Ketone Body Metabolism in the Neonate

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37 

Ketone Body Metabolism in the Neonate Baris Ercal  |  Peter A. Crawford

INTRODUCTION The metabolism of ketone bodies is evolutionarily conserved among all the domains of life on Earth.1-3 In mammals, ketone bodies are predominantly synthesized in the liver from acetyl coenzyme  A (CoA) derived from fatty acid oxidation, and

are then transported to peripheral tissues for oxidation during physiologic states consisting of limited carbohydrate and surplus fatty acid availability (reviewed by Robinson and Williamson,4 McGarry and Foster,5 and Cotter and colleagues6; Figure 37-1, A and B). During the neonatal period, starvation, and adherence to low-carbohydrate diets, ketone body oxidation contributes significantly to energy metabolism within numerous



Chapter 37 — Ketone Body Metabolism in the Neonate Hepatic Ketogenesis

Ketone Body Oxidation

Acyl-CoA

βOHB

β-Oxidation 2 Acetyl-CoA

AcAc

CoA-SH + ATP AcAc-CoA CoA-SH cThiolase AACS

NADH AcAc Succinyl-CoA

mThiolase

SCOT

CoA-SH

AcAc-CoA Acetyl-CoA HMGCS2 CoA-SH

HMGCL CO2

Succinate

2 Acetyl-CoA HCO–3 ACC + ATP Malonyl-CoA

AcAc-CoA CoA-SH mThiolase

HMG-CoA

Acetone

Nonoxidative Fates of Ketone Bodies

NAD+

BDH1

2 Acetyl-CoA

Fatty acid or glucose

Citrate synthase

Acetyl-CoA

AcAc

FAS Lipogenesis

TCA Cycle

HMGCS1

CoA-SH HMG-CoA HMGCR Mevalonate

NADH BDH1

NAD+

βOHB

A

371

B

C

Cholesterol synthesis

Figure 37-1  Pathways of ketone body metabolism. A, Hepatic pathway of mitochondrial ketogenesis via 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase 2 (HMGCS2). B, Oxidation of ketone bodies within mitochondria of peripheral tissues via succinyl coenzyme A:3-oxoacid coenzyme A transferase (SCOT). Substrate competition with acetyl coenzyme A (acetyl-CoA) derived from glycolysis or fatty acid oxidation is also shown. C, Nonoxidative metabolic fates of ketone bodies including cytoplasmic lipogenesis and cholesterol synthesis. AACS, Acetoacetyl coenzyme A synthetase; AcAc-CoA, acetoacetyl coenzyme A; ACC, acetyl coenzyme A carboxylase; ATP, adenosine triphosphate; BDH1, β-hydroxybutyrate dehydrogenase 1; βOHB, β-hydroxybutyrate; CoA, coenzyme A; CoA-SH, free coenzyme A; FAS, fatty acid synthase; HMGCL, HMG-CoA lyase; HMGCS1, HMG-CoA synthase 1; HMGCR, HMG-CoA reductase; NAD+, oxidized nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; PDH, pyruvate dehydrogenase; TCA, tricarboxylic acid; cThiolase, cytoplasmic thiolase; mThiolase, mitochondrial thiolase.

extrahepatic tissues (Figure 37-2). In these carbohydrate-limiting states, circulating ketone body concentrations can increase from approximately 50  µmol/L in a normal fed mature human to up to 7  mmol/L. In neonatal humans, or after a prolonged fast in healthy adults, circulating ketone body concentrations can rise to approximately 1  mmol/L. In certain pathologic states such as diabetic ketoacidosis, ketone body concentrations can reach as high as 20  mmol/L if the state is left untreated.2,4,7 Although ketone bodies often serve energetic roles, ketone body metabolism also provides substrates for de novo lipogenesis and sterol biosynthesis in many tissues, including the developing brain, lactating mammary gland, and liver8-11 (see Figure 37-1, C). Within the mitochondria, hepatic ketogenesis converges with the fundamental metabolic pathways of fatty acid β-oxidation, the tricarboxylic acid (TCA) cycle, and gluconeogenesis (Figure 37-3). In various disease states, including infantile ketoacidosis and type 1 diabetes, dysfunctional ketone body metabolism is observed and may even play a role in pathogenesis. Ketone body metabolism also shifts over the course of normal development and aging.1220 The dynamic role of ketone body metabolism in the neonatal period will be the primary focus of this chapter.

AVAILABILITY OF KETONE BODIES Under physiologic conditions, the rate of utilization of ketone bodies is directly proportional to their concentration in the circulation, which in turn represents a balance between production (ketogenesis) by the liver and disposal by peripheral tissues

(ketolysis). Ketone bodies are excreted in the urine (ketonuria) when the renal reabsorption threshold is exceeded. The relationship between production and disposal can be disturbed if the utilization of ketone bodies is inhibited by drugs,21,22 with congenital absence of key enzymes required for ketone body utilization,23 or in insulin-deficient states secondary to a metabolic defect in utilization.24 The concentration of ketone bodies in the blood is extremely sensitive to alterations in the physiologic state. The balance of ketone body production and disposal determines the steady-state circulating concentration of ketone bodies, and although it is inappropriate to apply the definitions universally among all individuals, in general terms, in humans normoketonemia is characterized by a serum total ketone body concentration of less than 0.2 mmol/L, ketonemia is characterized by a serum total ketone body concentration of greater than 0.2 mmol/L, and ketoacidosis is characterized by a serum total ketone body concentration of greater than 7 mmol/L.25 In adult humans, small but characteristic diurnal changes in blood ketone body concentrations have been observed.26 Larger increases in concentration occur with fasting (in both humans and rats), with consumption of a high-fat diet, after exercise, in late pregnancy, and during suckling (Table 37-1). In pregnancy, blood ketone bodies are available to the fetus in prevailing concentrations because the placenta appears to be freely permeable to the ketone bodies acetoacetate (AcAc) and β-hydroxybutyrate (βOHB).27,28

KETONEMIA IN PREGNANCY The major substrate of the mammalian fetus is glucose supplied by the mother.28 However, the concentration of ketone

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SECTION VI — Lipid Metabolism

Triglycerides Lipolysis βOHB AcAc

Fatty acids

Ketogenesis Terminal oxidation βOHB AcAc

Figure 37-2  Integrative physiology of ketone body metabolism. Intertissue coordination of hepatic ketogenesis and peripheral disposal of ketone bodies. AcAc, Acetoacetate; βOHB, β-hydroxybutyrate.

Fatty acids β-Oxidation Thiolase Acetyl-CoA

PDH Pyruvate PC

PK PEP

CS OAA

PEPCK

Glycolysis

SuccinylCoA

ACLY Ac-CoA + OAA ACC

TCA Cycle

Malonyl-CoA α-KG

Gluconeogenesis

HMG-CoA HMGCL

Citrate

ME

Malate

HMGCS2 AcAc-CoA

FAS

CO2 AcAc BDH1 βOHB

Acetone NADH NAD+

Lipogenesis

Glucose Figure 37-3  Integration of ketogenesis with hepatic mitochondrial metabolism. Ketogenesis is shown here interfacing with the hepatic mitochondrial metabolic pathways, including the tricarboxylic acid (TCA) cycle, lipogenesis, anaplerosis, cataplerosis, and glucose metabolism. AcAc, Acetoacetate; ACC, acetyl coenzyme A carboxylase; ACLY, adenosine triphosphate citrate lyase; BDH1, β-hydroxybutyrate dehydrogenase 1; βOHB, β-hydroxybutyrate; CoA, coenzyme A; CS, citrate synthase; FAS, fatty acid synthase; HMG, 3-hydroxy-3-methylglutaryl; HMGCL, 3-hydroxy-3-methylglutaryl coenzyme A lyase; HMGCS2, 3-hydroxy-3-methylglutaryl coenzyme A synthase 2; α-KG, α-ketoglutarate; ME, malic enzyme; NAD+, oxidized nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; OAA, oxaloacetate; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PEP, phosphenolpyruvate; PEPCK, phosphenolpyruvate carboxykinase; PK, pyruvate kinase.



Chapter 37 — Ketone Body Metabolism in the Neonate

Table 37-1  Range of Blood Ketone Body Concentrations in Humans and Rats Clinical/ Experimental Condition Fed normal diet Fed high-fat diet Fasted: 12-24 hr Fasted: 48-72 hr After exercise Late pregnancy Late pregnancy after 48-hr fast Neonate: 0-1 day Neonate: 5-10 days Hypoglycemia Untreated diabetes mellitus

Ketone Body Concentration (mmol/L)* Human

Rat

~0.1 ≤3 ≤0.3 2-3 ≤2 ≤1 4-6

≤0.3 4-5 1-2 2-3 ≤2 ≤0.3 6-15

0.2-0.5 0.7-1.0 1.5-5 ≤25

0.2 0.5-1.1 — ≤10

*Concentrations of total ketone bodies (acetoacetate plus 3-hydroxybutyrate measured by enzyme assay) in whole blood.

bodies in maternal blood is increased in the last trimester of pregnancy,25,27 and high concentrations are attained during delivery, particularly if it is prolonged (ketosis of labor).27,28 This increase in the concentration of blood ketone bodies may be related in part to the decrease in food intake around parturition because hyperketonemia develops more rapidly with fasting in women in the second trimester of pregnancy than in nonpregnant control subjects.29,30 Poorly controlled diabetes during pregnancy (including gestational diabetes) leads to wide fluctuations in the concentrations of blood ketone bodies.31

KETONEMIA IN THE NEONATAL PERIOD During the suckling period the neonate is presented with a relatively high-fat and low-carbohydrate diet. Both humans and rats have marked hyperketonemia in the early suckling period, compared with the respective adult fed values (see Table 37-1). By contrast, the concentrations of ketone bodies are not increased to the same extent in the blood of puppies,32 piglets,33 and lambs.34 Fatty acids (both long-chain fatty acids [LCFAs] and medium-chain fatty acids [MCFAs]) are the major precursors of ketone bodies, and one contributing reason for the species differences in neonatal ketonemia may be the fat content of the maternal milk (rat milk contains 14.8 g fat/100 g; sheep milk contains 5.3 g fat/100 g). Some mammalian species, including pigs, do not leverage ketogenesis as a high-capacity conduit for disposal of excess acetyl-CoA, instead using acetate to export acetyl-CoA carbons.35,36 Neonatal hyperketonemia in humans is a physiologic event, and thus any marked deviation in blood ketone body concentration likely indicates underlying disease.37 Increased concentrations of ketone bodies may accompany the hypoglycemia associated with inborn errors of metabolism, and a decrease may occur in hyperinsulinism. It is therefore advisable to measure blood ketone body concentrations by a specific enzymatic method38 in neonates presenting with hypoglycemia or any other abnormality in the concentration of circulating substrates. For example, infants born small for their gestational age have increased blood concentrations of gluconeogenic substrates, in association with decreased blood ketone body concentrations.37,39 The latter is due to defective development of the ketogenic capacity of the liver and decreased availability of peripheral

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adipose tissue to supply the key ketogenic substrate nonesterified fatty acids (see later).

REGULATION OF KETOGENESIS Insight into the regulation of ketogenesis has increased dramatically in recent years with the use of novel genomic, proteomic, and metabolomic analyses. Earlier investigations were based mainly on studies in rats—in particular, during the fed-to-starved transition, as well as from work on perfused livers or isolated hepatocytes from adult rats. Consequently, in the review of regulation of ketogenesis in the adult presented in this section, comparison is made with the neonate or fetus whenever information is available. Further details are available in more detailed reviews.5,6,40-43

EXTRAHEPATIC REGULATION The regulation of ketogenesis is both extrahepatic and intrahepatic (see Figure 37-2). The major precursors of ketone bodies in the postabsorptive state are LCFAs, and in all physiologic situations associated with hyperketonemia, including those of the neonatal period, the plasma concentrations of LCFAs are increased (see Table 37-1).44-47 Extraction of LCFAs by the liver is concentration dependent, and a direct relationship between their concentration and the rate of ketogenesis exists in both rats and humans.48

ADIPOSE TISSUE LIPOLYSIS In the adult, a key factor in determining the supply of LCFAs to the liver is their rate of release (lipolysis) from adipose tissue triacylglycerol stores. Lipolysis is initiated by activation of adipose tissue lipases, adipose triglyceride lipase, and hormonesensitive lipase.49 Glucagon, epinephrine, norepinephrine, and thyroxine increase enzyme activity, whereas insulin (as well as some prostaglandins) has the opposite effect.50 Isolated human adipocytes from neonates are very sensitive to the lipolytic effects of thyrotropin,51 which increases in concentration immediately after birth and may therefore be involved in the regulation of lipolysis in the perinatal period. A decrease in blood glucose concentration (e.g., in starvation, fat feeding) along with a concomitant decrease in plasma insulin concentration leads to an increase in lipolysis and efflux of nonesterified fatty acids from adipose tissue. Carbohydrate provision increases insulin concentrations and thereby inhibits lipolysis. The rate of ketogenesis can therefore respond in a reciprocal way to the availability of glucose in the circulation, so an alternative fuel for the brain is provided when needed (see Figure 37-2). During suckling in the rat, the plasma insulin-to-glucagon ratio is decreased, favoring lipolysis.52,53 Ketone bodies regulate their own formation by feedback mechanisms on adipose tissue to decrease lipolysis. Current evidence suggests there are two mechanisms: (1) direct inhibition of lipolysis by ketone bodies via binding to the G protein– coupled niacin receptor GPR109A and (2) an indirect effect mediated by stimulation of insulin secretion.4,54,55 Work with the perfused rat pancreas suggests that ketone bodies increase insulin secretion only at concentrations above 1 mmol/L.56 These feedback mechanisms have not been investigated in neonates. In the suckling neonate, the MCFAs (i.e., 12 or fewer carbons) of maternal milk are an important source of precursors for ketogenesis.57 These are in the form of triacylglycerols (one MCFA and two LCFAs per triacylglycerol) and are hydrolyzed by the action of lingual lipase. They are rapidly absorbed from the stomach into the portal venous system58 and are therefore directly available to the liver, unlike LCFAs derived from milk lipids, which are transported as triacylglycerols (within

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SECTION VI — Lipid Metabolism

chylomicrons) by way of the lymphatic system and initially enter the peripheral circulation. In the neonatal period, chylomicrons may provide a direct source of LCFAs for the liver, as suggested by the presence of lipoprotein lipase (responsible for the hydrolysis of triacylglycerols contained in chylomicrons to LCFAs) in neonatal rat liver.59 However, the total lipoprotein lipase activity in liver constitutes only a small percentage (3%) of that in the whole body,57 the rest being contributed by muscle and adipose tissue.

INTRAHEPATIC REGULATION The liver is the primary tissue capable of synthesis and release of ketone bodies into the circulation, primarily due to the relatively hepatocyte-specific expression of the fate-committing ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 2 (HMGCS2). Ketone bodies can also be formed by the intestinal mucosa of neonatal rats via enterocytederived HMGCS2.60,61 Expression of HMGCS2 in intestinal mucosa is suppressed in weaned suckling rats, but is regulated by the gut microbiome.62-64 The rate of intestinal ketogenesis is less than 10% of that in liver of suckling animals, but this mechanism does provide an additional strategy for supplying ketone bodies to developing tissues.57

HEPATIC FATTY ACID CATABOLISM LCFAs hydrolyzed from triacylglycerols in adipose tissue are transported in the plasma bound to albumin, cross the cell membrane as free fatty acids, and then bind again to cytosolic binding proteins.4 Within the liver, the LCFAs either can be reesterified to form triacylglycerols (for subsequent secretion as very-lowdensity lipoprotein) and phospholipids or can enter the mitochondria by way of the carnitine palmitoyltransferase (CPT) system to undergo β-oxidation. The resulting yield of acetyl-CoA can (1) be converted to the ketone bodies AcAc and βOHB via a series of four mitochondrial enzymatic reactions referred to as the HMG-CoA pathway (see Figure 37-1, A),65-68 (2) enter the TCA cycle, where it can undergo terminal oxidation, or (3) pass out of mitochondria as citrate to be used as a cytosolic substrate for fatty acid or sterol biosynthesis69 (see Figures 37-2 and 37-3). By contrast, MCFAs are not converted to triacylglycerols in mammalian liver, and directly traverse the inner mitochondrial membrane, bypassing the CPT system. Within the mitochondrial matrix, the MCFAs are converted to the corresponding acyl-CoA derivatives by acyl-CoA synthetases and undergo β-oxidation. Ketone bodies efflux from hepatocytes via SLC16 members of the monocarboxylate transporter (MCT) family, enter the circulation, are transported into cells of extrahepatic tissues through MCT-dependent mechanisms, and are ultimately oxidized.70 Mechanisms of ketone body transport into and out of mitochondria have not been definitively established.

MITOCHONDRIAL 3-HYDROXY-3-METHYLGLUTARYL COENZYME A SYNTHASE REGULATION The mitochondrial matrix enzyme HMGCS2 catalyzes the formation of HMG-CoA from acetoacetyl-CoA and acetyl-CoA. A cytosolic HMG-CoA synthase, HMG-CoA synthase 1 (HMGCS1), catalyzes the same reaction in the sterol biosynthetic pathway (see Figure 37-1, C). HMGCS2 diverged from HMGCS1 between 400 million and 900 million years ago, and this occurred, in part, to support the emergence of early vertebrates and the development of larger brains.71 In the 1960s, HMGCS2 was proposed by Williamson and colleagues72 to be the fate-committing enzyme of hepatic ketogenesis, and subsequent experiments by separate groups confirmed this.73 Expression of the Hmgcs2 gene and the encoded protein is dynamically regulated throughout various physiologic contexts, including the transition to extrauterine life, starvation, diabetes, aging, and during adherence to ketogenic diets.2,12,74-77

Transcriptional regulation of Hmgcs2 includes methylation of its 5′ regulatory sequences, which silences Hmgcs2 transcription in fetal liver as well as in nonketogenic adult tissues.78 At birth these regulatory regions of hepatic Hmgcs2 become hypomethylated, allowing it to respond to circulating hormones.78-80 Insulin and glucagon regulate Hmgcs2 transcription via sequestration of forkhead box A2 (FOXA2) from the nucleus or activation of cyclic adenine monophosphate (cAMP) regulatory element binding protein, respectively.64,67,76,79,81 Free fatty acids induce Hmgcs2 expression in a peroxisome proliferator–activated receptor α (PPARα) dependent manner.82,83 Inhibition of mammalian target of rapamycin complex 1 signaling has been identified as a primary mechanism responsible for relieving repression of PPARα transcriptional activity and thus inducing ketogenesis during the fasting period.12,84 HMGCS2 may translocate to the nucleus and play a role as a transcriptional cofactor for PPARα and induce its own gene transcription.85,86 Using an established PPARα knockout mouse model, a recent study assessed the impact of the loss of this transcription factor specifically in the livers of neonatal mice. Livers of neonatal PPARα-knockout mice exhibited surprisingly normal terminal fatty acid oxidation, despite decreased expression of acyl-CoA dehydrogenases. However, PPARα-deficient neonates exhibited impaired ketogenesis, with associated decreases in messenger RNA (mRNA) and protein abundances of both HMGCS2 and βOHB dehydrogenase 1 (BDH1). These knockout mice accumulated a substantial amount of triacylglyerols in their livers, thus implicating ketogenic deficiency in defective hepatic lipid metabolism.87 PPARα-dependent regulation of ketogenesis during the neonatal period and starvation is mediated via hepatic fibroblast growth factor 21, an important circulating biomarker and signaling molecule involved in glucose and lipid metabolism in a variety of tissues.82,88-90 HMGCS2 also appears to regulate fibroblast growth factor 21 in a positive feedback loop, although this observation requires further experimental evaluation.91 Since the resolution of the crystal structure of human HMGCS2,92 ongoing investigations have addressed the role of posttranslational modifications in regulating enzyme catalytic activity and the subsequent impact on ketogenesis among other cellular metabolic pathways. Recent studies have demonstrated that HMGCS2 is regulated by lysine acetylation and succinylation, as well as via serine phosphorylation. Two members of the sirtuin family of oxidized nicotinamide adenine dinucleotide dependent deacylases, sirtuin 3 and sirtuin 5, were further shown to deacetylate and desuccinylate HMGCS2, respectively. Lysine deacetylation and desuccinylation, and serine phosphorylation all increase HMGCS2 catalytic activity.93-96 Although these posttranslational modifications alter HMGCS2 activity in vitro, their roles and effects in vivo have yet to be determined. A transgenic mouse model of hepatic HMGCS2 overexpression revealed augmented ketogenesis, normoglycemia, normal plasma triglyceride concentrations, diminished concentrations of circulating fatty acids, and hyperketonemia.97 Unfortunately, this model was not explored in neonatal, dietary, or metabolic contexts other than the adult fed state. A more recent study revealed the roles of HMGCS2 in carbohydrate-laden, non– classically ketogenic states using an antisense oligonucleotide– based approach to knockdown Hmgcs2 to generate ketogenesis-insufficient mice. This model revealed alterations in TCA cycle activity and increased glucose and fatty acid synthesis in ketogenesis-insufficient animals, indicating redirection of acetyl-CoA handling. When given a high-fat diet, adult ketogenesisinsufficient mice exhibited features of nonalcoholic steato­ hepatitis, including liver damage, inflammation, and severely dysfunctional mitochondrial metabolism. These findings reveal unexpected roles for ketogenesis as a regulator of substrate flux through mitochondrial and cytosolic metabolic pathways even during physiologic states that are not classically ketogenic.98



Partial ketogenic insufficiency was also induced in neonatal mice. Even with modest reductions in HMGCS2 levels and the circulating levels of ketone bodies, a six-fold increase in hepatic triglyceride content was observed in mildly ketogenic-insufficient animals. This striking discrepancy reinforces ketogenesis as a potential target in dysfunctional fat metabolism during the neonatal period.

KETOGENIC ADAPTATIONS OF NEONATAL LIVER Livers of fetal rats have a low capacity for fatty acid catabolism and ketogenesis, but these functions rapidly increase after birth. Available evidence indicates that the capacity of the liver for LCFA catabolism increases in the suckling period. A number of the key enzymes show higher activities,99-101 particularly CPT I, the concentration of which increases at birth and attains values 3 to 6 times higher than those of the adult within a few days of parturition; it then declines rapidly on weaning.101 Increases in enzyme activity99-103 are paralleled by higher rates of ketogenesis from LCFAs in isolated hepatocytes from suckling rats than in cells from adult rats.102,104 These enzymatic changes also correlate with nutrient supply. During the prenatal period there is a steady supply of carbohydrates and protein via the placenta, whereas free fatty acids are either unable to cross this barrier or are unable to be metabolized by fetal tissues. After birth a shortterm starvation (which in human neonates stimulates lipolysis of peripheral adipose stores) precedes the suckling period, which is marked by ingestion of the high-fat, low-carbohydrate diet of maternal milk. These dietary changes correspond to hormonal levels, gene expression patterns, and ultimately substrate utilization during the fetal and neonatal periods (as reviewed by Girard and colleagues77). The rate of ketogenesis from MCFAs is not increased in hepatocytes from suckling rats compared with that in cells from adult animals.102 Increased ketogenesis from LCFAs during the neonatal period is connected with either their esterification or their entry into the mitochondria via CPT.

MALONYL COENZYME A In adult liver, CPT I is regulated by short-term changes in the concentration of carnitine (a cosubstrate) and malonyl-CoA, which is a potent inhibitor of CPT I.5 Malonyl-CoA is a key intermediate in the conversion of carbohydrate to fat, and the hepatic concentration is directly correlated with the rate of lipogenesis (de novo fatty acid synthesis).105 In liver, a major lipogenic precursor is pyruvate, formed from lactate returning to the liver as a product of glycolysis in peripheral tissues or from hepatic glucose through glucose uptake or glycogenolysis and then glycolysis. Therefore the rate of lipogenesis and the concentration of malonyl-CoA generally indicate the carbohydrate status of the liver: a high rate of lipogenesis is associated with an elevated malonyl-CoA concentration, inhibition of CPT I, and a decreased rate of ketogenesis. Conversely, a decrease in lipogenesis secondary to lack of substrate or hormonal inactivation of the malonyl-CoA–synthesizing enzyme acetyl-CoA carboxylase (ACC)106 results in a decrease in malonyl-CoA concentration and stimulation of ketogenesis due to increased entry of long-chain acyl-CoA into the mitochondria. In addition, the sensitivity of CPT I to inhibition by malonyl-CoA is affected by a change in the physiologic state107-111 The rate of lipogenesis in isolated hepatocytes from suckling animals102 or livers of suckling animals in vivo112 is low, partially due to dietary alterations but mainly due to the decrease in the activities of key lipogenic enzymes (e.g., ACC,113 fatty acid synthase114), a pattern that is rapidly reversed on weaning. Hepatic malonyl-CoA concentration is very low during suckling. This fact, together with the decreased sensitivity of CPT I to inhibition by malonyl-CoA,110,111 suggests that in the suckling neonate, regulation of ketogenesis depends on substrate supply, increased capacity of the mitochondria for fatty acid catabolism,

Chapter 37 — Ketone Body Metabolism in the Neonate

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particularly the entry of LCFA, and an increased expression of the key catalytic enzymes of ketone body production. Still to be determined is the nature of the signal or signals that bring about the stimulation of ketogenesis immediately after birth.77,80,115 One contributor is the sharp and rapid decrease in the insulin-toglucagon ratio,53,77 but recent observations also suggest that fattyacid-ligand-activated PPARα-dependent DNA demethylation regulates the fatty acid β-oxidation genes in the postnatal liver.116

INSULIN AND GLUCAGON Hormones regulate the supply of LCFAs to the liver, and insulin and glucagon can also act directly on the liver. Glucagon or dibutyryl cAMP—its second messenger—stimulates ketogenesis from LCFAs in perfused livers117,118 and isolated hepatocytes102 from adult rats. Dibutyryl cAMP or glucagon does not increase ketogenesis from MCFAs,119,120 suggesting that the site of action of glucagon is at the disposition of long-chain acyl-CoA between the pathways of esterification and β-oxidation. A possible mechanism for the effects of glucagon on ketogenesis is suggested by the finding that the hormone (or cAMP) inhibits hepatic lipogenesis in vivo121 and in vitro,121,122 and consequently decreases the concentration of malonyl-CoA.105 The site of inhibition is ACC, which is inactivated by glucagon through increased phosphorylation of the protein.106 A separate mechanism by which glucagon regulates ketogenesis is via direct activation of HMGCS2 by it desuccinylating the enzyme and thus relieving it of this inhibitory modification.66,67 Glucagon was also found to induce the acetylation and activation of the transcription factor FOXA2, inducing a gene expression profile of increased fatty acid oxidation and ketogenesis.123 Decreased esterification of LCFAs by microsomal fractions isolated from livers of fed rats perfused with dibutyryl cAMP has been reported.124 Activation of CPT I by glucagon (or cAMP) has been described.125 In hepatocytes from suckling rats, dibutyryl cAMP has no effect on ketogenesis.102 This lack of effect may be due to the elevation of cAMP concentration in neonatal hepatocytes28 and the consequent maximal stimulation of ketogenesis. Additional evidence that hepatocytes from neonatal rats are maximally stimulated is the finding that starvation of neonatal rats, in contrast with adult rats, does not further increase the rate of ketogenesis from LCFA in vitro.104 By analogy to its potent antilipolytic effect on adipocytes, insulin likely exerts a direct antiketogenic effect on the liver. Unlike glucagon, the mechanism of insulin-mediated suppression of ketogenesis remains incompletely delineated. Insulin stimulates the de novo synthesis of fatty acids, in part by activation of acetyl-CoA mediated by a change in LCFA supply to the liver.126 In addition, insulin-mediated sequestration of the transcription factor FOXA2 from the nucleus, resulting in decreased expression of fatty acid oxidation and ketogenic genes, including Hmgcs2.81 Livers of suckling rats55 and human neonates127 are resistant to the suppressive effects of insulin on glucose production, and this resistance may also extend to its effects on lipid metabolism. None of the putative direct hepatic actions of glucagon or insulin discussed earlier are likely to affect MCFA metabolism, which appears to be favored over LCFA metabolism in livers of suckling rats.128 This predilection may be important in the formulation of infant feedings.129 Thus a number of changes favor a high rate of ketogenesis in the suckling neonate (Table 37-2), which are reversed on weaning the neonate to a high-carbohydrate diet.

KETONE BODY UTILIZATION With availability of sufficient ketone bodies in the circulation, their rate of utilization is to a large extent regulated by the

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Table 37-2  Changes That Favor Ketogenesis in Suckling Rats Factor

Change* 52,53,77

Plasma insulin concentration Plasma glucagon concentration52,53,77 Insulin-to-glucagon ratio52,53,77 Plasma concentration of LCFAs46 Hepatic carnitine palmitoyltransferase I activity101 Sensitivity of carnitine palmitoyltransferase I to malonyl-CoA inhibition110,111 Hepatic carnitine concentration207 Hepatic lipogenesis102,112 Hepatic malonyl-CoA concentration

Decreased Increased Decreased Increased Increased Decreased Increased Decreased Decreased†

Data from cited references. CoA, Coenyme A, LCFA, long-chain fatty acid. *Reported changes are relative to determinations in the adult fed rat, which has a low rate of ketogenesis. † Malonyl-CoA has not been measured in neonatal rat liver but a reasonable assumption is that its level is low.

circulating concentration.130 Transport of ketone bodies through the plasma membrane or mitochondrial inner membranes is not considered to limit their utilization in muscle because the tissue concentration is linearly related to the plasma concentration.131 This may not be true for brain, however, because the ketone body concentrations in rat brain132 and human cerebrospinal fluid133 are considerably less than those in blood in hyperketonemic states. Within the cell, the metabolism of ketone bodies depends on the activities of the “initiating” enzymes, which allow their entry into the metabolic pathways of the cell. The acetyl-CoA formed from ketone bodies either is completely oxidized in the TCA cycle to provide energy (adenosine triphosphate) or can be used along with ketone bodies themselves as precursors of fatty acid or sterol (cholesterol) synthesis (see Figure 37-1). More detailed reviews of ketone body metabolism in peripheral tissues are available.4,6

MITOCHONDRIAL PATHWAY The major sites of ketone body utilization are the mitochondria. Before transport to mitochondria, ketone bodies must transverse the plasma membrane. MCT1 and MCT2 facilitate the uptake of ketone bodies from the circulation into cells, including across the blood-brain barrier.134,135 Once in the mitochondria, the primary circulating ketone body, D-(–)-βOHB, is oxidized to AcAc by BDH1, along with conversion of oxidized nicotinamide adenine dinucleotide to its reduced form. The reaction is reversible, but the equilibrium is in favor of AcAc reduction to D-(–)βOHB, and thus mass action favors BOHB oxidation. BDH1 is tightly bound to the inner mitochondrial membrane. The initiating enzyme for AcAc metabolism is the mitochondrial matrix enzyme succinyl-CoA:3-oxoacid-CoA transferase (SCOT; encoded by nuclear Oxct1), succinyl-CoA is derived from the TCA cycle, and because succinate is returned to the cycle, no loss of cycle intermediates occurs. The equilibrium of the reaction is in favor of AcAc formation, but mass action drives AcAc conversion to acetoacetyl-CoA, particularly in the setting of high citrate synthase activity (see Figure 37-1, B). Km (around 0.2 mmol/L) of SCOT for AcAc and its kinetic properties are similar in different rat tissues.136 SCOT of rat tissues is inhibited by AcAc at concentrations in excess of 5 mmol/L, which may suppress ketone body utilization in severely hyperketonemic, highly ketogenic states.137

To limit futile cycling, expression of SCOT is selectively excluded in hepatocytes, possibly due to specific silencing of Oxct1 mRNA via hepatic expression of the microRNA miR-122.138 Acetoacetyl-CoA formed by the SCOT reaction is cleaved by acetoacetyl-CoA thiolase to yield two molecules of acetyl-CoA (see Figure 37-1, B). The equilibrium of this reaction is strongly in favor of acetyl-CoA formation, and when this reaction is coupled with that of SCOT, utilization of AcAc is favored. Acetoacetyl-CoA thiolase, unlike SCOT, is present in both the mitochondrial matrix and the cytosol (see Figure 37-1, B and C).139 Unlike the hepatic HMG-CoA pathway for synthesis of ketone bodies, the mitochondrial utilization pathway is reversible, and thus utilization of ketone bodies depends on the prevailing concentrations of βOHB and AcAc and the rate of removal of acetylCoA. The latter depends on the activity of the TCA cycle and on the rate of acetyl-CoA formation from other substrates, mainly oxidation of fatty acids. This alternative acetyl-CoA source explains why it is possible to demonstrate net formation of ketone bodies in vitro when kidney slices are incubated with fatty acids.140 Thus in certain tissues, the free reversibility of the pathway can be viewed as a means of “buffering” the mitochondrial acetyl-CoA pool. Of course, the concentrations of cosubstrates (succinyl-CoA, succinate) and cofactors (CoA, oxidized nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide) also influence the utilization of ketone bodies. To explore the role of this pathway in the neonate, Oxct1−/− mice, which exhibit a congenital and global loss of SCOT, were generated. After use of stable isotopic tracers and NMR spectroscopy had confirmed that SCOT deficiency abrogated ketone body oxidation, it was revealed that these mice are born normal, but develop severe hyperketonemic hypoglycemia after the onset of suckling, and invariably die within 48 hours of birth. Unlike most states of hyperketonemia, these knockout mice displayed very high AcAc-to-βOHB ratios. Brains of neonatal SCOT-deficient mice exhibited increased concentrations of markers of cellular autophagy and oxidation of glucose, whereas skeletal muscle in these animals had increased lactate oxidation as a compensatory means of attempting to maintain metabolic homeostasis.141 Loss of extrahepatic SCOT was found to ultimately impact intermediary metabolism in the livers of neonatal mice that have fed after birth. On postnatal day 1, the livers of germline SCOT-deficient mice engage a gluconeogenic transcriptional program, but exhibit dysfunctional fatty acid oxidation, and demonstrate impaired de novo βOHB production, resulting in an oxidized hepatic redox potential (due to a high circulating AcAc-to-βOHB ratio), all of which occur in response to the ingestion of high-fat milk from the mother.142 These mice could not be completely rescued long term with carbohydrate feeding, although some benefit was achieved.141

CYTOSOLIC PATHWAY After efflux from mitochondria, ketone bodies contribute to key anabolic pathways in the cytosol as well. Ketone bodies serve as substrates for lipogenesis and sterol biosynthesis in developing brain, lactating mammary gland, and in the liver after the enzymatic activation of AcAc to AcAc-CoA by acetoacetyl-CoA synthetase (AACS)8-11 (see Figure 37-1, C). Although AACS enzyme activity is at most 10% that of SCOT, it has a high affinity (Km = 50 µmol/L) for AcAc and can be readily saturated even in the fed state. AcAc-CoA acts as a direct substrate for HMGCS1, the cytoplasmic HMG-CoA synthase, which catalyzes the fatecommitting step of sterol biosynthesis. However, entry of AcAcCoA into lipogenesis requires a thiolytic cleavage reaction to yield acetyl-CoA, which can then be carboxylated via ACC to form the lipogenic substrate malonyl-CoA.9,69,143-145 Recent studies support the physiologic importance of ketone bodies as anabolic



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substrates. By use of in vivo and in vitro AACS-knockout models, lack of cytosolic ketone body metabolism was found to lower total blood cholesterol concentration in mice, impair differentiation of primary mouse embryonic neuronal cells in culture, and inhibit adipocyte differentiation in immortalized cells.146-148 Although the anabolic fates of ketone bodies have been shown to serve important metabolic roles, these pathways are not essential for the disposal of ketone bodies because SCOT deficiency alone causes severe hyperketonemia.141,149-152 Further studies using genetic and functional disruption of AACS are warranted to thoroughly assess the physiologic roles for nonoxidative fates of ketone bodies in various cell and organ systems.

of pyruvate dehydrogenase. The overall effect is decreased glucose utilization. In addition, a higher proportion of any glucose that still undergoes glycolysis leaves the tissue as lactate and pyruvate and returns to the liver for gluconeogenesis. Neonatal SCOT-deficient mice exhibit increased glycolysis and glucose oxidation in the brain despite high circulating levels of ketone bodies, suggesting other regulatory mechanisms at the substrate flux level that may be independent of glycolytic enzyme inhibition.141,167 In normal children, however, an inverse correlation has been noted between glucose flux and the degree of ketonemia.168 This observation supports the view that glucose and ketone body interactions operate at the whole-body level.

L-(+)-Β-HYDROXYBUTYRATE

GLUCOSE PRODUCTION

Although hepatic ketogenesis produces only D-(–)-βOHB, the physiologic substrate used for oxidation, L-(+)-βOHB is measurable in ketolytic tissues but not in the circulation. L-(+)-βOHB is generated from the hydrolysis of the β-oxidation intermediate L-(+)-βOHB-CoA but is not a BDH1 substrate.153-156 SLC16A transporters in rat myocytes do not demonstrate stereoselectivity for βOHB.157 In brains of suckling rats, L-(+)-βOHB can be used for synthesis of fatty acids and sterols.91,92 These observations may be important when racemic DL-βOHB is administered to humans or used for experiments.

Ketone bodies can alter hepatic glucose production by their effects on the supply of two important precursors, glycerol and alanine. Ketone bodies decrease the flux of glycerol to the liver by their antilipolytic action on adipose tissue and apparently also on muscle triacylglycerol stores.169 Hypoalaninemia is present in a number of conditions associated with hyperketonemia, including starvation, diabetes, and ketotic hypoglycemia of childhood, and can be induced by infusion of βOHB.170 The mechanism by which ketone bodies decrease muscle release of alanine has not yet been established. Two possibilities are recognized: (1) inhibition of muscle glycolysis decreases pyruvate availability to form alanine by transamination or (2) a direct inhibition of muscle proteolysis.

SIGNALING ROLES OF KETONE BODIES Recent evidence has solidified the notion that metabolic status plays a crucial role in regulating cellular functions via signaling, transcriptional, and epigenetic pathways. As mentioned earlier, βOHB has been identified as an endogenous ligand for the niacin receptor GPR109A, which is not stereoselective for βOHB.54,158 Through an unknown mechanism, βOHB also modulates autonomic nervous system activity through GPR41, expressed primarily in sympathetic ganglia.159 βOHB inhibits class I histone deacetylases. Providing exogenous βOHB to mouse tissues led to increased histone acetylation, which was correlated with the induction of antioxidant gene expression, ultimately leading to improved handling of oxidative stress.160 βOHB supplementation has also been implicated in extending lifespan in Caenorhabditis elegans,161 as well as promoting neuroprotection, potentially via activation of a specific macrophage population.162,163 Finally, βOHB modulates macrophage signaling by inhibiting the NLRP3-dependent inflammasome, through incompletely defined mechanisms.164 The various mechanisms through which βOHB may act as a signaling metabolite via extracellular receptors or by ultimately altering epigenetic regulation of gene expression are explored in a review by Newman and Verdin.165 Although it remains possible that redox alterations or oxidative metabolism of βOHB may account for part of these signaling effects, the results are promising and pave the way for further understanding of the signaling roles ketone bodies may serve.

EFFECTS OF KETONE BODIES ON GLUCOSE METABOLISM GLUCOSE UTILIZATION

Physiologic concentrations of AcAc and βOHB decrease glucose utilization in a number of adult rat tissues, including heart, soleus muscle, kidney, brain, and lactating mammary gland.4 These tissues all exhibit high rates of glycolysis and utilization of the pyruvate formed from glucose 6-phosphate. Consequently, if glucose metabolism were not inhibited in conditions associated with relative carbohydrate deficit, the animal would rapidly become hypoglycemic. The mechanism for the inhibition of glucose metabolism by ketone bodies was established in studies by Randle and colleagues166 and involves inhibition of the enzymes phosphofructokinase and hexokinase and inactivation

ROLES OF KETONE BODIES IN NEONATAL BRAIN Ketone bodies are the primary alternative fuel source for cerebral metabolism when glucose is unavailable. This is due primarily to the brain’s poor ability to oxidize fatty acids, attributable in part to the low activity of 3-ketoacyl-CoA-thiolase in the brain.171 Permeability of the blood-brain barrier to ketone bodies depends on MCT1. In rats, permeability of the blood-brain barrier to βOHB increases by a factor of 7 during suckling before falling again after they have been weaned. This rise in permeability has been shown to be associated with a 25-fold increase in MCT1 expression while thy are suckling compared with adult levels. Additionally, there is regional variation in uptake and utilization of ketone bodies in the brain,10,172 and it appears that the telencephalon uses the most ketone bodies and the hind brain uses the least.172,173 Arteriovenous difference measurements across the brain of adult and suckling rats have shown that for a given arterial concentration of ketone bodies, the extraction by suckling rat brain is 3 to 4 times higher than that by adult brain.132,174 This finding is in agreement with the higher activities of the enzymes of ketone body utilization in suckling rat brain. A similar increase in ketone body extraction by brain has been demonstrated when human neonates and adults have been compared.175,176 However, no appreciable increase in the activities of the enzymes of ketone body utilization has been demonstrated in human neonatal brain.176,177 Therefore the increased utilization in the human neonatal brain is likely to be due to increased cerebral permeability to ketone bodies, as demonstrated in neonatal rat brain.178 The major route of ketone body utilization by brains of suckling rats is terminal oxidation via TCA cycle and electron transport chain activity for high-energy phosphate generation,179 which spares glucose. However, a portion of the carbon (5% to 10%) is converted to fatty acids and cholesterol.179-183 The newborn brain, which consumes up to 70% of the total energy expenditure at birth, is capable of oxidizing ketone bodies at a rate at least four-fold greater than that of adult brain, with ketone bodies accounting for as much as 12% of the cerebral oxygen consumption in neonates.2,184 Therefore studies of animal models congenitally deficient for ketone body oxidation in brain were

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expected to reveal metabolic abnormalities. Although germline SCOT-knockout mice exhibit hyperketonemic hypoglycemia and die in the first 2 days of life, tissue-specific SCOT-knockout models resulted in relatively normal development to adulthood and metabolic homeostasis. SCOT was selectively knocked out in neurons, cardiomyocytes, or skeletal myocytes, three tissues that most avidly oxidize ketone bodies for energetic needs. Mice from each of these tissue-specific knockout strains grew to adulthood, tolerated starvation with moderate hyperketonemia but not hypoglycemia, and were overtly normal. Neonatal neuronspecific SCOT-deficient mice exhibited altered glucose handling, exhibiting increased glycolysis and glucose oxidation in the brain.6 Together with the analysis of mice with congenital but global SCOT deficiency, these studies reveal an essential role for whole-body utilization of ketone bodies during neonatal development that cannot be attributed to any one particular ketolytic tissue, but that selective loss of ketone body oxidation forces metabolic adaptations that could lead to deficiencies in select environmental conditions.

HUMAN KETONE BODY   ENZYME DEFICIENCIES The increasing availability and decreasing cost of genomic sequencing opens the possibility of personalized medicine. Inborn errors of metabolism can have devastating consequences if left untreated, and potential mutations in the enzymes of ketogenesis and ketone body oxidation should be included in this category.

SCOT DEFICIENCY SCOT deficiency was first reported in 1972, with the postmortem tissue analysis of a 6-month-old child who had died after periods of persistent ketonemia and recurring bouts of severe ketoacidosis.23 In the following decades, numerous clinical cases of SCOT deficiency have been reported. These patients exhibit recurring attacks of ketoacidosis secondary to dysfunctional peripheral utilization of circulating ketone bodies. Most patients were neonates or young children who presented with hyperketonemia and metabolic acidosis of unknown cause, occasionally with concomitant hypoglycemia and cardiomyopathy. Enzyme activity assays and sequencing analysis ultimately revealed various mutations in the OXCT1 gene that rendered the encoded SCOT dysfunctional.150-152,185-190

MITOCHONDRIAL 3-HYDROXY-3-METHYLGLUTARYL COENZYME A SYNTHASE 2 DEFICIENCY Mutations in the fate-committing ketogenic enzyme HMGCS2 have been characterized in more than a dozen clinical cases in young patients. In each of these cases, the patient typically presents within the first few years of life with hypoketotic hypoglycemia, commonly after a prolonged fast secondary to a gastrointestinal tract infection. Urinary organic acid and plasma acylcarnitine profiles are frequently nonspecific or normal.191-197 The most recent description of human HMGCS2 deficiency identified a number of novel and known circulating biomarkers to assist in confirming the diagnosis. By retrospectively examining urine organic acid profiles, Pitt and colleagues198 concluded that a cutoff of adipic acid of more than 200 µmol/mmol creatinine and 4-hydroxy-6-methyl-2-pyrone of more than 20 µmol/mmol creatinine generated a positive predictive value of 80% for HMGCS2 deficiency.

3-HYDROXY-3-METHYLGLUTARYL COENZYME A LYASE DEFICIENCY HMG-CoA lyase is a ketogenic enzyme in the liver that catalyzes the formation of AcAc from HMG-CoA within the mitochondria.

However, it also plays a prominent role in the catabolism of the amino acid leucine. Because of this, HMG-CoA lyase deficiency not only causes hypoketotic hypoglycemia similar to that caused by HMGCS2 mutations but also leads to organic acid accumulation and metabolic acidosis due to altered leucine metabolism. This disorder also occurs in childhood and can be mistaken for Reye syndrome because of the overlapping symptoms, including vomiting, lethargy, and convulsions.199-202

MITOCHONDRIAL β-KETOTHIOLASE DEFICIENCY

Similarly to HMG-CoA lyase, mitochondrial β-ketothiolase functions as both an enzyme of ketone body metabolism and in the processing of the amino acid isoleucine. Mitochondrial β-ketothiolase is involved in the conversion of acetoacetylCoA to acetyl-CoA in the ketolytic pathway. Mitochondrial β-ketothiolase deficiency is an autosomal recessive disorder that occurs in young childhood with vomiting, hyperketonemic hypoglycemia, and accumulation of isoleucine breakdown products in the blood and urine, including 2-methylacetoacetate, 2-methyl-3-hydroxybutyrate, and tiglylglycine.203-207

KETONE BODY TRANSPORTER DEFICIENCY A recent study analyzed the genome of a patient with severe ketoacidosis, suspecting a defect in the extrahepatic ketolytic machinery, and discovered a homozygous mutation in the gene encoding MCT1 (SLC16A1). Numerous patients were subsequently found to have inactivating mutations in SLC16A1, all of whom presented with ketoacidosis after a period of fasting or infection in early childhood. Intravenous administration of glucose or dextrose with bicarbonate quickly reversed the ketoacidosis, while avoidance of fasting prevented future ketoacidotic episodes.70

SUMMARY Ketone body metabolism serves an essential metabolic role in the development of the neonate. Although ketone bodies are important energetic substrates, particularly in highly oxidative tissues such as the brain, the pathways of hepatic ketogenesis and peripheral ketolysis are dynamically regulated mitochondrial processes that impact cellular signaling and metabolic functioning in myriad ways. Disruption of ketone body metabolism in model organisms and in humans has severe clinical consequences, including steatohepatitis, ketoacidosis, and death in the neonatal period. Clinical assessment of metabolic abnormalities in the neonatal period should usually interrogate this pathway, and ongoing investigation will elucidate the mechanisms involved in how ketone body metabolism may ameliorate or exacerbate pathologic conditions.

ACKNOWLEDGMENTS The authors are most grateful to the authors of the previous edition of this chapter, including Paul S. Thornton, whose leadership and input on this chapter’s themes and data (particularly within the tables) were invaluable. Complete reference list is available at www.ExpertConsult.com.

REFERENCES 1. Aneja P, Dziak R, Cai GQ, et al: Identification of an acetoacetyl coenzyme A synthetase-dependent pathway for utilization of L-(+)-3-hydroxybutyrate in Sinorhizobium meliloti. J Bacteriol 184:1571–1577, 2002. 2. Cahill GF, Jr: Fuel metabolism in starvation. Annu Rev Nutr 26:1–22, 2006. 3. Krishnakumar AM, Sliwa D, Endrizzi JA, et  al: Getting a handle on the role of coenzyme M in alkene metabolism. Microbiol Mol Biol Rev 72:445–456, 2008.

4. Robinson AM, Williamson DH: Physiological roles of ketone bodies as substrates and signals in mammalian tissues. Physiol Rev 60:143–187, 1980. 5. McGarry JD, Foster DW: Regulation of hepatic fatty acid oxidation and ketone body production. Annu Rev Biochem 49:395–420, 1980. 6. Cotter DG, Schugar RC, Crawford PA: Ketone body metabolism and cardiovascular disease. Am J Physiol Heart Circ Physiol 304:H1060–H1076, 2013. 7. Johnson RH, Walton JL, Krebs HA, et al: Post-exercise ketosis. Lancet 2:1383– 1385, 1969. 8. Freed LE, Endemann G, Tomera JF, et al: Lipogenesis from ketone bodies in perfused livers from streptozocin-induced diabetic rats. Diabetes 37:50–55, 1988. 9. Endemann G, Goetz PG, Edmond J, et al: Lipogenesis from ketone bodies in the isolated perfused rat liver. Evidence for the cytosolic activation of acetoacetate. J Biol Chem 257:3434–3440, 1982. 10. Morris AA: Cerebral ketone body metabolism. J Inherit Metab Dis 28:109–121, 2005. 11. Robinson AM, Williamson DH: Utilization of D-3-hydroxy[3-14C]butyrate for lipogenesis in vivo in lactating rat mammary gland. Biochem J 176:635–638, 1978. 12. Sengupta S, Peterson TR, Laplante M, et al: mTORC1 controls fasting-induced ketogenesis and its modulation by ageing. Nature 468:1100–1104, 2010. 13. Soeters MR, Sauerwein HP, Faas L, et al: Effects of insulin on ketogenesis following fasting in lean and obese men. Obesity (Silver Spring) 17:1326–1331, 2009. 14. Neely JR, Rovetto MJ, Oram JF: Myocardial utilization of carbohydrate and lipids. Prog Cardiovasc Dis 15:289–329, 1972. 15. Lommi J, Kupari M, Koskinen P, et al: Blood ketone bodies in congestive heart failure. J Am Coll Cardiol 28:665–672, 1996. 16. Kupari M, Lommi J, Ventila M, et al: Breath acetone in congestive heart failure. Am J Cardiol 76:1076–1078, 1995. 17. Lommi J, Koskinen P, Naveri H, et al: Heart failure ketosis. J Intern Med 242:231–238, 1997. 18. Pittman JG, Cohen P: The pathogenesis of cardiac cachexia. N Engl J Med 271:403–409 CONTD, 1964. 19. Fery F, Balasse EO: Ketone body production and disposal in diabetic ketosis. A comparison with fasting ketosis. Diabetes 34:326–332, 1985. 20. Hall SE, Wastney ME, Bolton TM, et al: Ketone body kinetics in humans: the effects of insulin-dependent diabetes, obesity, and starvation. J Lipid Res 25:1184–1194, 1984. 21. Stacpoole PW, Moore GW, Kornhauser DM: Metabolic effects of dichloroacetate in patients with diabetes mellitus and hyperlipoproteinemia. N Engl J Med 298:526–530, 1978. 22. Williamson DH, Wilson MB: The effects of cyclopropane derivatives on ketonebody metabolism in vivo. Biochem J 94:19C–20C, 1965. 23. Tildon JT, Cornblath M: Succinyl-CoA: 3-ketoacid CoA-transferase deficiency. A cause for ketoacidosis in infancy. J Clin Invest 51:493–498, 1972. 24. Sherwin RS, Hendler RG, Felig P: Effect of diabetes mellitus and insulin on the turnover and metabolic response to ketones in man. Diabetes 25:776–784, 1976. 25. Williamson D: The production and utilization of ketone bodies in the neonate. In Jones CT, editor: Biochemical development of the fetus and neonate, Amsterdam, 1982, Elsevier Biomedical Press, pp 621–650. 26. Wildenhoff KE, Johansen JP, Karstoft H, et al: Diurnal variations in the concentrations of blood acetoacetate and 3-hydroxybutyrate. The ketone body peak around midnight and its relationship to free fatty acids, glycerol, insulin, growth hormone and glucose in serum and plasma. Acta Med Scand 195:25– 28, 1974. 27. Paterson P, Sheath J, Taft P, et al: Maternal and foetal ketone concentrations in plasma and urine. Lancet 1:862–865, 1967.

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28. Sabata V, Wolf H, Lausmann S: The role of free fatty acids, glycerol, ketone bodies and glucose in the energy metabolism of the mother and fetus during delivery. Biol Neonat 13:7–17, 1968. 29. Felig P, Lynch V: Starvation in human pregnancy: hypoglycemia, hypoinsulinemia, and hyperketonemia. Science 170:990–992, 1970. 30. Kim YJ, Felig P: Maternal and amniotic fluid substrate levels during caloric deprivation in human pregnancy. Metabolism 21:507–512, 1972. 31. Persson B, Lunell NO: Metabolic control in diabetic pregnancy. Variations in plasma concentrations of glucose, free fatty acids, glycerol, ketone bodies, insulin, and human chorionic somatomammotropin during the last trimester. Am J Obstet Gynecol 122:737–745, 1975. 32. Spitzer JJ, Weng JT: Removal and utilization of ketone bodies by the brain of newborn puppies. J Neurochem 19:2169–2173, 1972. 33. Gentz J, Bengtsson G, Hakkarainen J, et al: Metabolic effects of starvation during neonatal period in the piglet. Am J Physiol 218:662–668, 1970. 34. Varnam GC, Jeacock MK, Shepherd DA: Hepatic ketone-body metabolism in developing sheep and pregnant ewes. Br J Nutr 40:359–367, 1978. 35. Lin X, Adams SH, Odle J: Acetate represents a major product of heptanoate and octanoate beta-oxidation in hepatocytes isolated from neonatal piglets. Biochem J 318(Pt 1):235–240, 1996. 36. Duee PH, Pegorier JP, Quant PA, et al: Hepatic ketogenesis in newborn pigs is limited by low mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity. Biochem J 298(Pt 1):207–212, 1994. 37. Hawdon JM, Ward Platt MP, Aynsley-Green A: Patterns of metabolic adaptation for preterm and term infants in the first neonatal week. Arch Dis Child 67:357–365, 1992. 38. Williamson DH, Mellanby J, Krebs HA: Enzymic determination of D(-)-betahydroxybutyric acid and acetoacetic acid in blood. Biochem J 82:90–96, 1962. 39. Haymond MW, Karl IE, Pagliara AS: Increased gluconeogenic substrates in the small-for-gestational-age infant. N Engl J Med 291:322–328, 1974. 40. Fukao T, Lopaschuk GD, Mitchell GA: Pathways and control of ketone body metabolism: on the fringe of lipid biochemistry. Prostaglandins Leukot Essent Fatty Acids 70:243–251, 2004. 41. Sugden M, Williamson D: Short-term hormonal control of ketogenesis. In Hue L, Van de Werve G, editors: Short-term regulation of liver metabolism, Amsterdam, 1981, Elsevier/North Holland Biomedical Press, pp 291–309. 42. Zammit VA: Regulation of hepatic fatty acid oxidation and ketogenesis. Proc Nutr Soc 42:289–302, 1983. 43. Zammit VA: Mechanisms of regulation of the partition of fatty acids between oxidation and esterification in the liver. Prog Lipid Res 23:39–67, 1984. 44. Dahlquist G, Persson U, Persson B: The activity of D-β-hydroxybutyrate dehydrogenase in fetal, infant and adult rat brain and the influence of starvation. Biol Neonate 20:40–50, 1972. 45. Novak M, Melichar V, Hahn P, et al: Release of free fatty acids from adipose tissue obtained from newborn infants. J Lipid Res 6:91–95, 1965. 46. Page MA, Krebs HA, Williamson DH: Activities of enzymes of ketone-body utilization in brain and other tissues of suckling rats. Biochem J 121:49–53, 1971. 47. Persson B, Gentz J: The pattern of blood lipids, glycerol and ketone bodies during the neonatal period, infancy and childhood. Acta Paediatr Scand 55:353–362, 1966. 48. Garber AJ, Menzel PH, Boden G, et al: Hepatic ketogenesis and gluconeogenesis in humans. J Clin Invest 54:981–989, 1974. 49. Nielsen TS, Jessen N, Jorgensen JO, et al: Dissecting adipose tissue lipolysis: molecular regulation and implications for metabolic disease. J Mol Endocrinol 52:R199–R222, 2014. 50. Belfrage P: Hormonal control of lipid degradation. In Cryer A, Van R, editors: New perspectives in adipose tissue: structure, function and development, London, 1985, Butterworths, pp 121–144.



Chapter 37 — Ketone Body Metabolism in the Neonate

REFERENCES 1. Aneja P, Dziak R, Cai GQ, et al: Identification of an acetoacetyl coenzyme A synthetase-dependent pathway for utilization of L-(+)-3-hydroxybutyrate in Sinorhizobium meliloti. J Bacteriol 184:1571–1577, 2002. 2. Cahill GF, Jr: Fuel metabolism in starvation. Annu Rev Nutr 26:1–22, 2006. 3. Krishnakumar AM, Sliwa D, Endrizzi JA, et al: Getting a handle on the role of coenzyme M in alkene metabolism. Microbiol Mol Biol Rev 72:445–456, 2008. 4. Robinson AM, Williamson DH: Physiological roles of ketone bodies as substrates and signals in mammalian tissues. Physiol Rev 60:143–187, 1980. 5. McGarry JD, Foster DW: Regulation of hepatic fatty acid oxidation and ketone body production. Annu Rev Biochem 49:395–420, 1980. 6. Cotter DG, Schugar RC, Crawford PA: Ketone body metabolism and cardiovascular disease. Am J Physiol Heart Circ Physiol 304:H1060–H1076, 2013. 7. Johnson RH, Walton JL, Krebs HA, et al: Post-exercise ketosis. Lancet 2:1383– 1385, 1969. 8. Freed LE, Endemann G, Tomera JF, et al: Lipogenesis from ketone bodies in perfused livers from streptozocin-induced diabetic rats. Diabetes 37:50–55, 1988. 9. Endemann G, Goetz PG, Edmond J, et al: Lipogenesis from ketone bodies in the isolated perfused rat liver. Evidence for the cytosolic activation of acetoacetate. J Biol Chem 257:3434–3440, 1982. 10. Morris AA: Cerebral ketone body metabolism. J Inherit Metab Dis 28:109– 121, 2005. 11. Robinson AM, Williamson DH: Utilization of D-3-hydroxy[3-14C]butyrate for lipogenesis in vivo in lactating rat mammary gland. Biochem J 176:635–638, 1978. 12. Sengupta S, Peterson TR, Laplante M, et al: mTORC1 controls fasting-induced ketogenesis and its modulation by ageing. Nature 468:1100–1104, 2010. 13. Soeters MR, Sauerwein HP, Faas L, et al: Effects of insulin on ketogenesis following fasting in lean and obese men. Obesity (Silver Spring) 17:1326– 1331, 2009. 14. Neely JR, Rovetto MJ, Oram JF: Myocardial utilization of carbohydrate and lipids. Prog Cardiovasc Dis 15:289–329, 1972. 15. Lommi J, Kupari M, Koskinen P, et al: Blood ketone bodies in congestive heart failure. J Am Coll Cardiol 28:665–672, 1996. 16. Kupari M, Lommi J, Ventila M, et al: Breath acetone in congestive heart failure. Am J Cardiol 76:1076–1078, 1995. 17. Lommi J, Koskinen P, Naveri H, et al: Heart failure ketosis. J Intern Med 242:231–238, 1997. 18. Pittman JG, Cohen P: The pathogenesis of cardiac cachexia. N Engl J Med 271:403–409 CONTD, 1964. 19. Fery F, Balasse EO: Ketone body production and disposal in diabetic ketosis. A comparison with fasting ketosis. Diabetes 34:326–332, 1985. 20. Hall SE, Wastney ME, Bolton TM, et al: Ketone body kinetics in humans: the effects of insulin-dependent diabetes, obesity, and starvation. J Lipid Res 25:1184–1194, 1984. 21. Stacpoole PW, Moore GW, Kornhauser DM: Metabolic effects of dichloroacetate in patients with diabetes mellitus and hyperlipoproteinemia. N Engl J Med 298:526–530, 1978. 22. Williamson DH, Wilson MB: The effects of cyclopropane derivatives on ketone-body metabolism in vivo. Biochem J 94:19C–20C, 1965. 23. Tildon JT, Cornblath M: Succinyl-CoA: 3-ketoacid CoA-transferase deficiency. A cause for ketoacidosis in infancy. J Clin Invest 51:493–498, 1972. 24. Sherwin RS, Hendler RG, Felig P: Effect of diabetes mellitus and insulin on the turnover and metabolic response to ketones in man. Diabetes 25:776– 784, 1976. 25. Williamson D: The production and utilization of ketone bodies in the neonate. In Jones CT, editor: Biochemical development of the fetus and neonate, Amsterdam, 1982, Elsevier Biomedical Press, pp 621–650. 26. Wildenhoff KE, Johansen JP, Karstoft H, et al: Diurnal variations in the concentrations of blood acetoacetate and 3-hydroxybutyrate. The ketone body peak around midnight and its relationship to free fatty acids, glycerol, insulin, growth hormone and glucose in serum and plasma. Acta Med Scand 195:25– 28, 1974. 27. Paterson P, Sheath J, Taft P, et al: Maternal and foetal ketone concentrations in plasma and urine. Lancet 1:862–865, 1967. 28. Sabata V, Wolf H, Lausmann S: The role of free fatty acids, glycerol, ketone bodies and glucose in the energy metabolism of the mother and fetus during delivery. Biol Neonat 13:7–17, 1968. 29. Felig P, Lynch V: Starvation in human pregnancy: hypoglycemia, hypoinsulinemia, and hyperketonemia. Science 170:990–992, 1970. 30. Kim YJ, Felig P: Maternal and amniotic fluid substrate levels during caloric deprivation in human pregnancy. Metabolism 21:507–512, 1972. 31. Persson B, Lunell NO: Metabolic control in diabetic pregnancy. Variations in plasma concentrations of glucose, free fatty acids, glycerol, ketone bodies, insulin, and human chorionic somatomammotropin during the last trimester. Am J Obstet Gynecol 122:737–745, 1975. 32. Spitzer JJ, Weng JT: Removal and utilization of ketone bodies by the brain of newborn puppies. J Neurochem 19:2169–2173, 1972. 33. 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Chapter 37 — Ketone Body Metabolism in the Neonate

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