Heart ketone metabolism under acute ketone supplementation in ZDF rats, a type 2 diabetes heart failure model

Abstract

Background

In non-insulin-dependent, type 2, diabetes mellitus (T2D), glucose metabolism is compromised, and the heart loses its metabolic flexibility. The Zucker Diabetic Fatty rat (ZDF) model, which replicates the pathophysiology of T2D in patients, shows that as T2D progresses so does heart failure. Heart ketone metabolism seems to play a role in mitigating the heart failure process. This study assesses ketone metabolism in a ZDF heart failure model using cardiac PET imaging.

Methods

Six lean ZDF rats (CTRL) and six diabetic obese ZDF rats (T2D) were evaluated for coronary flow reserve (CFR) using [¹³N]ammonia ([¹³N]NH₃) cardiac PET. In addition, rats were evaluated with [¹¹C]acetoacetate ([¹¹C]AcAc) PET during rest and stress conditions to assess ketone metabolism, both at baseline and under an acute exogenous ketone ester oral supplementation. Blood chemistry, cardiac function and hemodynamic parameters were also evaluated under these conditions.

Results

CFR was impaired in the T2D model (CTRL: 1.8 ± 0.5; T2D: 1.4 ± 0.2, p < 0.05) suggesting the development of heart failure in the T2D model. Blood ketones increased more than 2-fold after supplementation. The [¹¹C]AcAc heart ketone uptake values with and without ketone supplementation were similar for the CTRL group, and these values were higher than for T2D rats. For the T2D group, the uptake decreased by 20% at rest under ketone supplementation vs. no supplementation (p < 0.05) and remained unchanged under stress with and without supplementation. Because of this decrease at rest, the stress/rest ratio after supplementation increases to the level observed in CTRL. [¹¹C]AcAc heart ketone metabolism showed a slight decrease under stress for the CTRL group, but not for the T2D. Under ketone supplementation, the metabolism stress/rest ratio increased only in T2D (1.25 ± 0.29, p = 0.03 compared to baseline).

Conclusion

In a rat model of T2D and CFR impairment, we were able to measure changes in ketone metabolism using [¹¹C]AcAc PET at rest and under stress with and without acute ketone supplementation. Our findings suggest that the heart ketone metabolism of T2D rats is impaired during the heart failure process. Ketone supplementation may have the potential to restore this cardiac reserve.

Introduction

There is a growing interest in the therapeutic potential of ketones. This previously neglected fuel source is now the focus of many studies, investigating, for example, cognitive decline, weight loss, athletic performance and heart failure [1–4]. Ketones could serve as an alternative and efficient fuel in the context of energy scarcity. This could potentially improve cardiac function after a loss of metabolic flexibility that is characteristic of conditions such as heart failure [5–9]. Since heart failure remains a leading cause of death, ketogenic interventions could be a valuable therapeutic option to improve patient outcomes.

Ketone metabolism can be investigated in vivo with positron emission tomography (PET) using the ketone radiotracers [¹¹C]acetoacetate ([¹¹C]AcAc) and [¹¹C]beta-hydroxybutyrate ([¹¹C]BHB) [10–14]. [¹¹C]AcAc has been used extensively to assess the role of ketones as an alternative fuel for the brain in cognitive decline, a condition in which glucose metabolism is impaired [4, 15]. Recently, ketone radiotracers were used to investigate human heart and kidney metabolism in combination with ketogenic interventions [11]. PET has a high translational potential, which allows for simultaneous or sequential studies to be conducted in animal models and humans to identify key metabolic mechanisms and test their potential as therapeutic targets. In heart failure, preclinical and clinical studies of ketogenic interventions can shed light on metabolic shifts and their impact on heart function [6, 10].

In preclinical research, the Zucker Diabetic Fatty (ZDF) rat model of type 2 diabetes (T2D) is well-known for the investigation of heart failure progression. This rat model shows several hallmarks of heart failure, such as anomalies in metabolic substrate utilization, heart function, and coronary vascular function. However, it has never been investigated with a cardiac PET stress/rest imaging protocol [16–18].

Cardiac PET assessments of myocardial blood flow (MBF) and energy metabolism represent valuable tools to characterize cardiac pathologies. In heart failure patients, impaired coronary flow reserve (CFR), derived from the MBF at rest and under stress, is indicative of a higher risk of cardiac complications. Moreover, nearly half of the patients hospitalized for heart failure also have T2D, which impairs the metabolic flexibility of the heart.

The aim of this study is to investigate the role of ketone metabolism in the failing heart. To this end, the ZDF T2D rats were validated as an appropriate model of heart failure by assessing MBF and CFR using cardiac PET imaging with [¹³N]NH₃. The metabolic flexibility of the failing heart was investigated using measures of hemodynamic parameters, heart function and blood chemistry with and without acute oral ketone supplementation. Ketone uptake and metabolism were assessed using cardiac PET with [¹¹C]AcAc, providing insight into potential therapeutic interventions for heart failure.

Materials and methods

Animals

All animal experiments were performed in accordance with the Canadian Council on Animal Care guidelines and were approved by the institutional animal research ethics board. Twelve male rats, six ZDF T2D (ZDF-Lepr^fa/fa) and six ZDF lean controls (ZDF-Lepr^fa/+; CTRL) (Charles River Laboratories; Kingston, NY), were fed ad libitum with regular chow, and their glycemia was followed-up every week with a glucometer (Precision Xtra™, Abbott Laboratories, USA), starting at 10 weeks of age, to assess the progression of glucose intolerance in the T2D. No treatment was administered for diabetes.

Study protocol

PET imaging of the rats was started at 23 weeks of age. Imaging consisted of three stress/rest cardiac acquisitions: (1) [¹³N]NH₃ assessment of MBF and CFR to determine cardiac status without ketone supplementation; (2) [¹¹C]AcAc assessment of ketone uptake and metabolism without ketone supplementation, and (3) [¹¹C]AcAc assessment of ketone uptake and metabolism with acute ketone supplementation (Fig. 1). Acquisitions were performed at least 72 h apart in randomized order and were all completed within 2 weeks. [¹³N]NH3 was obtained by cyclotron bombardment of H₂O by the nuclear reaction 16O(p,α)13N. [¹¹C]AcAc was synthesized by carboxylation of lithium isopropenolate anions, produced by the addition of isopropenyl acetate to methyllithium [10].

Fig. 1.

Study protocol of cardiac PET at rest and under pharmacological stressor (adenosine)

Every imaging session was initiated with a 0.6 mL gavage of water or ketone ester (0.6 g/kg of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate) 30 min prior to radiotracer injection [19]. Ten minutes before radiotracer injection, anesthesia was induced with 2.0% isoflurane in 1.5 L/min of oxygen, and five minutes later, a 250 µL blood sample was withdrawn from the tail artery for blood chemistry. Two venous cannulas were inserted, one in each side of the tail, to administer the radiotracer and the pharmacological stressor adenosine [20].

During the imaging session, rats were maintained under light anesthesia delivered by a nose cone (1.0–2.0% isoflurane in O₂ at 1.5 L/min). Heart rate, body temperature, and respiratory rate were monitored throughout the imaging session using a Model 1025T Monitoring & Gating System (SA Instruments, Inc., Stony Brook, NY, USA). Animals were kept warm using a heating pad system.

Imaging protocol

Imaging was performed on a small-animal PET scanner with a 7.5 cm axial field of view, spatial resolution of 1.2 mm, and an energy window setting of 250–650 keV (LabPET/Triumph; Gamma Medica, Northridge, CA, USA) [21, 22]. The cardiac PET stress/rest imaging protocol consisted of two 10-minute acquisitions in dynamic gated list mode. The scans were obtained within 20 min of each other, the first scan being performed under a steady-state conditions (rest) and the second scan at maximum coronary vasodilatation conditions (stress). Cardiac PET acquisition started at the time of radiotracer injection. The injected activity was ∼ 50 MBq of [¹³N]NH₃ in 0.3 mL at 0.9 mL/min or ∼ 40 MBq of [¹¹C]AcAc in 0.3 mL at 0.9 mL/min. For the stress acquisition, the scan was started 3 min after the beginning of the adenosine infusion (140 µg/kg/min), and the adenosine was stopped 3 min after radiotracer injection [23].

PET images were reconstructed with a 3D maximum likelihood expectation maximization algorithm with 20 iterations. The matrix size was 160 × 160 × 128 with a voxel size of 0.5 × 0.5 × 0.6 mm³. Temporal segmentation was as follows: 18 × 5 s, 5 × 30 s, and 6 × 60 s. All PET images were corrected for radionuclide decay, dead time, and random coincidences. Typical [¹¹C]AcAc cardiac PET images, summed over 90–300 s, for the CTRL and T2D rats under the four conditions (rest/stress, with/without ketone supplementation) are shown in Fig. 2.

Fig. 2.

Typical sum images (90–300 s) of [¹¹C]acetoacetate cardiac PET. In the four conditions– CTRL: ZDF lean (^fa/+), T2D: ZDF obese T2D (^fa/fa)

PET kinetic analysis

The kinetic analysis was performed with the PCARD cardiac module of PMOD (version 3.9, Zürich, Switzerland). The image-derived input function (IDIF) was extracted from the left ventricle with a nonnegative matrix factorization (NNMF) method to avoid partial volume and spill-in effects [24]. The IDIF was corrected for the presence of metabolites [10]. For the [¹³N]NH₃ studies, a 2-tissue compartment kinetic model was used to extract the MBF [25]. For the [¹¹C]AcAc, the one-tissue [¹¹C]acetate model was adapted to derive the myocardial uptake rate constant (K₁; hereafter called ‘uptake’) and the metabolic rate (k₂) [10, 26].

Heart function

ECG-gated [¹¹C]AcAc data were selected from a subset of the data (t = 2 min to t = 6 min) and separated in 8 gates defined by fractions of the R-R intervals. Reorientation, assessment of the left ventricular volume (end-systole, end-diastole), and assessment of the left ventricular ejection fraction (LVEF) were performed with Corridor4DM (INVIA, Ann Arbor, USA). Unfortunately, because of a hardware issue, R-R interval data were only available for 3 rats per group for these analyses.

Blood chemistry assays

Plasma cholesterol and free fatty acids were analyzed retrospectively on frozen samples of 4 rats per group. Plasma acetoacetate, beta-hydroxybutyrate (BHB), glucose, cholesterol and triglycerides were analyzed by automated colorimetric assay on a clinical chemistry analyzer (Dimension Xpand Plus; Siemens Healthcare Diagnostics, Deerfield, IL, USA) using commercial kits and in-house for acetoacetate [27].

Statistical analysis

Data are presented as mean ± standard deviation. Blood chemistry, hemodynamic parameters, and kinetic parameters were analyzed using a two-tailed paired Student’s t-test for baseline vs. acute ketone ester supplementation, and for rest vs. stress. A two-tailed unpaired Student’s t-test was used to compare measures of CTRL vs. T2D rats. No statistical analysis was performed for heart function because of the small sample size. The threshold for significance was p ≤ 0.05.

Results

Blood chemistry and metabolites pre- and post-intervention

At 23 weeks of age, the T2D rats were significantly heavier and their blood glucose was twice as high compared to CTRL rats (Table 1). Blood acetoacetate and cholesterol levels were also twice as high in T2D vs. CTRL, and triglycerides were elevated by a factor of ∼ 10 (p < 0.05). Under acute ketone ester supplementation, the total plasma ketone (acetoacetate + BHB) levels doubled in CTRL, while they nearly tripled in T2D.

Table 1.

Animal characteristics

	CTRL		T2D
	Baseline	Ketone supplementation	Baseline	Ketone supplementation
Body weight (g)	377 ± 24	380 ± 23	436 ± 49#	433 ± 45#
Glucose (mmol/L)	19.2 ± 1.6	20.4 ± 1.5	39.5 ± 3.9#	41.9 ± 3.0#
Beta-hydroxybutyrate (mmol/L)	0.44 ± 0.15	0.98 ± 0.17*	0.67 ± 0.37	2.15 ± 0.86*#
Acetoacetate (mmol/L)	0.25 ± 0.13	0.45 ± 0.11*	0.52 ± 0.22#	1.18 ± 0.46*#
Ketones (mmol/L)	0.69 ± 0.27	1.43 ± 0.26*	1.20 ± 0.57	3.33 ± 1.27*#
Triglycerides (mmol/L)	0.4 ± 0.2	0.5 ± 0.2	4.8 ± 1.4#	3.6 ± 1.8#
Cholesterol (mmol/L)τ	2.3 ± 0.9	2.0 ± 0.8	5.5 ± 0.5#	5.5 ± 0.5#
Free fatty acids (mmol/L)τ	0.3 ± 0.1	0.2 ± 0.0	0.4 ± 0.0	0.3 ± 0.1

CTRL: ZDF Lean (^fa/+), T2D: ZDF Obese T2D (^fa/fa), τ: N = 4 per group, *: p < 0.05 two-tailed paired Student’s t-test baseline vs. ketone supplementation, #: p < 0.05 two-tailed unpaired Student’s t-test CTRL vs. T2D

Hemodynamic parameters and heart function

In T2D, the baseline heart rate was lower by ∼ 20%, and the respiration rate was ∼ 20% higher compared to CTRL (Table 2). These vital signs were similar under ketone ester supplementation. Under pharmacological adenosine stressor, heart and respiratory rates decreased, but this reduction was more important in CTRL compared to T2D by a factor of ∼ 2 (Table 2). The left ventricular volumes were generally greater for T2D, both at rest and stress, but not under ketone supplementation, and the LVEF seemed improved by ∼ 5% under acute ketone ester supplementation at stress (no statistical analysis was performed due to a technical issue leading to N = 3 per group instead of N = 6).

Table 2.

Hemodynamic and heart function

		CTRL		T2D
		Baseline	Ketone supplementation	Baseline	Ketone supplementation
Rest
	Beats per minute (BPM)	344 ± 20	349 ± 13	289 ± 19*	263 ± 23*
	Breaths per minute	53 ± 15	46 ± 9	62 ± 23	58 ± 21
	End-diastolic volume (EDV) µLτ	372 ± 72	353 ± 13	395 ± 35	321 ± 40
	End-systolic volume (ESV) µLτ	111 ± 2	135 ± 39	158 ± 44	132 ± 45
	% Left ventricle ejection fraction (LVEF)τ	70 ± 6	62 ± 12	61 ± 8	59 ± 13
Stress
	Beats per minute (BPM)	259 ± 10#	267 ± 14#	237 ± 21*#	241 ± 26#
	Breaths per minute	32 ± 8#	33 ± 6#	48 ± 15*	48 ± 9*
	End-diastolic volume (EDV) µLτ	475 ± 21	432 ± 11	470 ± 48	316 ± 80
	End-systolic volume (ESV) µLτ	170 ± 15	153 ± 13	189 ± 46	106 ± 14
	% Left ventricle ejection fraction (LVEF)τ	64 ± 1	65 ± 2	61 ± 7	66 ± 4

CTRL: ZDF lean (^fa/+), T2D: ZDF obese T2D (^fa/fa), τ: N = 3 per group, no statistical analysis were performed. There is no significant difference between baseline and. acute ketone supplementation.^*: p < 0.05, two-tailed unpaired Student’s t-test CTRL vs. T2D. ^#p < 0.05 two-tailed unpaired Student’s t-test rest vs. stress

Cardiac PET MBF with [13N]NH3

The gold-standard [¹³N]NH₃ MBF was similar at rest between both groups (Table 3; Fig. 3). Under pharmacological adenosine stress, the MBF increased by ∼ 40% for CTRL (p < 0.01) and by ∼ 25% for T2D (p = 0.11). This blunted MBF response to stress in T2D is indicative of impaired CFR (+ 84% CTRL vs. +34% T2D, p = 0.047), a hallmark of heart failure [28].

Table 3.

Cardiac PET myocardial blood flow and coronary flow reserve with [¹³N]ammonia

	CTRL	T2D
Rest (mL/g/min)	2.7 ± 0.8	2.7 ± 0.9
Stress (mL/g/min)	4.8 ± 0.9	3.6 ± 1.9*
CFR (stress/rest)	1.8 ± 0.5	1.3 ± 0.2*

CTRL: ZDF lean (^fa/+), T2D: ZDF obese T2D (^fa/fa), ^*: p < 0.05, two-tailed unpaired Student’s t-test CTRL vs. T2D

Fig. 3.

Cardiac PET myocardial blood flow (MBF) with [¹³N]ammonia at rest and during stress (adenosine). CTRL: ZDF lean (^fa/+) rats (N = 6) and T2D: ZDF obese (^fa/fa) rats (N = 6). *: p < 0.05, two-tailed unpaired Student’s t-test CTRL vs. T2D

Cardiac PET ketone metabolism with [11C]AcAc

In CTRL rats, acute ketone supplementation did not change the [¹¹C]AcAc uptake at rest and under adenosine stress. However, pharmacological stress increased the uptake by ∼ 30% vs. rest (27% without supplement and 32% with supplement; p < 0.03) (Table 4; Fig. 4). Uptake was lower in T2D vs. CTRL in all conditions. However, acute ketone supplementation further decreased the uptake in T2D at rest (-20% vs. no supplementation, p < 0.05), but not under stress. Because of this, the stress/rest ratio of uptake in T2D goes from 1.07 ± 0.04 without supplementation to 1.35 ± 0.24 with supplementation (p = 0.04). This latter ratio is similar to what is observed in CTRL rats. Figure 2 shows typical [¹¹C]AcAc cardiac PET images summed over 90–300 s.

Table 4.

Cardiac PET uptake (K₁) of [¹¹C]acetoacetate

		Water supplementation	Ketone supplementation
CRTL
	Rest (ml/g/min)	2.2 ± 0.4	2.2 ± 0.3
	Stress (ml/g/min)	2.8 ± 0.3	2.9 ± 0.3
	Uptake ratio (stress/rest)	1.30 ± 0.29	1.29 ± 0.13
T2D
	Rest (ml/g/min)	1.5 ± 0.2	1.2 ± 0.3*
	Stress (ml/g/min)	1.6 ± 0.2	1.6 ± 0.5
	Uptake ratio (stress/rest)	1.07 ± 0.04	1.35 ± 0.24*

CTRL: ZDF lean (^fa/+), T2D: ZDF obese T2D (^fa/fa), ^*: p < 0.05, two-tailed unpaired Student’s t-test CTRL vs. T2D

Fig. 4.

Cardiac PET uptake (K₁) of [¹¹C]acetoacetate at rest and during stress (adenosine). (A) CTRL: ZDF lean (^fa/+) rats (N = 6); (B) T2D: ZDF obese (^fa/fa) rats (N = 6). *: p < 0.05, two-tailed unpaired Student’s t-test CTRL vs. T2D

In CTRL rats, ketone metabolism (k₂) was not significantly affected by adenosine stress but was increased by acute ketone supplementation (rest: +17%, p = 0.01; stress: +20%, p = 0.004) (Table 5; Fig. 5). In T2D rats, there was no significant effect of stress or ketone supplementation on ketone metabolism. However, we observed a trend toward lower metabolism at rest and higher metabolism under stress with ketone supplementation. These two effects become significant in the T2D, when combined into the stress/rest metabolic ratio which is higher with supplementation vs. no supplementation (p = 0.047).

Table 5.

Cardiac PET ketone, [¹¹C]acetoacetate metabolism (k₂)

		Water supplementation	Ketone supplementation
CRTL
	Rest (min− 1)	1.1 ± 0.1	1.3 ± 0.1
	Stress (min− 1)	1.0 ± 0.1	1.2 ± 0.1
	k2 ratio (stress/rest)	0.89 ± 0.13	0.93 ± 0.12
T2D
	Rest (min− 1)	0.9 ± 0.2	0.8 ± 0.2
	Stress (min− 1)	0.8 ± 0.2	0.9 ± 0.2
	k2 ratio (stress/rest)	0.96 ± 0.26	1.25 ± 0.29*

CTRL: ZDF lean (^fa/+):, T2D: ZDF obese T2D (^fa/fa), ^*: p < 0.05, two-tailed unpaired Student’s t-test CTRL vs. T2D

Fig. 5.

Cardiac PET metabolism (k₂) of [¹¹C]acetoacetate at rest and during stress (adenosine). (A) CTRL: ZDF lean (^fa/+) (N = 6); (B) T2D: ZDF obese (^fa/fa) (N = 6)

Discussion

To our knowledge, this is the first direct assessment of cardiac ketone metabolism with PET under acute ketone ester supplementation in T2D. At 23 weeks of age, the ZDF T2D rats represent an excellent model for the investigation of impaired metabolic flexibility and imminent heart failure in diabetes [29–31]. ZDF rats with T2D exhibited elevated blood glucose, triglycerides, and cholesterol levels, as well as greater left ventricular volume and lower left-ventricular ejection fraction. This animal model is thus representative of the metabolic abnormalities observed in adult T2D patients [32]. The CFR was decreased in the T2D model compared to the CTRL rats, but MBF was similar at rest. This is suggestive of endothelial dysfunction, a feature previously identified in this rat model, which is a hallmark of coronary microvascular disease [33, 34] and which mirrors the CFR abnormality seen in T2D heart failure patients [35]. Any improvement in CFR will usually lower the disease burden [33].

Like most studies of rodent models, this study was conducted under anesthesia. Isoflurane is known to impact several biochemical and physiological parameters, such as glycemia, heart rate and blood pressure [36, 37]. In previous rest/stress studies, we compared the effects of isoflurane and propofol [25]. We observed an equivalent drop of the heart rate using adenosine as a pharmacologic stressor. Also, isoflurane and other anesthetic agents increase blood glucose [38–40]. In our study, glycemia was measured 5 min after isoflurane induction, and results show equivalence (CTRL ∼ 20 mmol/L, T2D ∼ 40mmol/L) with and without ketone supplementation. However, no blood sample was taken at the end of the imaging session, which limits our assessment of the effect of anesthesia on blood glucose. Although anesthesia modifies heart function and blood chemistry values, great care was taken to control the level of anesthesia through monitoring of heart and respiratory rates to minimize anesthesia-related fluctuations during the experiment.

In this study, exogenous ketone supplementation in the T2D model surprisingly reduced the myocardial ketone uptake at rest. This led to an increase in the uptake ratio (K₁ stress/rest), restoring it to values observed in CTRL. The k₂ metabolic ratio (stress/rest) also increased with acute supplementation in the T2D group indicating an improved response to the energy demand brought by pharmacological stress. Therefore, ZDF T2D rats with heart failure at 23 weeks of age may benefit from the ketone supplementation. Further investigation is needed to determine the underlying mechanisms and therapeutic potential.

In a healthy population supplemented acutely or in the long-term with exogenous ketones, there is generally an increase in blood ketones, combined with a reduction in blood glucose and lipid biomarkers [41]. In our T2D model, the energy substrates were imbalanced at baseline [42, 43]. Based on the acute effects observed in this study, long-term ketone supplementation in combination with T2D treatment could potentially slow down the development of heart failure or improve heart function in patients.

Heart failure has been broadly investigated by cardiac PET imaging with [¹¹C]acetate, [¹³N]NH₃ and [¹⁵O]H₂O, proving useful to study impaired CFR and oxygen metabolism. [¹¹C]palmitate, ¹⁸F-FTHA, [¹¹C]glucose, and [¹⁸F]FDG have also been used to study cardiac flexibility to fatty acid metabolism and glucose oxidation [25, 44–46]. Our group has used [¹¹C]AcAc to assess heart ketone metabolism in a rat model with doxorubicin-induced heart failure, and found a decrease in both uptake and metabolism [10]. With [¹¹C]acetate in the same heart failure model, we have also shown a stress-induced decrease in MBF, oxygen consumption and their respective ratio and reserve [44]. In the present study, cardiac [¹³N]NH₃ PET corroborates and validates the previous CFR impairment finding [33]. Furthermore, we were able to evaluate ketone metabolism under the different conditions with the ketone radiotracer [¹¹C]AcAc, demonstrating potential benefits related to ketone supplementation [47].

The concept that the failing heart has increased ketone body oxidation is generally accepted, but the exact role of ketones and the impact of pharmacological interventions on this specific metabolism remain to be elucidated [48]. Myocardial ketone oxidation in T2D has not been investigated, probably due to concerns about high blood ketone level in diabetes. However, using the arterio-venous method, Mizuno and colleagues observed that, in T2D, the heart consumes more ketones [8]. T2D is characterized by insulin resistance that reduces glucose uptake but increases fatty acid uptake by the heart [49]. When a racemic BHB salt was infused in healthy volunteers, a decrease in heart glucose metabolism was observed, while fatty acid consumption remained unchanged [6]. In the mouse isolated perfused heart model, ketone supplementation increased ketone and glucose oxidation without affecting fatty acid consumption [7]. However, in the T2D mouse perfused heart model, in which energy production is also reduced, an increase in ketone supply only increased ketone oxidation [49]. An interesting result of the present study is that, in the T2D failing heart, the ratio between stress/rest (i.e. cardiac reserve) for ketone uptake and metabolism increased after exogenous ketone ester supplementation. This finding supports further investigation of the long-term effects of ketone supplementation for the treatment of T2D. Such investigation would be required to confirm the hypothesis that ketone supplementation can increase cardiac reserve and improve cardiac function in the failing T2D heart.

Note that the ZDF T2D rats in this study did not receive any treatment for diabetes. Therefore, they are not representative of patients followed in the clinic. It would be highly relevant to study T2D-related heart failure in cohorts undergoing diabetes treatments, notably with the promising SGLT2 inhibitors, with and without ketone supplementation, in order to determine the true potential of ketone therapy [50].

Fatty acid and glucose metabolism will also need to be evaluated, along with ketones, in the T2D heart failure model with and without treatment. The mitochondrial utilization of ketones and the anaplerotic reactions required for energy production through the Krebs cycle also need to be further investigated [51]. The impact of exogenous ketones on the Krebs cycle substrate reserves, and their replacement could deprive the energy system [47]. It is thus conceivable that the efficiency of energy supplementation could be enhanced by combining it with other by-products that are essential for the production of ATP.

Conclusion

This study has demonstrated that acute administration of exogenous ketones has an effect on myocardial consumption in a T2D model of heart failure. Exogenous supplementation increased the ketone uptake and metabolism ratios (cardiac reserve) in the failing heart. The ZDF T2D heart failure model presents the same broad risk factors as the human population with T2D and heart failure. However, further long-term investigation of this ketone intervention and heart failure model for potential therapeutic options is required, in particular in settings of chronic ketone supplementation with standard treatment for T2D. Cardiac PET imaging is a powerful tool for these types of in vivo studies, notably because it allows the investigation of multiple energy substrates and it has the amazing potential for rapid translation from animal models to humans.

Author contributions

EC: Conceptualization, methodology, analysis, writing and editing. GR: Formal analysis, methodology, writing and editing. PPH, ER, SC, BG: Formal analysis and editing. VDP, OS, SP, ST: Methodology and editing. RL: Conceptualization, formal analysis, writing and editing.

Funding

This study was supported in-house by the institution.

Data availability

The data is available upon request. Please send an email to Etienne.croteau@usherbrooke.ca to request access.

Declarations

Ethical approval

All animal experiments were performed in accordance with the Canadian Council on Animal Care guidelines and were approved by the institutional animal research ethics board.

Consent for publication

Not applicable.

Competing interests

The author(s) declare the following interests/personal relationships: Stephen C. Cunnane consults for Nestle Health Science and Cerecin. He has received financial support and materials for research from Nestle Health Science. The author(s) declare no others conflict of interest and financial support.