In vivo assessment of the recovery of myocardial pyruvate dehydrogenase activity following a ketogenic diet

Figures

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  Figure 1. Longitudinal monitoring of skewed cardiac pyruvate metabolism by KD using HP [1-13C]pyruvate. (A) Cardiac metabolism altered by KD and the pathway of labelled carbon when HP [1-13C]pyruvate is injected. The solid circle and empty circle represent labelled (13C) and unlabelled (12C) carbons, respectively. The yellow arrow indicates metabolic changes induced by a KD. (B) Body weights of the rats during a KD and a ND. (C) Accumulative 13C spectra averaged from the KD group at baseline and after 5 weeks of KD. (D) Time courses of HP [1-13C]lactate, [1-13C]alanine, and H13CO3− at baseline and after 5 weeks of KD. The shaded spectra in (C) and shaded time-courses in (D) indicate +/− standard deviation. Changes of relative metabolite levels to total HP products from the CT (n = 6) and KD groups (n = 5): (E) H13CO3−/TP, (F) [1-13C]lactate/TP, and (G) [1-13C]pyruvate/TP. Two-way ANOVA tests were used (α = 0.05). ** indicates P < 0.01. TP, total HP 13C products; KD, ketogenic diet; ND, normal diet.

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  Figure 2. Recovery of in vivo myocardial PDH flux by reverting KD to ND. (A) In vivo imaging timeline for the KD-to-ND revert group (n = 7) and the ND-fed control group (n = 7). (B) Body weight monitored during the timeframe. (C) Time-averaged cardiac 13C spectra acquired from a representative rat at Day 0, 1, 2, 5, and 8 after changing the diet. Reverting to ND gradually recovered the PDH flux (H13CO3−/TP). (D) Age-matching CT group had non-significant changes. (E) HP H13CO3−/TP, (F) [1-13C]lactate/TP, and (G) [1-13C]alanine/TP were longitudinally monitored. Unpaired t-tests were used (two tailed, α = 0.05). * and ** indicate P < 0.05 and P < 0.01, respectively. TP, total HP 13C products.

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  Figure 3. NMR isotopomer analysis using [3-13C]pyruvate and [1,2-13C2]acetate. (A) Metabolic pathways of labelled substrates. (B) Representative 13C (27.0–35.0 ppm) and 1H (1.1–1.6 ppm) NMR spectra from control (CT, n = 5), ketogenic diet (KD, n = 4), and reverted (RT, n = 4) rat hearts. (C) Relative concentrations of [2-13C]acetyl-CoA isotopomer and [1,2-13C2]acetyl-CoA were calculated from 13C NMR. (D) 13C enrichments of lactate and alanine were calculated from 1H NMR. Unpaired t-tests were used (two tailed, α = 0.05). * and ** indicate P < 0.05 and P < 0.01, respectively. Suc, succinate; Glu, glutamate; CT, control; KD, ketogenic diet; RT, reverted diet.

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  Figure 4. Enzymatic analysis of PDH. (A) Active and inactive forms of PDH are regulated by PDK and PDP. (B) PDK, (C) PDH activity, (D) phosphorylated PDH (p-PDH), (E) CoASH, (F) Acetyl-CoA, and (G) Acetyl-CoA/CoASH levels were measured ex vivo from the cardiac tissue of the control (CT, n = 3), ketogenic diet (KD, 5 weeks, n = 4), and KD-to-ND reverted (RT, 5 weeks of KD followed by 9 days of ND, n = 3) groups. Unpaired t-tests were used (two tailed, α = 0.05). * and ** indicate P < 0.05 and P < 0.01, respectively. PDH, pyruvate dehydrogenase; PDK, pyruvate dehydrogenase kinase; PDP, pyruvate dehydrogenase phosphatase.

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  Figure 5. Plasma levels of ketones, glucose, and NEFA. Circulating ketones, glucose, and NEFA were measured from blood longitudinally at baseline, during KD up to 5 weeks, and after reversion to ND up to 8 days. (A) The elevated level of ketones by a KD remained consistent during the KD and decreased to the control level within 2 days of reverted diet. (B) Glucose level dropped with a KD, then recovered to the control level within 2 days of reverted diet. (C) The NEFA level peaked at 2 weeks of KD then gradually decreased over the period of KD, then decreased to the control level within 2 days of reverted diet. Two-way ANOVA tests were used (α = 0.05). * and ** indicate P < 0.05 and P < 0.01, respectively. KD, ketogenic diet; ND, normal diet; NEFA, non-esterified fatty acids.