Microplate with mesh grids placed on top of specimen to prevent floating and probe interference. Next day, hearts were incubated in Seahorse XF Base Medium Minimal DMEM (0mM Glucose) supplemented with pyruvate (1mg/mL), ribose (1mg/mL), or control (no supplement) for 2 h. Basal OCR was measured at 6 min intervals over a 72 min using a Seahorse XF e Biosciences system. Rotenone (5μM) and antimycin A (20μM) were added simultaneously to block mitochondrial electron transport (76).
Statistics -Data are expressed as means ± SD. Student's t-tests were performed, unless otherwise noted, to compare means between adrenergiccompetent and adrenergic-deficient groups, with p < 0.05 required to reject the null hypothesis.   Student's t-test was used to compare means between competent and deficient groups. **p < 0.01.

Figure 2. LC-MS metabolomics analysis of isolated adrenergic-deficient embryonic hearts.
Columns A1-A4 represents the adrenergic-competent controls with three embryonic hearts analyzed per group. Columns B1-B4 represents the adrenergic-deficient group with three embryonic hearts analyzed per group. Data are presented as fold-change compared to competent control littermates. *p < 0.05; **p < 0.01; ***p < 0.001.     Steady-state metabolites involved in pentose phosphate pathway and nucleotide biosynthesis metabolism in E11.5 adrenergic-deficient hearts compared to controls (dotted line). Schematic representation of pentose phosphate pathway and related pathways provided as reference. (B) G-6-PDH activity in adrenergic-competent (•) and adrenergic-deficient (□) embryonic hearts, expressed as milliunits (mU) of enzyme per mL of sample and NADH produced per min, respectively. mU/mL is calculated as [(nmol NADH)/(min)(mL sample)]. (C and D) G-6-PDH protein concentrations (~50-60kDa) in adrenergicdeficient embryonic trunks (□) compared to controls (•), normalized to β-actin. The β-actin panel is reused as a representative image of six replicated experiments probing for GAPDH, G-6-PDH and PDH. Numerical values below x-axes refer to the number (n) of samples analyzed. Student's t-test was used to compare means between competent and deficient groups. *p < 0.05, **p < 0.01. Relative phospho-and acetyl-GAPDH and G-6-PDH protein concentrations in adrenergic-competent (•) and adrenergic-deficient (□) embryonic trunks, normalized to total GAPDH protein or G-6-PDH protein.
Numerical values below x-axes refer to the number (n) of samples analyzed. Student's t-test was used to compare means between competent and deficient groups. *p < 0.05, **p < 0.01.  Oxygen consumption rates in E11.5 adrenergic-competent (•) and adrenergicdeficient (□) embryonic hearts after addition of pyruvate (■), ribose (♦), and rotenone/antimycin A treatment, expressed as percentage compared to adrenergic-competent control baseline. (C) Steady-state ATP concentrations in adrenergic-competent and adrenergic-deficient embryonic hearts after addition of pyruvate and ribose. Numerical values below x-axes refers to the number (n) of samples analyzed. Data are represented as a percentage compared to competent controls. One-way ANOVA was performed with Dunnett's multiple comparison test used to evaluate differences between treated groups and control groups. *p < 0.05, **p < 0.01, ***p < 0.001 compared to adrenergic-competent controls. # p < 0.05, ### p < 0.001 compared to adrenergic-deficient controls. Figure 11. Summary of metabolic pathways and targets affected by adrenergic deficiency during embryonic heart development. Schematic representation of metabolites and enzymes of PPP, glycolysis, and TCA cycle. Metabolites and enzymes significantly lower in adrenergic deficient embryos compared to competent controls indicated in red or , respectively.