Fig 1.
Graphical representation of experimental timeline.
Experimental procedures and biopsy sampling timepoints are shown.
Table 1.
PLS-DA model diagnostics.
Fig 2.
PLS-DA scores plots for the S45-B time interval.
PLS-DA scores plots show model discrimination between FS and CPF animals during the response to shock (S45-B) in each of the four compartments (liver, muscle, serum, urine). Models are of varying quality and statistical significance as reported in Table 1 but indicate that there is a difference in response to shock according to feeding status.
Fig 3.
Heatmap of key differences in energy dependence between fasted and fed animals.
Metabolomics data from four compartments demonstrates that animals enter the experiment in different metabolic states depending on feeding status (FS or CPF). At baseline (leftmost column), FS animals show a reliance on internal reserves demonstrated by liver, muscle, and serum levels of BCAAs while CPF animals process glucose from the pre-feed. During the response to shock (middle column), both groups demonstrate an increased reliance on internal fuel reserves. Increases in glucose in liver, muscle, and serum show mobilization of glucose in both groups for use as fuel. Evidence of increased proteolysis is demonstrated as well in increasing levels of BCAAs. During resuscitation (rightmost column), the reliance on fuel resources is diminished in both groups, with greater ATP degradation in CPF animals.
Table 2.
VIP (variable importance in projection) metabolites.
Fig 4.
Metabolic profile associated with feeding status at Baseline.
a) Higher levels of glucose are observed in liver, muscle, and serum of CPF animals. Urine choline and betaine as well as a reduced level of muscle ATP support the hypothesis of enhanced glucose-associated biosynthesis. Formation of a mitochondrial megachannel that allows for exit of citric acid cycle intermediates is suggested by elevated levels of fumarate, succinate, and citrate in the urine of CPF animals. b) Levels of liver, muscle, and serum BCAAs (isoleucine, leucine, and valine) as well as serum AA (tyrosine, serine, and threonine) are all higher in fasted animals at baseline suggesting increased proteolysis. The difference in the levels of serum urea (#) approach significance (p = 0.09) further supporting this hypothesis. * = VIP metabolite where levels observed in CPF animals > than that observed in FS animals. ◆ = VIP metabolite where levels observed in FS animals > than that observed in CPF animals. NV = metabolite not visible in that compartment.
Fig 5.
Biosynthesis involving the 1-carbon pool.
Metabolomics evidence from four compartments suggests prioritization of biosynthetic activities by CPF animals at baseline. Glucose provision is associated with biosynthetic activities that involve the transfer of a methyl group. The methyl group is donated by conversion of betaine to dimethylglycine (DMG) or from the folate cycle, both actions generating S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). Biomolecules generated include purines, proteins, creatine, taurine, and glutathione. Metabolites in red were identified as VIP metabolites by PLS-DA analysis. Letters in superscript indicate the compartment the metabolite was observed in: liver (L), muscle (M), serum (S), or urine (U).
Fig 6.
Purine degradation/salvage pathway.
Metabolomics evidence from four compartments suggests alternate routes for observed differences in purine abundances. ATP is degraded in a series of reactions to uric acid or allantoin. Under non-stress conditions, this degradation process proceeds at a low level. When physiologic conditions change, intermediates in the pathway can be diverted to meet these demands. For example, during fasting, IMP can be salvaged for the production of ATP thus reducing the level of HX. In our study, HX levels are higher at baseline in liver of CPF animals. We propose that this observation is a result of increased salvage in FS animals. During ischemia 5'-nucleotidase acts on AMP to generate adenosine, a potential vasodilator. In our study, adenosine levels are ~3X higher in the liver of CPF animals when compared to FS animals suggesting a greater need for vasodilation. Metabolites in red were identified as VIP metabolites by PLS-DA analysis. Letters in superscript indicate the compartment the metabolite was observed in: liver (L), muscle (M), serum (S), or urine (U).
Fig 7.
Metabolic profile associated with the response to shock.
Regardless of pre-trauma dietary state, both CPF and FS animals exhibit a similar metabolic response to shock. Glucose levels increase from baseline in the liver, muscle, and serum. This is attributed to the breakdown of glycogen. The increase is greater in CPF animals presumably due to the enhanced glycogen stores in CPF animals. Both tissues also exhibit increased levels of BCAA (isoleucine, leucine, valine), suggesting more proteolysis during shock than at baseline. Greater increases in BCAA levels are observed in FS animals. Muscle creatine phosphate (PCr) levels decrease in both groups during shock compared to baseline but the decrease is greater by almost 4-fold in FS animals when compared to CPF animals. Since muscle ATP levels are not differentiating between the two groups, FS animals appeared to rely more heavily on this non-oxidative mode of ATP generation. Alanine levels (^) increase to a greater extent in the muscle of FS animals. This difference could reflect the time lag necessary to shift the metabolic machinery dedicated to glucose use in CPF animals at baseline to that necessary to process the greater load of amino acids at shock. The urine metabolome is not profiled at this time interval. During shock, animals minimize fluid loss and metabolite levels would not accurately reflect metabolic activities. * = VIP metabolite where levels observed in CPF animals > than that observed in FS animals. ◆ = VIP metabolite where levels observed in FS animals > than that observed in CPF animals.