Measuring changes in substrate utilization in the myocardium in response to fasting using hyperpolarized [1-(13)C]butyrate and [1-(13)C]pyruvate

Abstract

Cardiac dysfunction is often associated with a shift in substrate preference for ATP production. Hyperpolarized (HP) (13)C magnetic resonance spectroscopy (MRS) has the unique ability to detect real-time metabolic changes in vivo due to its high sensitivity and specificity. Here a protocol using HP [1-(13)C]pyruvate and [1-(13)C]butyrate is used to measure carbohydrate versus fatty acid metabolism in vivo. Metabolic changes in fed and fasted Sprague Dawley rats (n = 36) were studied at 9.4 T after tail vein injections. Pyruvate and butyrate competed for acetyl-CoA production, as evidenced by significant changes in [(13)C]bicarbonate (-48%), [1-(13)C]acetylcarnitine (+113%), and [5-(13)C]glutamate (-63%), following fasting. Butyrate uptake was unaffected by fasting, as indicated by [1-(13)C]butyrylcarnitine. Mitochondrial pseudoketogenesis facilitated the labeling of the ketone bodies [1-(13)C]acetoacetate and [1-(13)C]β-hydroxybutyryate, without evidence of true ketogenesis. HP [1-(13)C]acetoacetate was increased in fasting (250%) but decreased during pyruvate co-injection (-82%). Combining HP (13)C technology and co-administration of separate imaging agents enables noninvasive and simultaneous monitoring of both fatty acid and carbohydrate oxidation. This protocol illustrates a novel method for assessing metabolic flux through different enzymatic pathways simultaneously and enables mechanistic studies of the changing myocardial energetics often associated with disease.

Figure 1. Metabolism of [1-¹³C]pyruvate and [1-¹³C]butyrate in the myocardium in vivo.

Metabolic scheme indicating the propagation of the ¹³C label from pyruvate to its downstream metabolites with red arrows and those of butyrate with blue arrows. The ¹³C label of [1-¹³C]pyruvate will not enter the TCA cycle. After an overnight fast, ketone body uptake will increase the intracellular acetoacetate and β -hydroxybutyrate concentrations. Detectable metabolite ¹³C resonances are indicated with grey boxes. CAT: carnitine acetyltransferase; CS: citrate synthase; AAT: acetoacetyl-CoA thiolase; OAT: 3-oxoacid CoA transferase; PT: pyruvate transporter; PDH: pyruvate dehydrogenase, LDH: lactate dehydrogenase; ALT: alanine transaminase; BHBDH: β -hydroxybutyrate dehydrogenase; CA: carbonic anhydrase; V_X: transport and conversion to glutamate.

Figure 2. In vivo myocardial ¹³C spectra acquired during hyperpolarized MR experiments.

(a) In vivo cardiac ¹³C spectrum recorded after the injection and metabolism of hyperpolarized [1-¹³C]pyruvate in a fed animal. (b) In vivo cardiac ¹³C spectra recorded after the injection and metabolism of hyperpolarized [1-¹³C]butyrate in the fed and fasted state, without the presence of pyruvate. (c) Spectral time course of myocardial metabolism in vivo following the co-injection of hyperpolarized [1-¹³C]butyrate and [1-¹³C]pyruvate. (d) A sum spectrum with expansion to show the downstream metabolites of HP butyrate and pyruvate in the case of co-injection.

Figure 3. Ratios of measured metabolite signals following hyperpolarized pyruvate metabolism.

Signal ratios of lactate (a) bicarbonate (b) and alanine (c) relative to pyruvate where hyperpolarized [1-¹³C]pyruvate was either injected separately or co-injected with hyperpolarized [1-¹³C]butyrate, in both fed and fasted animals. *P =  0.02 in co-injection compared with single injection groups, ^§P =  0.03 in fasted groups compared with fed groups, ^***P =  0.001 in fasted groups vs. fed groups.

Figure 4. Ratios of measured metabolite signals following hyperpolarized butyrate metabolism.

In vivo metabolite ratios of ¹³C labeled acetylcarnitine (a), glutamate (b), butyrylcarnitine (c), and acetoacetate (d) relative to their hyperpolarized metabolic precursor butyrate. Animals were exposed to a single injection of butyrate, or a co-injection of butyrate and pyruvate. *P =  0.01 in co-injection compared with single injection groups, **P =  0.006 in co-injection compared with single injection groups, ^§P =  0.05 in fasted groups compared with fed groups, ^‡P =  0.09 in fasted groups vs. fed groups. Butyrylcarnitine cannot be directly observed during co-injection due to spectral overlap.

Figure 5. Diagrams of the metabolic pathways probed by the HP experiments.

(a) Diagram of butyrate metabolism in the fed and fasted case. Fasting lowers the apparent glutamate signal while raising the acetoacetate signal. As carbohydrates and ketones are not labeled, they are grayed out. (b) The impact of substrate competition in the fed state. The extra source of acetyl units drives the acetylcarnitine pool size up, while the simultaneous presence of butyrate and pyruvate drives down the appearance of [5-¹³C]glutamate and [¹³C]bicarbonate. It is hypothesized that the decreased acetoacetate signal is ascribed to the effects of antiporting associated with the mitochondrial pyruvate transporter. (c) Substrate competition in the fasted state mirrors that of the fed state, though fasting restores some of the [1-¹³C]acetoacetate production. Acetylcarnitine is not significantly different between panels a and c. (d) A comparison of the fed versus fasted state (Panel b versus Panel c). Fasting produces an increase in the acetoacetate signal but a decrease in the acetylcarnitine signal. Fasting also results in a lower [¹³C]bicarbonate signal whenever it is compared to the fed state.