Ketone metabolism in the failing heart

The high energy demands of the heart are met primarily by the mitochondrial oxidation of fatty acids and glucose. However, in heart failure there is a decrease in cardiac mitochondrial oxidative metabolism and glucose oxidation that can lead to an energy starved heart. Ketone bodies are readily oxidized by the heart, and can provide an additional source of energy for the failing heart. Ketone oxidation is increased in the failing heart, which may be an adaptive response to lessen the severity of heart failure. While ketone have been widely touted as a “thrifty fuel”, increasing ketone oxidation in the heart does not increase cardiac efficiency (cardiac work/oxygen consumed), but rather does provide an additional fuel source for the failing heart. Increasing ketone supply to the heart and increasing mitochondrial ketone oxidation increases mitochondrial tricarboxylic acid cycle activity. In support of this, increasing circulating ketone by iv infusion of ketone bodies acutely improves heart function in heart failure patients. Chronically, treatment with sodium glucose co-transporter 2 inhibitors, which decreases the severity of heart failure, also increases ketone body supply to the heart. While ketogenic diets increase circulating ketone levels, minimal benefit on cardiac function in heart failure has been observed, possibly due to the fact that these dietary regimens also markedly increase circulating fatty acids. Recent studies, however, have suggested that administration of ketone ester cocktails may improve cardiac function in heart failure. Combined, emerging data suggests that increasing cardiac ketone oxidation may be a therapeutic strategy to treat heart failure.     

ICS II protects against cardiac hypertrophy by regulating metabolic remodelling,not by inhibiting autophagy

Cardiac hypertrophy is characterized by a shift in metabolic substrate utilization. Therefore, the regulation of ketone body uptake and metabolism may have beneficial effects on heart injuries that induce cardiac remodelling. In this study, we investigated whether icariside II (ICS II) protects against cardiac hypertrophy in mice and cardiomyocytes. To create cardiac hypertrophy animal and cell models, mice were subjected to transverse aortic constriction (TAC), and embryonic rat cardiomyocytes (H9C2) were stimulated with angiotensin II, a neurohumoral stressor. Both the in vivo and in vitro results suggest that ICS II treatment ameliorated pressure overload–induced cardiac hypertrophy and preserved heart function. In addition, apoptosis and oxidative stress were reduced in the presence of ICS II. Moreover, ICS II inhibited excess autophagy in TAC-induced hearts and angiotensin II–stimulated cardiomyocytes. Mechanistically, we found that ICS II administration regulated SIRT3 expression in cardiac remodelling. SIRT3 activation increased ketone body transportation and utilization. Collectively, our data show that ICS II attenuated cardiac hypertrophy by modulating ketone body and fatty acid metabolism, and that this was likely due to the activation of the SIRT3-AMPK pathway. ICS II treatment may provide a new therapeutic strategy for improving myocardial metabolism in cardiac hypertrophy and heart failure.     

CaMKII and GLUT1 in heart failure and the role of gliflozins

Empagliflozin, a selective sodium-glucose co-transporter 2 (SGLT2) inhibitor, has been shown to reduce mortality and hospitalization for heart failure in diabetic patients in the EMPA-REG-OUTCOME trial (Zinman et al., 2015). Surprisingly, dapagliflozin, another SGLT2 inhibitor, exerted comparable effects on clinical endpoints even in the absence of diabetes mellitus (DAPA-HF trial) (McMurray et al., 2019). There is a myriad of suggested underlying mechanisms ranging from improved glycemic control and hemodynamic effects to altered myocardial metabolism, inflammation, neurohumoral activation and intracellular ion homeostasis.Here, we review the effects of gliflozins on cardiac electro-mechanical coupling with an emphasis on novel CaMKII-mediated pathways and on cardiac glucose and ketone metabolism in the failing heart. We focus on empagliflozin as it is the gliflozin with the most abundant experimental evidence for direct effects on the heart. Where useful, we aim to compare empagliflozin to other gliflozins. To facilitate understanding of empagliflozin-induced alterations, we first give a short summary of the pathophysiological role of CaMKII in heart failure, as well as cardiac changes of glucose and ketone body metabolism in the failing heart.     

Brain uptake and metabolism of ketone bodies in animal models

As a consequence of the high fat content of maternal milk, the brain metabolism of the suckling rat represents a model of naturally occurring ketosis. During the period of lactation, the rate of uptake and metabolism of the two ketone bodies, beta-hydroxybutyrate and acetoacetate is high. The ketone bodies enter the brain via monocarboxylate transporters whose expression and activity is much higher in the brain of the suckling than the mature rat. beta-Hydroxybutyrate and acetoacetate taken up by the brain are efficiently used as substrates for energy metabolism, and for amino acid and lipid biosynthesis, two pathways that are important for this period of active brain growth. Ketone bodies can represent about 30-70% of the total energy metabolism balance of the immature rat brain. The active metabolism of ketone bodies in the immature brain is related to the high activity of the enzymes of ketone body metabolism. Thus, the use of ketone bodies by the immature rodent brain serves to spare glucose for metabolic pathways that cannot be fulfilled by ketones such as the pentose phosphate pathway mainly. The latter pathway leads to the biosynthesis of ribose mandatory for DNA synthesis and NADPH which is not formed during ketone body metabolism and is a key cofactor in lipid biosynthesis. Finally, ketone bodies by serving mainly biosynthetic purposes spare glucose for the emergence of various functions such as audition, vision as well as more integrated and adapted behaviors whose appearance during brain maturation seems to critically relate upon active glucose supply and specific regional increased use.     

Differential action of thyroid hormones on the activity of certain enzymes in rat kidney and brain.

In rat kidney several mitochondrial and soluble enzyme activities are stimulated by thyroid hormones and the mitochondrial membrane fluidity is also increased. However, the ketone metabolism enzyme activities of D-3-hydroxybutyrate dehydrogenase and of 3-oxoacid CoA-transferase are not significantly affected by the hyperthyroid state and the ketone body concentration is not greatly changed. Therefore, in hyperthyroid rats the response of the kidney, as far as the ketone bodies and their metabolizing enzymes are concerned, is at variance with that of the liver and the heart. In the brain of young rats, age 8-9 weeks, the activities of the enzymes of ketone body metabolism and those responsible for other metabolic pathways are not influenced by the hyperthyroid state. In these animals, however, the activities of two enzymes, NAD-isocitrate dehydrogenase and pyruvate kinase, are still stimulated by 28 and 41%, respectively. This can be probably related to the higher energy requirement for definitive brain maturation in young hyperthyroid rats.     

肝细胞的异质性：葡萄糖和酮体在大鼠肝脏的代谢

用大鼠肝脏门静脉或肝静脉周围的肝细胞来研究葡萄糖和酮体生成的区域分布。肝细胞通过毛地黄皂苷－胶原酶灌流技术分离。门静脉周围肝细胞的γ谷氨酰转肽酶的活性比肝静脉周围肝细胞高２．４倍;而谷氨酰胺合成酶的活性则相反,肝静脉周围肝细胞高出５６倍。门静脉周围肝细胞的内源性葡萄糖合成比肝静脉周围肝细胞高１．５７倍。给予刺激葡萄糖异生的底物,门静脉周围肝细胞的葡萄糖合成则增加１．７－２．１倍。肝静脉周围肝细胞的内源性酮体生成比门静脉周围肝细胞高１．３倍。给予能明显刺激酮体生成的辛酸盐,肝静脉周围肝细胞的酮体生成仅略为增加。我们的结果证实,在基础和刺激的条件下,葡萄糖的异生在门静脉周围肝细胞中优先,而酮体生成仅在肝静脉周围肝细胞占微弱的优势。     

Relationship between exogenous fuel availability and performance by teleost and elasmobranch hearts

Summary Performance by perfused isolated hearts of sea raven (Hemitripterus americanus) and skate (Raja erinecea), representatives of teleost and elasmobranch fishes, respectively, was monitored over a 30 min period under conditions of variable metabolic fuel availability. In both preparations initial cardiac output and hence fuel delivery to the myocardia were comparable to in vivo levels. Pressure development and hence overall work rate of the sea raven heart was also similar to in vivo levels.Fuel deprived sea raven hearts entered into a modest but significant contractile failure which could be prevented by the inclusion of 10 mM glucose or 1.0 mM palmitate in the perfusion medium. Addition of the glycolytic inhibitor iodoacetate to the medium resulted in rapid heart failure. Performance in the presence of iodoacetate could be improved by the inclusion of palmitate, lactate, or acetoacetate in the perfusion media but only high physiological levels of palmitate could completely alleviate the effect of iodoacetate.The inclusion of 1.0 mM palmitate in the perfusion medium of skate hearts resulted in a significant decrease in performance relative to fuel deprived hearts. Addition of iodoacetate to the medium resulted in rapid contractile failure. Hearts perfused with medium containing both iodoacetate and acetoacetate performed as well as fuel deprived hearts, indicating that this ketone body is an effective metabolic fuel.The performance data reported here are consistent with a previously established biochemical framework. The teleost heart has the capability of utilizing exogenous fatty acid as a metabolic fuel and this substrate may be able to support the contractile process independently. In contrast, fatty acid metabolism in the elasmobranch heart is poorly developed and appears to be more dependent upon the catabolism of blood borne ketone bodies.     

Versatility of microglial bioenergetic machinery under starving conditions

Microglia are highly dynamic cells in the brain. Their functional diversity and phenotypic versatility brought microglial energy metabolism into the focus of research. Although it is known that microenvironmental cues shape microglial phenotype, their bioenergetic response to local nutrient availability remains unclear.In the present study effects of energy substrates on the oxidative and glycolytic metabolism of primary – and BV-2 microglial cells were investigated. Cellular oxygen consumption, glycolytic activity, the levels of intracellular ATP/ADP, autophagy, mTOR phosphorylation, apoptosis and cell viability were measured in the absence of nutrients or in the presence of physiological energy substrates: glutamine, glucose, lactate, pyruvate or ketone bodies.All of the oxidative energy metabolites increased the rate of basal and maximal respiration. However, the addition of glucose decreased microglial oxidative metabolism and glycolytic activity was enhanced. Increased ATP/ADP ratio and cell viability, activation of the mTOR and reduction of autophagic activity were observed in glutamine-supplemented media. Moreover, moderate and transient oxidation of ketone bodies was highly enhanced by glutamine, suggesting that anaplerosis of the TCA-cycle could stimulate ketone body oxidation.It is concluded that microglia show high metabolic plasticity and utilize a wide range of substrates. Among them glutamine is the most efficient metabolite. To our knowledge these data provide the first account of microglial direct metabolic response to nutrients under short-term starvation and demonstrate that microglia exhibit versatile metabolic machinery. Our finding that microglia have a distinct bioenergetic profile provides a critical foundation for specifying microglial contributions to brain energy metabolism.     

A Histochemical Study of the Distribution of β-Hydroxybutyrate Dehydrogenase in Developing Rat Cerebellum

The distribution of beta-hydroxybutyrate dehydrogenase (3-hydroxybutyrate dehydrogenase, EC 1.1.1.30) in the developing rat cerebellum has been determined using a histochemical method. Staining of Purkinje cells, particularly the soma, was seen at all ages examined. Intense staining of the proximal portions of Purkinje dendrites was noted at 8-11 days postnatally, with less prominent staining of Purkinje dendrites and surrounding structures of the molecular layer seen at later times. Development of glomeruli in the granule cell layer could also be observed due to the intense staining of these structures. (Although noncerebellar structures were not the focus of this study, intense staining of the choroid plexus of the fourth ventricle was also noted.) the transient external germinal layer of the cerebellum did not show appreciable staining. Since beta-hydroxybutyrate dehydrogenase is required for ketone body metabolism, the apparent low level of this enzyme in the external germinal layer suggests that the cells of this layer are not particularly well adapted for utilization of ketone bodies. Thus these results do not provide support for the suggestion that ketone bodies may serve as major substrates for energy metabolism in the external germinal layer of the developing cerebellum. Indeed, the rather restricted distribution of this enzyme in both developing and mature cerebellum (and presumably elsewhere in brain) suggests that ketone body metabolism may be largely confined to relatively few specific cellular compartments.     

Lipid, ketone body and oxidative metabolism in the African lungfish, Protopterus dolloi following 60 days of fasting and aestivation

The potential importance of lipids and ketone bodies as fuels in the African lungfish, Protopterus dolloi, and the role of oxidative metabolism, were examined under control, fasted and aestivated conditions. In aestivating but not fasting lungfish, the activities of citrate synthase (CS) and cytochrome c oxidase (CCO) (enzymes of oxidative metabolism) showed tissue-specific changes. Significant reductions in CS activity occurred in the kidney, heart, gill and muscle, and in CCO in the liver and kidney tissues. Aestivation, but not fasting, also had a tissue-specific effect on mitochondrial state 3 respiration rates (using succinate as a substrate), with a >50% reduction in the liver, yet no change within muscle mitochondria. There is no indication that enzymes involved in lipid catabolism are up-regulated during periods of fasting or aestivation; however, both 3-hydroxyacyl CoA dehydrogenase (HOAD) and carnitine palmitoyl CoA transferase (CPT) activities were sustained in the liver despite the approximately 42% reduction in CCO activity, potentially indicating lipid metabolism is of importance during aestivation. Lungfish are able to utilize both the d- and l-stereoisomers of the ketone body beta-hydroxybutyrate (beta-HB); however, beta-HB does not appear to be an important fuel source during aestivation or fasting as no changes were observed in beta-HB tissue levels. This study demonstrates that an important aspect of metabolic depression during aestivation in lungfish is the tissue-specific down regulation of enzymes of aerobic metabolism while maintaining the activities of enzymes in pathways that supply substrates for aerobic metabolism.     

Empagliflozin improves left ventricular diastolic function of db/db mice

ObjectivesInvestigation of the effect of SGLT2 inhibition by empagliflozin on left ventricular function in a model of diabetic cardiomyopathy.BackgroundSGLT2 inhibition is a new strategy to treat diabetes. In the EMPA-REG Outcome trial empagliflozin treatment reduced cardiovascular and overall mortality in patients with diabetes presumably due to beneficial cardiac effects, leading to reduced heart failure hospitalization. The relevant mechanisms remain currently elusive but might be mediated by a shift in cardiac substrate utilization leading to improved energetic supply to the heart.MethodsWe used db/db mice on high-fat western diet with or without empagliflozin treatment as a model of severe diabetes. Left ventricular function was assessed by pressure catheter with or without dobutamine stress.ResultsTreatment with empagliflozin significantly increased glycosuria, improved glucose metabolism, ameliorated left ventricular diastolic function and reduced mortality of mice. This was associated with reduced cardiac glucose concentrations and decreased calcium/calmodulin-dependent protein kinase (CaMKII) activation with subsequent less phosphorylation of the ryanodine receptor (RyR). No change of cardiac ketone bodies or branched-chain amino acid (BCAA) metabolites in serum was detected nor was cardiac expression of relevant catabolic enzymes for these substrates affected.ConclusionsIn a murine model of severe diabetes empagliflozin-dependent SGLT2 inhibition improved diastolic function and reduced mortality. Improvement of diastolic function was likely mediated by reduced spontaneous diastolic sarcoplasmic reticulum (SR) calcium release but independent of changes in cardiac ketone and BCAA metabolism.     

Purification and properties of a pig heart thiolase with broad chain length specificity and comparison of thiolases from pig heart and Escherichia coli.

A thiolase (acetyl CoA acyltransferase, EC 2.3-1.16) which acts on substrates of various chain lengths (thiolase I) has been purified from pig heart muscle 366-fold to near homogeneity as judged by gel electrophoresis. Its molecular weight was estimated to be 200,000 in the absence and 46,000 in the presence of sodium dodecyl sulfate. Kinetic measurements with acetoacetyl coenzyme A, 3-ketohexanoyl-CoA, 3-ketooctanoyl-CoA, and 3-ketodecanoyl-CoA yielded apparent Km values of 16, 8.3, 2.4, and 1.8 micron, respectively, whereas apparent Vmax values of 65 to 69 mumol/min/mg were obtained with all substrates except for acetoacetyl-CoA, with which a value of 26.5 mumol/min/mg was observed. Antibodies prepared against this thiolase were used to demonstrate that thiolase I and acetoacetyl-CoA thilase (thiolase II) from pig heart mitochondria are immunologically unrelated. The antibodies cross-reacted, however, with thiolase I from beef heart. Kinetic constants (Km, Vmax) were also determined for thiolases I and II from Escherichia coli, as were the native and subunit molecular weights of E. coli thiolase II. Although the E. coli thiolases were found to be immunologically distinct from the pig heart enzymes, their physical and kinetic properties are strikingly similar to those of the heart thiolases. In view of this finding and in view of the known physiological functions of the E. coli thiolases, it is proposed that thiolase I from pig heart is only involved in fatty acid metabolism, whereas thiolase II functions solely in ketone body degradation.     

Acetoacetate,d-3-hydroxybutyrate and glucose utilization by capillaries isolated from developing rat brain

Isolated cerebral capillaries from developing rats utilize glucose as well as ketone bodies essentially for oxidative metabolism. However, CO₂ production from [U-¹⁴C]glucose was significantly greater than from ketone bodies (except at 5 mM). Ketone body utilization (in the presence of 5 mM glucose in the incubation medium) was concentration-dependent (up to 5 mM). Lipid synthesis from ketone bodies was comparable to that from glucose up to 1 mM. At concentrations ⩾ 1 mM, acetoacetate incorporation into total lipids and fatty acids was higher than other substrates, however, this difference was statistically significant only at 5 mM. Incorporation of substrates into sterols was very low (> 1 pmol/h/mg protein).     

酮体代谢与阿尔茨海默病

阿尔茨海默病(Alzheimer's disease,AD)是老年痴呆症的一种主要类型,也是神经退行性疾病中发病率最高的一种疾病.随着我国老龄人口的持续上升,AD患者人数也呈增长趋势.研究表明,脑内葡萄糖代谢的降低远早于β淀粉样沉淀发生,而酮体是脑内替代葡萄糖的主要能量来源.因此,脑中能量代谢底物转换为酮体是AD早期代谢特征.目前,AD病理进程中酮体调控的机制还不清楚.深入了解AD发生、发展过程中酮体代谢的分子机制,对于寻找AD早期诊断标志物、探索AD的防治方法具有重要意义.本文就酮体代谢及其在AD中的研究进展进行综述.     

Enzyme activities support the use of liver lipid-derived ketone bodies as aerobic fuels in muscle tissues of active sharks

Few data exist to test the hypothesis that elasmobranchs utilize ketone bodies rather than fatty acids for aerobic metabolism in muscle, especially in continuously swimming, pelagic sharks, which are expected to be more reliant on lipid fuel stores during periods between feeding bouts and due to their high aerobic metabolic rates. Therefore, to provide support for this hypothesis, biochemical indices of lipid metabolism were measured in the slow-twitch, oxidative (red) myotomal muscle, heart, and liver of several active shark species, including the endothermic shortfin mako, Isurus oxyrinchus. Tissues were assayed spectrophotometrically for indicator enzymes of fatty acid oxidation (3-hydroxy-o-acyl-CoA dehydrogenase), ketone-body catabolism (3-oxoacid-CoA transferase), and ketogenesis (hydroxy-methylglutaryl-CoA synthase). Red muscle and heart had high capacities for ketone utilization, low capacities for fatty acid oxidation, and undetectable levels of ketogenic enzymes. Liver demonstrated undetectable activities of ketone catabolic enzymes but high capacities for fatty acid oxidation and ketogenesis. Serum concentrations of the ketone beta-hydroxybutyrate varied interspecifically (means of 0.128-0.978 micromol mL(-1)) but were higher than levels previously reported for teleosts. These results are consistent with the hypothesis that aerobic metabolism in muscle tissue of active sharks utilizes ketone bodies, and not fatty acids, derived from liver lipid stores.     

Ketone bodies and two-compartment tumor metabolism: Stromal ketone production fuels mitochondrial biogenesis in epithelial cancer cells

We have previously suggested that ketone body metabolism is critical for tumor progression and metastasis. Here, using a co-culture system employing human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts, we provide new evidence to directly support this hypothesis. More specifically, we show that the enzymes required for ketone body production are highly upregulated within cancer-associated fibroblasts. This appears to be mechanistically controlled by the stromal expression of caveolin-1 (Cav-1) and/or serum starvation. In addition, treatment with ketone bodies (such as 3-hydroxy-butyrate, and/or butanediol) is sufficient to drive mitochondrial biogenesis in human breast cancer cells. This observation was also validated by unbiased proteomic analysis. Interestingly, an MCT1 inhibitor was sufficient to block the onset of mitochondrial biogenesis in human breast cancer cells, suggesting a possible avenue for anticancer therapy. Finally, using human breast cancer tumor samples, we directly confirmed that the enzymes associated with ketone body production (HMGCS2, HMGCL and BDH1) were preferentially expressed in the tumor stroma. Conversely, enzymes associated with ketone re-utilization (ACAT1) and mitochondrial biogenesis (HSP60) were selectively associated with the epithelial tumor cell compartment. Our current findings are consistent with the “two-compartment tumor metabolism” model. Furthermore, they suggest that we should target ketone body metabolism as a new area for drug discovery, for the prevention and treatment of human cancers.     

Acetoacetate, d-3-hydroxybutyrate and glucose utilization by capillaries isolated from developing rat brain

Isolated cerebral capillaries from developing rats utilize glucose as well as ketone bodies essentially for oxidative metabolism. However, CO₂ production from [U-¹⁴C]glucose was significantly greater than from ketone bodies (except at 5 mM). Ketone body utilization (in the presence of 5 mM glucose in the incubation medium) was concentration-dependent (up to 5 mM). Lipid synthesis from ketone bodies was comparable to that from glucose up to 1 mM. At concentrations 1 mM, acetoacetate incorporation into total lipids and fatty acids was higher than other substrates, however, this difference was statistically significant only at 5 mM. Incorporation of substrates into sterols was very low (> 1 pmol/h/mg protein).     

Stoichiometric traps in the tricarboxylic acid cycle. I. Self-inhibition and triggering phenomena

The steady-state oxidation of 2 mM pyruvate in pigeon and rat heart mitochondria in the presence of ADP-glucose-hexokinase load can be strongly inhibited by excess (10-40 mM) of pyruvate or beta-hydroxybutyrate. This inhibition is accompanied by the accumulation of alpha-ketoglutarate and a decrease of malate. The mechanism of such substrate inhibition may be associated with the limitation of the tricarboxylic acid cycle flux by low levels of oxaloacetate and free CoA due to their being trapped as alpha-ketoglutarate and acetyl-CoA. Contrary to pyruvate, the ketone bodies in the absence of other substrates produce self-inhibition of their oxidation at as low concentrations as 0.5-1 mM. At 10-15 mM of acetoacetate, a complete suppression of respiration may develop. At a high load (preset by ADP or the uncoupler CCCP), the suppression is characterised by the accumulation of malate and a decrease of alpha-ketoglutarate. At low loads, the reverse distribution of the intermediates takes place. It is concluded that the system of ketone body oxidation in heart mitochondria is an example of biochemical triggers (systems with two alternative stable states).