Hyperpolarized 13 C MRI Reveals Large Changes in Pyruvate Metabolism During Digestion in Snakes

Abstract

Purpose: Hyperpolarized ¹³ C MRI is a powerful technique to study dynamic metabolic processes in vivo; but it has predominantly been used in mammals, mostly humans, pigs, and rodents.

Methods: In the present study, we use this technique to characterize the metabolic fate of hyperpolarized [1-¹³ C]pyruvate in Burmese pythons (Python bivittatus), a large species of constricting snake that exhibits a four- to tenfold rise in metabolism and large growth of the visceral organs within 24-48 h of ingestion of their large meals.

Results: We demonstrate a fivefold elevation of the whole-body lactate-to-pyruvate ratio in digesting snakes, pointing to a large rise in lactate production from pyruvate. Consistent with the well-known metabolic stimulation of digestion, measurements of mitochondrial respiration in hepatocytes in vitro indicate a marked postprandial upregulation of mitochondrial respiration. We observed that a low SNR of the hyperpolarized ¹³ C produced metabolites in the python, and this lack of signal was possibly due to the low metabolism of reptiles compared with mammals, preventing quantification of alanine and bicarbonate production with the experimental setup used in this study. Spatial quantification of the [1-¹³ C]lactate was only possible in postprandial snakes (with high metabolism), where a statistically significant difference between the heart and liver was observed.

Conclusion: We confirm the large postprandial rise in the wet mass of most visceral organs, except for the heart, and demonstrated that it is possible to image the [1-¹³ C]pyruvate uptake and intracellular conversion to [1-¹³ C]lactate in ectothermic animals.

Keywords: MRI; [1-13C]pyruvate; metabolism; postprandial; python; reptile.

FIGURE 1

(A) Illustration of the experimental setup: (1) schematic of the dynamic nuclear polarization hyperpolarizer Spinlab (GE Healthcare, Brøndby, Denmark) (positioned in a separate room ∼5 m from MR system) used for hyperpolarization of [1‐ ¹³ C]pyruvate; (2) neonate resuscitator bag, used for manual oxygen ventilation every fifth minute; (3) feedback‐controlled heating unit setup to maintain a body temperature of 30°C; (4) air hoses for the heating unit; (5) thermoregulated polystyrene box (with a python inside); and (6) surface coils for ¹ H MRI and ¹³ C‐MRS. (B) The thermoregulated Styrofoam box containing 1 Python bivittatus (body mass: 1341 g, total length 165 cm). (C) Anatomical ¹ H image overlaid with a time‐averaged ¹³ C image of [1‐ ¹³ C]lactate. Annotation in (B, C): (1) head; (2) tail; (3) heart; (4) lungs; (5) liver; and the cranial‐to‐caudal position of the transition into the (6) stomach (containing undigested rats), (7) small intestine, and (8) colon, respectively, and (9) feces

FIGURE 2

(A) Summed [1‐¹³C]pyruvate spectra (blue) and AMARES fitted result (red) ([1‐¹³C]pyruvate: 171 ppm), showing a substantial [1‐¹³C]lactate peak (at 183 ppm) in postprandial animals with a 183% increased absolute lactate signal compared to the fasted animals (left), whereas the [1‐¹³C]lactate peak from fasted animals (right) is almost absent. (B) Plot of the lactate‐to‐pyruvate ratio for all snakes, showing a statistically significant increased lactate production in postprandial (fed) animals (P < 0.0001). (C) Plot showing a statistical significantly increased lactate production in the liver, compared to the heart of postprandial animals (P = 0.003). (D, E) Time series of [1‐¹³C]pyruvate (D) and [1‐¹³C]lactate (E) and at 0, 6, 9, 12, 15, 18, and 21 s after administration of 4 mL 125 mM hyperpolarized [1‐¹³C]pyruvate in 1 postprandial python (legends display raw spectral MR‐signal). [1‐¹³C]lactate (D) and [1‐¹³C]pyruvate (E) images are overlaying anatomical ¹H images (for annotation compare with the animal in Figure 1C). These time series show that [1‐¹³C]pyruvate was distributed in the whole body, with hotspots in the heart and the proximal part of the colon, and that [1‐¹³C]lactate was accumulated mainly in the liver and heart (E) over the 21 s after administration of the [1‐¹³C]pyruvate‐tracer

FIGURE 3

(A) From left to right: Anatomical images, the summed [1‐¹³C]pyruvate distributional patterns in % signal to maximum signal, first‐order moments (FM) [0–70] in seconds, and TTP [0–70] in seconds were similar in in the fed (top) and fasted (bottom) animals. (B) TTP regional analysis of the heart and liver revealed a statistically significant difference between the organs (P = 0.002) but no response to feeding (P = 0.32), indicating no significant perfusion difference with feeding status. Also, no interaction was observed (organ × feeding, P = 0.79). (C) First‐order moment analysis of the heart and liver revealed a statistically significant difference between the organs (P = 0.004), whereas neither the response to feeding P = 0.12 nor an interaction were significant (P = 0.13, organ × feeding). An asterisk denotes interorgan statistical significance. N = 5 fasted and N = 6 fed animals. TTP, time‐to‐peak