POTENTIAL OF ANAPLEROTIC TRIHEPTANOIN FOR THE TREATMENT OF

LONG-CHAIN FATTY ACID OXIDATION DISORDERS

by

LEI GU

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Dissertation Advisor: Dr. Henri Brunengraber

Department of Nutrition

School of Medicine

CASE WESTERN RESERVE UNIVERSITY

May, 2010 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

______LEI GU candidate for the ______degreeDoctor of Philosophy *.

(signed)______Edith Lerner, PhD (chair of the committee)

______Henri Brunengraber, MD, PhD

______Janos Kerner, PhD

______Colleen M. Croniger, PhD

______Michelle A. Puchowicz, PhD

______

(date) ______Nov 30, 2009

*We also certify that written approval has been obtained for any proprietary material contained therein.

Table of Contents

Table of Contents ...... iii

List of Tables ...... ix

List of Figures ...... x

Acknowledgements ...... xii

List of Abbreviations ...... xiii

Abstract ...... xvi

1. LIPOLYSIS ...... 1

1.1 Introduction of Lipolysis ...... 1

1.2 Introduction of Hormone-Sensitive Lipase ...... 3

1.3 Regulation of Adipocyte Lipolysis ...... 4

1.4 Lipolysis under Stress ...... 8

1.5 Measurement of Lipolysis...... 9

2. FATTY ACID OXIDATION ...... 10

2.1 Introduction of Fatty Acid Oxidation Pathway ...... 10

2.1.1 Cellular Uptake of Fatty Acids ...... 10 iii

2.1.2 Activation of Fatty Acids ...... 12

2.1.3 Carnitine Palmitoyltransferase System ...... 14

2.1.4 Mitochondrial β-oxidation Spiral ...... 16

2.1.5 Fatty Acid α- and ω-oxidation ...... 18

2.2 Regulation of Fatty Acid Oxidation ...... 19

2.2.1 Regulation of Carnitine Palmitoyltransferase I by Malonyl-CoA ..... 19

2.2.2 Redox Regulation ...... 21

2.2.3 Feedback Regulation of Fatty Acid Oxidation ...... 22

2.2.4 Regulation of Fatty Acid Oxidation by CoA ...... 23

2.3 Ketone Body Synthesis ...... 24

2.3.1 C4-ketogenesis ...... 24

2.3.2 C5-ketogenesis ...... 26

2.3.3 HMG-CoA synthase ...... 26

2.3.4 Interrelations between C4- and C5-ketogenesis ...... 31

2.4 Ketone Body Utilization ...... 32

2.4.1 Ketolysis Pathway ...... 32

2.4.2 Utilization of Ketone Bodies in Brain ...... 32

2.4.3 Utilization of Ketone Bodies in Heart ...... 34

2.4.4 Utilization of Ketone Bodies in Kidney ...... 35

3. FATTY ACID OXIDATION DISORDERS ...... 36

3.1 Introduction of Fatty Acid Oxidation Disorders (FOD) ...... 36

iv

3.1.1 Disorders of Plasma Membrane Functions ...... 36

3.1.2 Disorders of the Carnitine Palmitoyltransferase System ...... 37

3.1.3 Long-chain Fatty Acid Oxidation Disorders ...... 38

3.1.4 Disorders of Medium-chain Fatty Acid Oxidation ...... 39

3.1.5 Disorders of Short-chain Fatty Acid Oxidation ...... 40

3.2 Pathogenesis of Fatty Acid Oxidation Disorders ...... 41

3.3 Diagnosis of Fatty Acid Oxidation Disorders ...... 42

3.4 Treatment of Fatty Acid Oxidation Disorders ...... 43

3.4.1 Carnitine Supplementation ...... 43

3.4.2 Decreased Dietary Fat intake ...... 44

3.4.3 Increased Dietary Carbohydrate ...... 44

3.4.4 Minimizing Fat Mobilization ...... 45

3.4.5 Infusion of Glucose and Insulin as Acute Treatment ...... 45

3.4.6 Medium-chain Triglycerides (MCT) ...... 46

3.4.7 Other Strategies ...... 47

3.5 Triheptanoin as the Treatment for Long-chain Fatty Acid Oxidation

Disorders ...... 47

3.5.1 Introduction of Triheptanoin ...... 47

3.5.2 Metabolism of Triheptanoin ...... 49

3.5.3 Triheptanoin versus Trioctanoin ...... 51

3.5.4 Previous Studies of Triheptanoin ...... 54

v

4. ANAPLEROSIS ...... 55

4.1 Introduction of Anaplerosis ...... 55

4.2 Anaplerotic Substrates ...... 58

4.2.1 Pyruvate ...... 58

4.2.2 Glutamate/Glutamine ...... 59

4.2.3 Precursors of Propionyl-CoA ...... 60

4.3 Measurement of Anaplerosis ...... 63

5. GLYCERONEOGENESIS ...... 66

5.1 Introduction of Glyceroneogenesis ...... 66

5.2 Regulation of Glyceroneogenesis ...... 69

5.3 Measurement of Glyceroneogenesis ...... 69

5.4 Problems in Measuring Glyceroneogenesis in vivo ...... 72

6. STATEMENT OF PURPOSES ...... 74

7. HYPOTHESES ...... 75

8. EXPERIMENTAL PROCEDURES ...... 76

8.1 Animals ...... 76

8.2 Protocol ...... 77

8.3 Tracer Studies ...... 81

vi

8.4 Rationale for Selection of Infusion Groups ...... 81

8.5 Assay of Glucose and Glycerol in the Plasma ...... 82

8.6 Assay of Heptanoate, C4-ketone Bodies, C5-ketone Bodies, and Oleate in the Plasma ...... 84

8.7 Assay of Short-chain and Medium-chain Acyl-CoAs in the Liver ...... 85

8.8 Calculations ...... 87

8.9 Statistics ...... 88

9. RESULTS ...... 89

9.1 Plasma Heptanoate Concentrations ...... 89

9.2 Plasma C5-ketone Bodies Concentrations ...... 92

9.3 Plasma C4-ketone Bodies Concentrations ...... 94

9.4 Mid-potential of the BHP/BKP System ...... 96

9.5 Plasma Glycerol Concentrations ...... 96

9.6 Plasma Glucose Concentrations ...... 99

9.7 Liver Glycogen Contents ...... 99

9.8 Plasma Oleate Concentrations and Enrichments ...... 102

9.9 Plasma Insulin Levels ...... 105

9.10 Liver Acyl-CoAs Concentrations ...... 105

10. DISCUSSION ...... 108

10.1 Heptanoate Metabolism and C4- and C5-Ketogenesis ...... 108

vii

10.2 Activation of Lipolysis in Triheptanoin Infusions ...... 109

10.3 Fatty Acids Released from Lipolysis Are Re-esterified in Triheptanoin

Infusions ...... 111

10.4 Sources of Glycerol-3-Phosphate for Fatty Acid Re-esterification ...... 113

10.5 Fluxes of Glucose Production ...... 113

10.6 Profiles of Medium-chain and Short-chain Acyl-CoAs in the Liver ...... 117

11. CONCLUSIONS AND CLINICAL IMPLICATIONS ...... 118

12. FURTHER DIRECTIONS ...... 119

REFERENCES ...... 122

viii

List of Tables

Table 1. Rates of administration of substrates and isotopic tracers ...... 80

Table 2. Liver glycogen contents and endogenous Ra of glucose, glycerol and oleate ...... 101

Table 3. Liver acyl-CoA concentrations ...... 107

ix

List of Figures

Figure 1. Lipolysis in the adipocyte ...... 2

Figure 2. Regulation of lipolysis in the adipocyte ...... 7

Figure 3. Transport of fatty acids into the cell ...... 13

Figure 4. The carnitine palmitoyltransferase system ...... 15

Figure 5. Mitochondrial β-oxidation spiral and the sites of feedback control .. 17

Figure 6. C4-ketone body metabolism ...... 29

Figure 7. C5-ketone body metabolism ...... 30

Figure 8. Formula of triheptanoin ...... 48

Figure 9. Hepatic oxidation of heptanoate ...... 50

Figure 10. Metabolism of heptanoate versus even-chain fatty acids ...... 53

Figure 11. Sources and anaplerotic pathway of propionyl-CoA ...... 62

Figure 12. The glyceroneogenesis pathway ...... 68

Figure 13. Experimental protocol ...... 78

Figure 14. Profile of heptanoate concentrations in rat plasma ...... 91

Figure 15. Profile of total C5-ketone body concentrations in rat plasma ...... 93

Figure 16. Profile of total C4-ketone body concentrations in rat plasma ...... 95

Figure 17. Profile of glycerol concentrations in rat plasma ...... 98

Figure 18. Profile of glucose concentrations in rat plasma ...... 100

Figure 19. Profile of oleate concentrations in rat plasma ...... 103

Figure 20. Profile of M18 oleate enrichments in rat plasma ...... 104

x

Figure 21. Comparison between the endogenous glucose Ra, the rate of liver glycogen synthesis over control and the potential glucose productions ...... 116

xi

Acknowledgements

First and foremost, I want to especially thank my mentor and dissertation advisor, Dr. Henri Brunengraber, for what has been undoubtedly the greatest educational experience of my life. You gave me so much support and guidance with your endless patience. You are brilliant and my life is better for having known you.

I am very grateful to the members of my dissertation committee, Dr. Colleen

Croniger, Dr. Michelle Puchowicz, Dr. Janos Kerner, and Dr. Edith Lerner for their great kindness, valuable advices, support and encouragements.

I also want to thank all the members of the Brunengraber lab who became my good friends and my support network. In particular, I am deeply thankful of Dr.

Guo-Fang Zhang, for your kind words and profound scientific expertise. I want to express my sincerely thanks to Dr. Rajan S. Kombu, Shuang, John, Sophie,

Stephanie, Fred and Lan for your help and most importantly your friendship. It was a joy to have worked with you all.

The dissertation is lovingly dedicated to my parents, without whom I could not have accomplished this goal.

The past three and half years in my PhD study were a wonderful period, with laughs, with pains, with challenges, and with gains. It is always an unforgettable part of my life.

Thank you all, named and unnamed.

xii

List of Abbreviations

AcAc Acetoacetate

ACC Acetyl-CoA carboxylase

AMPK AMP-activated protein kinase

ATGL Adipose triglyceride lipase

BHB β-hydroxybutyrate

BHP β-hydroxypentanoate

BKP β-ketopentanoate

CAC Citric acid cycle

CACT Carnitine:acylcarnitine translocase

CaSR Calcium –sensing receptor

CNS Central nervous system

CoA Coenzyme A

CPT Carnitine palmitoyltransferase

DG Diglyceride

ERK Extracellular signal-regulated kinase

FA Fatty acid

FABP Fatty acid binding protein

FAT Fatty acid translocase

FATP Fatty acid transport protein

FOD Fatty acid oxidation disorder

xiii

GABA γ-aminobutyric acid

HEG-CoA 3-hydroxy-3-ethylglutaryl-CoA

HMG-CoA 3-hydroxy-3-methylglutaryl-CoA

HSL Hormone-sensitive lipase

JNK c-Jun N-terminal kinase

LCHAD Long-chain 3-hydroxyacyl-CoA dehydrogenase

MAPK Mitogen-activated protein kinase

MCAD Medium-chain acyl-CoA dehydrogenase

MCD Malonyl-CoA decarboxylase

MCKAT Medium-chain 3-ketoacyl-CoA thiolase

MCT Medium-chain triglycerides

MG Monoglyceride

MGL Monoglyceride lipase

MID Mass isotopomer distribution

MTP Mitochondrial trifunctional protein

NPY Neuropeptide Y

PEPCK Phosphoenolpyruvate carboxykinase

PKA Protein kinase A

PKG Protein kinase G

PPAR Peroxisome proliferator-activated receptor

PYY Peptide YY

Ra Rate of appearance

xiv

SCAD Short-chain acyl-CoA dehydrogenase

TG Triglyceride

TH Triheptanoin

TNF-α Tumor necrosis factor alpha

VLCAD Very-long-chain acyl-CoA dehydrogenase

VLDL Very low-density lipoprotein

xv

Potential of Anaplerotic Triheptanoin for the Treatment of Long-chain Fatty

Acid Oxidation Disorders

Abstract

by

LEI GU

Long-chain fatty acid oxidation disorders are the genetic and metabolic diseases in which the body is unable to break down long-chain fatty acids to make energy. The anaplerotic medium-odd-chain triglyceride, triheptanoin, is used in clinical trials for the chronic dietary treatment of patients with long-chain fatty acid oxidation disorders. We previously showed that the intravenous infusion of triheptanoin increases lipolysis traced by the turnover of glycerol (Kinman RP et al Am. J. Physiol. 291: E860-E866, 2006). In the present study, we tested whether lipolysis induced by triheptanoin infusion is accompanied by the potentially harmful release of long-chain fatty acids.

Overnight fasted rats were infused with heptanoate ± glycerol, or triheptanoin ±

(glucose + insulin), through parenteral or enteral route. The liver metabolism of heptanoate β-oxidation has two main fates: (i) anaplerotic gluconeogenesis from the propionyl moiety, and (ii) formation of C5-ketone bodies. Intravenous infusion of heptanoate alone or with glycerol did not affect endogenous xvi glycerol Ra and oleate Ra, but increased endogenous glucose Ra from gluconeogenic precursors. Intravenous infusion of triheptanoin at 40% of caloric requirements markedly increased endogenous glycerol turnover but not endogenous oleate turnover. Thus, the activation of lipolysis was balanced by fatty acid re-esterification in the same cells. The provision of glycerol-3-phosphate for fatty acid re-esterification is most likely derived from hyperglycemia and the increased glucose turnover during intravenous infusion of triheptanoin. Intravenous infusion of triheptanoin under hyperglycemia and hyperinsulinemia conditions inhibited heptanoate utilization and C4-,

C5-ketogenesis, as well as halved endogenous glycerol turnover and oleate turnover. Intraduodenal infusion of triheptanoin did not activate lipolysis. The liver acyl-CoA profile showed the accumulation of intermediates of heptanoate

β-oxidation and C5-ketogenesis, and a decrease in free CoA, but no evidence of metabolic perturbations of liver metabolism.

Our data suggest that triheptanoin, administered either intravenously or intraduodenally, could be used for intensive care and nutritional support of metabolic decompensated patients including long-chain fatty acid oxidation disorders.

xvii

1. LIPOLYSIS

1.1 Introduction of Lipolysis

Triglycerides are the major fuel store in the body. Triglycerides yield more than twice as much energy (9 kcal/g) as carbohydrates (3.75 kcal/g) and proteins (4 kcal/g). In mammals, white adipose tissue is the most important storage organ of triglycerides (1). During fasting or under periods of increased energy demand, triglycerides stored in the adipose tissue are mobilized for hydrolysis.

Hydrolysis of adipocyte triglycerides is called lipolysis. The lipolytic pathway proceeds in a regulated and orderly manner, requiring the activation of different enzymes acting at each step (2). Lipolysis in adipocytes is illustrated in Fig 1. Triglycerides are hydrolyzed through three consecutive reactions catalyzed by three enzymes: adipose triglyceride lipase (ATGL) (3; 4), hormone-sensitive lipase (HSL) (5), and monoglyceride lipase (MGL) (6).

ATGL catalyzes the hydrolysis of triglycerides, and HSL catalyzes the hydrolysis of triglycerides as well as diglycerides, whereas MGL is required for the complete hydrolysis of monoglycerides. Hydrolysis of one mole of triglyceride in the adipocyte results in the release of one mole of glycerol and three moles of fatty acid into the blood.

The process of lipolysis may also occur extracellularly. Very low-density lipoproteins (VLDLs) are secreted from the liver into the circulation where they are hydrolyzed by endothelial cell associated lipoprotein lipase (1; 7). 1

Figure 1. Lipolysis in the adipocyte

TG: triglyceride; DG: diglyceride; MG: monoglyceride; ATGL: adipose triglyceride lipase; HSL: hormone-sensitive lipase; MGL: monoglyceride lipase;

FA: fatty acid

2

1.2 Introduction of Hormone-Sensitive Lipase

HSL is the key enzyme in adipocyte lipolysis. HSL is an 84 to 130 kDa protein

(5; 8; 9). HSL comprises three major domains: a catalytic domain, a regulatory domain encoding several phosphorylation sites, and an N-terminal domain involved in protein-protein and protein-lipid interactions (8). HSL hydrolyzes triglycerides and diglycerides, showing a preference for the sn 1-ester or

3-ester bonds (8; 10). Consequently, MGL acting in the adipocytes always has

2-ester specificity to give complete breakdown of triglycerides (6). It was recently demonstrated that activation of HSL requires two steps: protein phosphorylation, and binding to the PAT-1 domain of lipid droplet coat proteins

(11). Perilipin is the major PAT protein associated with the lipid droplet in mature adipocytes (11-13). Upon lipolytic stimulation, HSL is phosphorylated by cAMP-dependent protein kinase, with perilipin also phosphorylated and dissociated from the droplet allowing access of HSL to the lipid (8; 14). HSL also exhibits lipolytic activity against cholesteryl esters (15) and retinyl esters

(16; 17).

For more than 30 years after its discovery (18), HSL had been believed to be the only rate-limiting enzyme for lipolysis in the adipose tissue. However, recently, this notion was challenged by studies performed on HSL knock-out mice (19). Osuga et al. (19) reported that white adipose tissue mass in HSL -/- mice remained unchanged compared to the wild-type mice. Moreover, HSL -/- mice were not obese (19). Harada ea al. (20) reported that HSL -/- mice were 3 resistant to high-fat diet induced obesity. Zimmermann et al. (21) ascribed the reason that HSL knock-out mice were not obese and had unchanged white adipose tissue mass to markedly decreased cellular fatty acid re-esterification.

However, HSL -/- mice retained 40% of triglyceride lipase activities in white adipose tissue compared to the wild-type mice (19). Furthermore, triglyceride contents were decreased in white adipose tissue of HSL -/- mice (20). Also,

Wang et al. (22) that reported HSL -/- mice had significantly lower liver triglyceride content compared to controls after overnight fasting. These results suggest that HSL is not the rate-limiting enzyme for triglycerides hydrolysis.

Additional enzymes acting as triglyceride hydrolases present in adipocytes when HSL is absent. In 2004, ATGL (also called desnutrin) was identified as one of novel triglyceride specific lipase (3). In contrast, Haemmerle et al. (23) reported that HSL -/- mice accumulated diglycerides in white adipose tissue, indicating that HSL is only rate-limiting in the hydrolysis of cellular diglycerides.

Studies on HSL null mice also suggest HSL is involved in the determination of white versus brown adipocytes during adipocyte differentiation (24).

1.3 Regulation of Adipocyte Lipolysis

Lipolysis is tightly regulated by hormones, neurotransmitters, and other effectors. HSL is the major target of this regulation (9). During fasting, catecholamines bind to G protein coupled β-adrenergic receptors to activate

4 adenylate cyclase, which leads to an increased cAMP level and subsequently activates protein kinase A (PKA) (9; 25). PKA phosphorylates HSL and activates it. PKA also phosphorylates perilipin, which is displaced by HSL from the lipid droplet (8; 9). Thus, the lipid droplet is exposed to the active form of

HSL and lipolysis is stimulated. See Fig 2. (26).

Xu et al. (27) reported that glucocorticoids directly stimulate lipolysis.

Sengenes et al. (28) reported in 2003 that natriuretic peptides also have the lipolytic effect. However, instead of activate adenylate cyclase, increase cAMP and activate PKA, natriuretic peptides active guanylyl cyclase, increase cGMP level and subsequently activate protein kinase G (PKG). PKG phosphorylates and activates HSL, inducing lipolysis (28).

Growth hormone is found to be essential for the increased lipolysis (29-31).

However, the mechanism is not clear yet (32). We should note that isoflurane, a usual laboratory anesthesia agent for animals, is shown to stimulate growth hormone secretion and thus increase lipolysis (33).

Tumor necrosis factor alpha (TNF-α) induces lipolysis through a reduction in perilipin levels in adipocytes (35-37). This is achieved by activating mitogen-activated protein kinase (MAPK) signaling cascade via extracellular signal-regulated kinases 1 and 2 (ERK 1/2) and c-Jun N-terminal kinases (JNK)

(34-36).

In the fed state, insulin activates phosphodiesterase 3B, which degrades cAMP to 5’ AMP (26; 32) (Fig 2). Then the activation of PKA by cAMP is lost.

5

Thus, lipolysis is decreased through a reduction in the phosphorylation of HSL and perilipin. In addition, insulin may also phosphorylate and activate protein phosphatase-1. Activated protein phosphatase-1 rapidly dephosphorylates

HSL to inactive form, resulting in an inhibition of lipolysis (26; 32).

Muller et al. reported that palmitate, H2O2, and the antidiabetic sulfonylurea drug, glimepiride inhibit lipolysis by cAMP degradation (37). The inhibition of lipolysis by palmitate can also be considered as a negative feedback control.

That is also why under lipolytic condition, fatty acids are removed quickly from adipocytes by fatty acid binding proteins (FABPs), preventing accumulation and resultant product inhibition (8).

Receptors coupled to inhibitory G (Gi) proteins have antilipolytic effect (32).

These receptors involve α2-adrenergic receptors (32; 38; 39), adenosine (32;

40), extracellular calcium-sensing receptor (CaSR) (41), nicotinic acid (32; 42), prostaglandins (32; 43), neuropeptide Y (NPY) (32; 44), peptide YY (PYY) (32;

44), and the ketone body β-hydroxybutyrate (45; 46).

6

Figure 2. Regulation of lipolysis in the adipocyte

(26)

7

1.4 Lipolysis under Stress

Stress conditions usually include fever, burn injury, infection, trauma, surgery, and malignancy. Klein et al. (47) reported that in critically ill patients, both rates of appearance for glycerol and fatty acid were almost twice as those of in normal volunteers, suggesting lipolysis is accelerated in response to severe stress. Wolfe et al. (48) reported that in patients with severe burn injury, glycerol Ra and fatty acid Ra were also significantly higher than values in normal volunteers. Furthermore, the rate of triglyceride/fatty acid cycling was increased by 450% in burned patients (48). Similarly, Shaw et al. (49) reported in severely septic patients and weight-losing gastrointestinal cancer patients, glycerol Ra and fatty acid Ra were significantly elevated compared to basal Ra values. In addition, the increased rates of lipolysis could not be suppressed in these septic and cancer patients with glucose infusion (49). The same group

(50) reported that patients with blunt trauma had significantly elevated rate of glycerol turnover and fat oxidation in the basal state. Vallerand et al. (51) reported that humans suffering from cold stress at 5 ºC for 3 h greatly increased lipolysis and plasma fatty acid Ra. Farias-Silva et al. (52) reported that footshock stressed rats increased basal lipolysis in white adipocytes.

The increased β-adrenergic activity, which is indicated by elevated plasma catecholamines, is a possible explanation for the stimulation of lipolysis in stressed patients (53). Moreover, massive activation of lipolysis under stress conditions can also be attributed to insulin resistance (53). It is believed that in 8 stressed conditions, insulin action is inhibited by increased sympathetic drive as well as stress hormones (54).

1.5 Measurement of Lipolysis

Lipolysis can be measured by arteriovenous (A-V) differences. A-V difference method is specifically used for the investigation of lipolysis in individual tissues, for example, mostly forearm muscle and subcutaneous adipose tissue (54).

One catheter is inserted into a vein draining the tissue, while the other catheter is inserted into an artery. The calculation of lipolysis is simply based on the measurement of concentration difference for fatty acid and glycerol over the tissue.

Lipolysis can also be measured systemically using radioactive or stable isotopes. Whole-body lipolysis is measured based on the blood glycerol turnover when labeled glycerol tracer is infused (55). Through calculating the difference in the enrichment of glycerol tracer before and after infusion into the system, the extent of glycerol production from lipolysis is obtained from the dilution of the glycerol tracer. Due to the absence of glycerol kinase in the adipose tissue, all the glycerol produced from adipocyte lipolysis is released to the circulation (54). Thus, glycerol Ra reflects the maximal lipolysis (54).

However, fatty acid re-esterification occurs before the release of fatty acid from adipose tissue into blood, which means the rate of fatty acid release should not

9 exactly equal to 3 times of glycerol Ra from lipolysis (54). Therefore, in order to fully understand lipolysis, a labeled fatty acid tracer is required to be infused in addition to the labeled glycerol tracer. However, ubiquitous contaminations of palmitate and stearate make the measurement of fatty acid Ra difficult to be

13 precise. Alternatively, in my study, [U- C18]oleate tracer, which is not a general contaminant, is utilized for measuring the rate of fatty acid release from adipose tissue into the circulation.

2. FATTY ACID OXIDATION

2.1 Introduction of Fatty Acid Oxidation Pathway

2.1.1 Cellular Uptake of Fatty Acids

Most circulating free fatty acids derived from adipocyte lipolysis or plasma triglycerides such as VLDLs and chylomicrons are complexed to albumin.

Long-chain fatty acids can passively diffuse through the phospholipid bilayer into the cell (56). Additionally, the transport of long-chain fatty acids across cellular membranes is facilitated by three membrane-associated proteins (Fig

3): (i) fatty acid translocase (FAT/CD36) (57); (ii) the plasmalemmal fatty acid-binding protein (FABPpm) (58); and (iii) the fatty acid transport protein

(FATP) (59). It has been described that FAT/CD36 null mice have reduced fatty acid uptake in muscle and adipose tissue, as well as decreased triglyceride

10 synthesis as a result of limiting fatty acid supply (60). Ibrahimi et al. (61) reported that mice with muscle-specific overexpression of FAT/CD36 greatly enhanced fatty acid oxidation in response to muscle contraction, and had lower plasma fatty acids and triglycerides, resulting in sparing of glucose and increased insulin level. Clarke et al. (62) reported that rats with muscle overexpression of FABPpm increased the rates of palmitate transport and the rates of palmitate oxidation. There are six FATP isoforms in the fatty acid transport protein family (63). Wu et al. (64) reported that FATP1 null adpipocytes completely inhibited insulin-stimulated long-chain fatty acid uptake. Similarly, FATP1 null mice decreased 80% insulin-stimulated fatty acid uptake in skeletal muscle (64). Garcia-Martinez et al. (65) reported that overexpression of FATP1 in primary cultured human muscle cells enhanced palmitate and oleate uptake at various fatty acid concentrations. It has also been described that FAT/CD36 is more effective than FABPpm and FATP1 in facilitating fatty acid transport while FABPpm and FAT/CD36 are more effective than FATP1 in increasing the rates of long-chain fatty acid oxidation (66). Once inside the cell, long-chain fatty acids are transported from the plasmalemma to outer mitochondrial membrane by cytoplasmic fatty acid-binding proteins

(FABPc) (67).

11

2.1.2 Activation of Fatty Acids

Long-chain fatty acids are activated to long-chain acyl-CoAs by long-chain acyl-CoA synthetase in the outer mitochondrial membrane. The reaction is:

- - RCOO + CoASH + ATP RCOSCoA + AMP + PPi

Since this reaction requires free CoA and ATP, lack of CoASH and cytosolic

ATP would impair the activity of long-chain acyl-CoA synthetase and

β-oxidation flux (68). Long-chain acyl-CoA synthetase can also be inhibited by long-chain acyl-CoA as a feedback regulation (68; 69). FATP isoforms have the similar activity as long-chain acyl-CoA synthetase (70; 71).

12

Figure 3. Transport of fatty acids into the cell

(Bian F. Novel Aspects of Fatty Acid Oxidation Uncovered by the Combination of Mass Isotopomer Analysis and Metabolomics. OhioLINK ETD, 2006)

13

2.1.3 Carnitine Palmitoyltransferase System

Activated long-chain acyl-CoAs are transported into mitochondria matrix by the carnitine palmitoyltransferase (CPT) system (Fig 4 (72)). CPT I, located on the outer mitochondrial membrane, catalyzes the transfer of acyl groups from long-chain acyl-CoAs to carnitine to produce acylcarnitine. There are three tissue-specific CPT I isoforms: (i) liver (L-CPT I) (73), (ii) muscle (M-CPT I) (74), and (iii) brain (C-CPT I) (75), respectively. CPT I is inhibited by malonyl-CoA

(76) so that CPT I is believed to be the rate-limiting step of fatty acid oxidation.

A high level of malonyl-CoA correlates with decreased hepatic CPT I activity whereas a low level of malonyl-CoA correlates with increased hepatic CPT I activity. Acylcarnitine passes through the inner mitochondrial membrane via carnitine-acylcarnitine translocase (CACT) by a 1:1 exchange with free carnitine. CPT II is located on the matrix side of inner mitochondrial membrane.

There is only one CPT II isoform (77). CPT II regenerates long-chain acyl-CoA from acylcarnitine for β-oxidation.

14

Figure 4. The carnitine palmitoyltransferase system

(72)

15

2.1.4 Mitochondrial β-oxidation Spiral

Once inside the mitochondria, long-chain acyl-CoAs undergo β-oxidation.

Mitochondrial β-oxidation is the repeated process that sequentially removes two-carbon fragments from the carboxyl end of the long-chain acyl-CoA. One cycle of β-oxidation includes enzymatic dehydrogenation, hydration, oxidation and thiolysis (Fig 5 (78)). There are four main chain length specific enzymes catalyzing β-oxidation: acyl-CoA dehydrogenase, 2-enoyl-CoA hydratase,

3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase. The mitochondrial trifunctional protein (MTP) complex is closely bound to the inner mitochondrial membrane and is unique for long-chain fatty acid β-oxidation (79;

80). This complex comprises the last three consecutive enzymes. Long-chain

2-enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase are found on the α-subunit of the MTP complex, whereas long-chain

3-ketoacyl-CoA thiolase is located on the β-subunit. The overall reaction of one round of β-oxidation is:

+ Cn Acyl-CoA + FAD + H2O + NAD + CoASH Cn-2 Acyl-CoA +

+ FADH2 + NADH + H + Acetyl-CoA

Recurring β-oxidation cycles continue until the entire chain is completely cleaved into final product acetyl CoA.

16

Figure 5. Mitochondrial β-oxidation spiral and the sites of feedback control

(78)

17

2.1.5 Fatty Acid α- and ω-oxidation

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a multi-branched fatty acid (81). There is no endogenous synthesis of phytanic acid in man (82;

83), so it can only be derived either from the conversion of dietary phytol (81;

83) or direct dietary phytanic acid sources, for example, dairy products, meat, fish, fish oil, and vegetable oil (81).

Because phytanic acid has a methyl group at 3-position, it cannot be broken down by β-oxidation (83). Instead, phytanic acid undergoes α-oxidation. Fatty acid α-oxidation is the removal of the carboxyl end of the fatty acid. Phytanic acid is activated to corresponding CoA ester in peroxisomes by either phytanoyl-CoA ligase (84) or long-chain acyl-CoA synthetase (85). The first step of α-oxidation pathway is the hydroxylation of phytanoyl-CoA to

2-hydroxyphytanoyl-CoA, catalyzed by phytanoyl-CoA 2-hydroxylase (83).

Next, the second step is the decarboxylation of 2-hydroxyphytanoyl-CoA to an aldehyde pristanal and a formyl-CoA, catalyzed by 2-hydroxyphytanoyl-CoA lyase (83). Formyl-CoA is then converted to CoA and formate (83). Formate is subsequently converted to CO2 (83). The following step is the dehydrogenation of pristanal to pristanic acid, catalyzed by aldehyde dehydrogenase (83). Pristanic acid can be activated to its CoA ester and undergoes further peroxisomal β-oxidation (83).

Mutations of the phytanoyl-CoA 2-hydroxylase gene causes phytanic acid accumulation, which leads to Refsum disease (86). 18

There is also an alternative ω-oxidation for phytanic acid (87). Phytanic acid

ω-oxidation pathway is the degradation of phytanic acid from the ω-end of the fatty acid. Reactions of phytanic acid ω-oxidation involve the hydroxylation to

ω-hydroxyphytanic acid catalyzed by CYP enzyme superfamily, alcohol oxidation to carboxylic acid, and thioesterification with CoASH (88). The formed dicarboxyl-CoA undergoes subsequent β-oxidation to produce

3-methyladipic acid (88), which is excreted in the urine (89).

Wierzbicki et al. (88) reported that ω-oxidation functions as an important backup for catabolizing phytanic acid in patients with Refsum disease.

ω-oxidation can also occur with regular fatty acids. Medium-chain fatty acids are the main substrates for ω-oxidation to generate dicarboxylic acids.

2.2 Regulation of Fatty Acid Oxidation

2.2.1 Regulation of Carnitine Palmitoyltransferase I by Malonyl-CoA

The inhibition of CPT I by malonyl-CoA is very important in regulating fatty acid

β-oxidation flux. Malonyl-CoA is the first committed intermediate of fatty acid synthesis (90). It has been thought that the cytosolic concentration of malonyl-CoA is primarily determined by the activities of acetyl-CoA carboxylase (ACC) and malonyl-CoA decarboxylase (MCD) (91). A high level of malonyl-CoA correlates with decreased hepatic CPT I activity whereas a low level of malonyl-CoA correlates with increased hepatic CPT I activity. 19

ACC gene expression is stimulated by insulin and a high carbohydrate, low fat diet (92). ACC is also activated by citrate (91; 93). Activation of ACC results in increased malonyl-CoA concentration, which inhibits CPT I and the entry of fatty acids into mitochondria before oxidation. ACC is inhibited by AMPK phosphorylation and long-chain acyl-CoAs (91-93). Inactivation of ACC results in decreased malonyl-CoA concentration, which releases the suppression of

CPT I, facilitating the transport of fatty acids for mitochondrial oxidation.

Increased fatty acid oxidation flux controlled by CPT I can also result from decreased malonyl-CoA concentration through its degradation by MCD (94).

However, the mechanism of MCD regulation by AMPK is still not clear. For example, Saha et al. (95) reported the activation of AMPK phosphorylates and activates MCD during muscle contraction. Similarly, Park et al. (96) reported

MCD activity is increased by exercise and injection of AMPK activator. In contrast, Habinowski et al. (97) reported that MCD is not a substrate for AMPK phosphorylation.

Louet et al. reported that L-CPT I is induced by peroxisomes proliferators and long-chain fatty acids at the transcriptional level (98).

M-CPT I is much more sensitive to malonyl-CoA than L-CPT I (99). The concentration of malonyl-CoA in the heart and skeletal muscle is in the range of 1 – 10 μM (68; 100). Malonyl-CoA concentrations in these tissues are at most 1000 times of IC50 of malonyl-CoA on M-CPT I (68; 100-102). However, heart and skeletal muscle always have particularly high energy requirements

20 and active fatty acid oxidative capacities at all times. How can β-oxidation still take place in these tissues presenting M-CPT I? Eaton et al. (68; 100) proposed several possibilities: (i) there are insensitive splicing isoforms of

M-CPT I located on the outer mitochondrial membrane in muscle allowing

β-oxidation to proceed even in the extremely high concentration of malonyl-CoA (103; 104), (ii) much of malonyl-CoA is present inside the mitochondria rather than cytosol because of the carboxylation of acetyl-CoA catalyzed by mitochondrial propionyl-CoA carboxylase (105), (iii) 16% of mitochondrial MCD is overt and thus MCD is able to dispose cytosolic malonyl-CoA in heart (106), (iv) some of malonyl-CoA is bound to cytosolic binding proteins (107) or mitochondrial low-affinity sites (108) preventing the inhibition on M-CPT I, and (v) perhaps M-CPT I is not rate-limiting for

β-oxidation in heart and skeletal muscle. Schmidt et al. (109) also reported that the regulation of M-CPT I by malonyl-CoA can be affected by age and cold exposure.

2.2.2 Redox Regulation

Each cycle of fatty acid β-oxidation produces one mole NADH at the

3-hydroxyacyl-CoA dehydrogenase step. Thus, redox regulation is crucial in control of β-oxidation flux. A high ratio of NADH/NAD+ inhibits

3-hydroxyacyl-CoA dehydrogenase and also mitochondrial β-oxidation

21 pathway. For example, oxidation of ethanol to acetaldehyde and acetate increases the redox ratio and impairs β-oxidation. Enhanced NADH/NAD+ redox ratio by ethanol metabolism also inhibits other NAD+-dependent enzymes in CAC cycle such as isocitrate dehydrogenase and α-ketoglutarate dehydrogenase (110). A decrease in CAC cycle midly decreases fatty acid

β-oxidation (110). Moreover, ethanol inhibits CPT I in rat hepatocytes (111).

Also, Rabinowitz et al. (112) reported that chronic ethanol feeding in rats affects octanoate oxidation more than palmitate oxidation, indicating that the defect is in medium-chain acyl-CoA dehydrogenase. Van der Vusse et al. (113) reported that there is the competition for oxidation between lactate and fatty acids in energy metabolism. According to a similar mechanism as ethanol, the consequence of lactate oxidation to pyruvate increases mitochondrial redox ratio, resulting in the decreased β-oxidation activity (113).

2.2.3 Feedback Regulation of Fatty Acid Oxidation

There is a feedback control system in the regulation of β-oxidation flux (Fig 5

(78)). The enzymes in β-oxidation spiral can be inhibited by their respective products. For instance, 3-ketoacyl-CoA thiolase is inhibited by acetyl-CoA or an increased [acetyl-CoA]/[CoASH] ratio (68). In addition, 2-enoyl-CoA hydratase (114) and acyl-CoA dehydrogenase (115) with all kinds of chain-length specificities can be inhibited by the accumulation of

22

3-ketoacyl-CoA. Thus, the accumulation of 3-ketoacyl-CoA seems to inhibit the entire β-oxidation spiral (68).

2.2.4 Regulation of Fatty Acid Oxidation by CoA

Several reactions in fatty acid oxidation pathway require CoA. Both CPT II and

3-ketoacyl-CoA thiolase are CoASH dependent. As mentioned above, the accumulation of 3-ketoacyl-CoA thiolase can inhibit the entire β-oxidation and it is strictly regulated by [acetyl-CoA]/[CoASH] ratio. Therefore, the rate of

β-oxidation could be controlled by the availability of free CoA. It has been shown that the total free CoA pool is limited in size (68; 116). Trapping of free

CoA would result in very bad consequence in energy metabolism. Lack of mitochondrial CoASH inhibits 3-ketoacyl-CoA thiolase and subsequently the entire β-oxidation pathway, thus acyl-CoAs accumulate and trap even more of free CoA (68). CASTOR (CoA sequestration, toxicity or redistribution) could be triggered by many drugs like organic acids or drugs metabolized to organic acids, for example, propionic acid, valproic acid (117; 118), phenylbutyric acid

(118), benzoic acid (118), aspirin (salicylic acid) (117; 118), hypoglycin (117), amineptine (117), tianeptine (117), and 2-arylpropionic acid (117). Recycling of mitochondrial CoASH can result from the reactions catalyzed by carnitine acyltransferase (68; 119), acyl-CoA:glycine-N-acyltransferase (68; 120), citrate synthase (68), ketogenic acetoacetyl-CoA thiolase (68), acyl-CoA thioesterase

23

(68), as well as the breakdown of fatty acyl-CoAs catalyzed by acyl-CoA synthetase (68).

2.3 Ketone Body Synthesis

2.3.1 C4-ketogenesis

Ketone bodies are byproducts of fatty acid β-oxidation. Ketone body synthesis is in the liver mitochondria. C4-ketone body synthesis begins with the condensation of acetoacetyl-CoA (AcAc-CoA) derived from even-chain fatty acid degradation and acetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA

(HMG-CoA), catalyzing by HMG-CoA synthase. HMG lyase then cleaves

HMG-CoA to yield acetoacetate (AcAc). AcAc is reduced to β-hydroxybutyrate

(BHB) by BHB dehydrogenase. See Fig 6.

During the postprandial period, the concentration of circulating C4-ketone bodies is usually less than 0.1 mM (121; 122). During prolonged fasting, the concentration goes up to 6 mM (121-123). In diabetic ketoacidosis, the concentration of C4-ketone bodies can reach 25 mM (121; 122).

C4-ketogenesis is highly stimulated by glucagon (124; 125) and inhibited by insulin (124). High insulin levels markedly increase the [BHB]/[AcAc] ratio, which is an indicator of mitochondrial [NADH]/[NAD+] ratio (124). As mentioned earlier, the elevated mitochondrial redox ratio inhibits fatty acid oxidation pathway and also the CAC cycle. In addition, the redox ratio can also affect the 24 ketone body ratio in perfused rat liver (126). Wu et al. (126) reported that high

- [CO2] or [HCO3 ] decreased the mitochondrial redox ratio, thus resulting in a decreased [BHB]/[AcAc] ratio from octanoate in perfused livers from fasted rats, whereas extracellular pH did not alter total ketogenesis.

Fernandez-Figares et al. (124) reported that high levels of dexamethasone stimulated C4-ketogenesis whereas leptin had no effect on C4-ketogenesis in primary cultured porcine hepatocytes. Vega et al. (127) reported that

C4-ketogenesis can be enhanced in adult men by the short-term administration of oxandrolone, which increases hepatic lipase and fatty acid oxidation. The same group (128) also reported that moderately obese patients with endogenous hypertriglyceridemia had impaired C4-ketogenesis as shown by reduced BHB level during either fasting or after a fatty meal. Beylot et al. (129) reported that TNF-α injection inhibited hepatic C4-ketogenesis in rats as demonstrated by lower ketone bodies concentrations and appearance rates, even though the plasma free fatty acids concentrations increased. Nomura et al. (130) reported that C4-ketogenesis from oleate can be inhibited by the nitric oxide donor NOR 3 in isolated rat hepatocytes. Connolly et al. (131) reported that at low norepinephrine infusion rate C4-ketogenesis was not affected in conscious dogs, while at high norepinephrine infusion rate C4-ketogenesis was significantly stimulated.

25

2.3.2 C5-ketogenesis

C5-ketone bodies are formed from the oxidation of odd-chain fatty acids (Fig 7).

C5-ketone body synthesis uses the same enzymes in the HMG-CoA cycle as used by C4-ketogenesis (132). C5-ketogenesis begins with the condensation of

β-ketopentanoyl-CoA (BKP-CoA) derived from odd-chain fatty acid degradation and acetyl-CoA, catalyzed by HMG-CoA synthase, to form

3-hdroxy-3-ethylglutaryl-CoA (HEG-CoA). Subsequently, C5-ketone bodies:

β-hydroxypentanoate (BHP) and β-ketopentanoate (BKP) can be formed via

HMG-CoA lyase and BHB dehydrogenase.

Kinman et al. (133) reported that intravenous and intraduodenal infusion of triheptanoin (a medium odd-chain triglyceride) at 40% of the caloric requirement in rats increased blood concentration of C5-ketone bodies to about 0.15 mM and 0.22 mM, respectively, compared to rats infused with saline (undetectable). This is because odd-chain fatty acid precursors for

C5-ketone bodies are absent from the diet of non-ruminant mammals (132).

Leclerc et al. (134) reported that C5-ketone bodies can also be derived from liver oxidation of a potential nutrient, R,S-1,3-pentanediol. In addition,

C5-ketone bodies are very good anaplerotic precursors.

2.3.3 HMG-CoA synthase

Besides the control points at levels of supply of fatty acids into the liver 26

(adipocyte lipolysis) and entry of fatty acids into liver mitochondria (CPT I), ketogenesis is regulated through the rate-limiting step catalyzed by HMG-CoA synthase (121). HMG-CoA synthase is only largely present in the liver (121) so that liver is the primary site of ketone body synthesis. There are two HMG-CoA synthase isoforms (135): the cytosolic isoform and the mitochondrial isoform.

Cytosolic HMG-CoA synthase is involved in cholesterol synthesis, while mitochondrial HMG-CoA synthase is involved in ketone body synthesis (135).

Mitochondrial HMG-CoA synthase is regulated by (i) succinylation and desuccinylation in the short term (136; 137), and (ii) transcriptional control in the long term (137; 138). Succinyl-CoA inhibits HMG-CoA synthase and succinylates the enzyme by a covalent reaction (137; 139). Why is ketogenesis increased by fasting? It can be explained as glucagon secretion is stimulated during fasting and glucagon decreases both succinyl-CoA concentration and the extent of succinylation of HMG-CoA synthase (137; 137; 139; 140). Thus the activity of HMG-CoA synthase is increased during fasting.

Starvation also increases the transcription and protein amount of HMG-CoA synthase (137; 141). Moreover, prolonged exercise (137), administration of dibutyryl cAMP (121; 137), high fat feeding (141), suckling (142), and diabetes

(141) upregulate the gene expression of HMG-CoA synthase. In contrast, insulin (138; 143), weaning (121; 142), and the hepatocytes nuclear factor 4

(HNF-4) (144) downregulate the gene expression of HMG-CoA synthase.

Furthermore, HMG-CoA synthase can be induced by fatty acids at the

27 transcriptional level because the HMG-CoA synthase gene promoter contains a PPAR responsive element, which regulates the gene expression of the enzyme by fatty acids itself (137; 145; 146). Patel et al. (147) reported that although HMG-CoA synthase is inactivated by forming an adduct with

4-hydroxynonenal, a lipid peroxidant generated by chronic ethanol consumption, rats fed ethanol at 36% of total calorie requirement for 31 days have unaffected HMG-CoA synthase activity in the live. And the protein level of

HMG-CoA synthase is even increased, probably due to a compensatory mechanism (147).

28

Figure 6. C4-ketone body metabolism

29

Figure 7. C5-ketone body metabolism

30

2.3.4 Interrelations between C4- and C5-ketogenesis

Roe et al. (148) reported that three patients with very-long-chain acyl-CoA dehydrogenase deficiency had substantially increased plasma C4-ketone bodies and C5-ketone bodies during meals with triheptanoin. This suggests that the acetyl moieties contained in heptanoate can be used for both C4- and

C5-ketogenesis, and perhaps C5-ketone bodies may inhibit the utilization of

C4-ketone bodies in peripheral tissues. Kinman et al. (133) reported that the intravenous infusion of triheptanoin at 40% of caloric requirement in rats increased blood C5-ketone bodies concentration but decreased blood

C4-ketone bodies concentration. This suggests that C5-ketogenesis may interfere with the hepatic synthesis of C4-ketone bodies. Thus, it seems that there is a competition between C4- and C5-ketogenesis.

In 2009, Deng et al. (132) first reported the interrelationship between C4- and

C5-ketogenesis in rat livers perfused with different labeled octanoate and/or heptanoate. Deng et al. (132) reported that the uptake of octanoate is very similar to the uptake of heptanoate when octanoate or heptanoate was individually perfused but the flux of C4-ketogenesis is more rapid than that of

C5-ketogenesis. This is because propionyl moieties of heptanoate are not only used for C5-ketogenesis but are also channeled to the CAC cycle and gluconeogenesis (132). However, when octanoate and heptanoate were perfused together, the competition favored octanoate uptake over heptanoate uptake (132). In the presence of octanoate, C5-ketogenesis from heptanoate 31 was much lower compared to heptanoate perfusion alone. Deng et al. (132) concluded that in the perfusion of both octanoate and heptanoate,

C4-ketogenesis is predominant over C5-ketogenesis. Also, octanoate inhibits anaplerosis and gluconeogenesis from heptanoate.

2.4 Ketone Body Utilization

2.4.1 Ketolysis Pathway

Ketone bodies are excellent energy sources for many peripheral tissues. Brain, heart and kidney have the greatest capacity for utilizing ketone bodies (149).

C4- and C5-ketone bodies formed in the liver are exported to extrahepatic organs, and converted back to acetyl-CoA and propionyl-CoA by the same enzymes: BHB dehydrogenase, 3-oxoacid transferase, and AcAc-CoA thiolase

(Fig 6 and 7, right half). The key enzyme involved in the ketolysis pathway is mitochondrial AcAc:succinyl-CoA transferase (3-oxoacid-CoA transferase), which is highly expressed in almost all the tissues except liver (150). This is why the liver cannot use ketone bodies synthesized by itself.

2.4.2 Utilization of Ketone Bodies in Brain

Ketone bodies are transported across blood brain barrier by the monocarboxylate transporter (151; 152). The permeability of ketone bodies

32 into the brain is largely determined by the circulating concentration of ketone bodies. Thus, any condition that increases the concentration of ketone bodies present in the blood, for example, fasting, enhances ketone body influx into the brain (153). Except for the role of preferential use for energy supply, another function of ketone bodies in the brain is to provide AcAc-CoA and acetyl-CoA for the synthesis of cholesterol and lipids which are important for brain development (154). AcAc-CoA synthetase was described to activate ketone bodies for the incorporation into cholesterol and fatty acids (155). Recently, it was reported that there is a high expression of cytosolic AcAc-CoA synthetase for ketone body utilization in human brain (156). Ketone bodies are associated with the improvement of epilepsy in brain. The proposed mechanism is that the utilization of ketone bodies favors the production of γ-aminobutyric acid

(GABA). Daikhin et al. (157) reported that the utilization of ketone bodies in the brain produces sufficient amount of acetyl-CoA for the CAC cycle. It pulls the reaction toward the formation of citrate by the combination of oxaloacetate and acetyl-CoA. This leads to less oxaloacetate undergoing the reaction catalyzed by aspartate aminotransferase so that glutamate transamination is decreased

(157; 158). Relatively more glutamate is converted to more GABA by glutamate decarboxylase (157; 158). Since GABA is the major inhibitory neurotransmitter and a potent anti-epileptic agent in the mammalian brain (158;

159), ketone bodies in the brain have anticonvulsant effect through increasing

GABA synthesis (160).

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2.4.3 Utilization of Ketone Bodies in Heart

The heart is an organ requiring extremely high energy because it is comprised by continuously active cardiac muscle. Sultan (161) reported that BHB utilization is stimulated by physiological insulin concentration in hearts isolated from normal and diabetic rats. Sultan (162) also reported that anaplerotic propionate inhibited utilization of BHB but stimulated its oxidation in perfused non-working rat heart. Lippolis et al. (163) reported that hyperthyroid rats had reduced activities of BHB dehydrogenase and 3-oxoacid-CoA transferase in heart mitochondria. Thus, the utilization of ketone bodies in the heart is lower.

In heart mitochondria of diabetic rats, the activities of these two enzymes in ketone body utilization were decreased (164). In addition, Lukivskaya et al.

(165) reported that intragastrally ethanol feeding at 3 g/kg for 40 days also decreased the activities of these two ketone body utilizing enzymes in the rat heart. Forsey et al. (166) reported that ketone bodies competed with fatty acids and inhibited the oxidation of oleate and octanoate in isolated perfused heart.

Similarly, Vanoverschelde (167) et al. reported that the infusion of BHB competed with palmitate and decreased myocardial palmitate oxidation in humans and dogs. In perfused rat heart, ketone bodies inhibit glucose uptake and oxidation (168). 4-Bromocrotonic acid which inhibits AcAc-CoA thiolase inhibits ketone body degradation in rat heart mitochondria (169).

34

2.4.4 Utilization of Ketone Bodies in Kidney

Kidney is an organ using ketone bodies. Little et al. (170) reported that ketone bodies can account for more than 50% of the renal oxygen consumption if they are completely oxidized. And net ketone body uptake by the kidney is strongly correlated with the arterial concentration in dogs (170). Angielski et al. (171) reported that in rats under normal pH, the contribution of kidney to utilize ketone bodies is 28%, whereas in metabolic alkalosis, the kidney cannot utilize ketone bodies. Lietz et al. (172) reported that renal gluconeogenesis is activated by 5 mM BHB or AcAc, which is incubated in the presence of alanine and glycerol in isolated rabbit renal cortical tubules. Ikeda et al. (173) reported that the uptake of ketone bodies is not impaired by any of starving, thyrotoxic, or diabetic condition in perfused rat kidney. However, Sapir et al. (174) and

Owen et al. (123) reported that renal reabsorption of ketone bodies increased during prolonged starvation in obese subjects even though there is still a net uptake. And they believe that the increased reabsorption maintains a high circulating concentration of ketone bodies, thus decreasing ammonium excretion, which could minimize body protein loss (174).

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3. FATTY ACID OXIDATION DISORDERS

3.1 Introduction of Fatty Acid Oxidation Disorders (FOD)

Fatty acids are an important fuel for most tissues, particularly heart and skeletal muscle (175). Although glucose is a good energy source, the availability of glucose is very limited. During fasting and illness, the body would try to use stored fat. At least 25 enzymes and specific transport proteins are involved in mitochondrial fatty acid oxidation (176; 177). Due to genetic defects, certain transporter or enzyme which is responsible for fatty acid oxidation is either missing or not working correctly, causing fatty acid oxidation disorders

(FODs) in humans. FOD patients thus are unable to breakdown fatty acids to make energy, leading to severe health problems and even death.

FOD can affect (i) plasma membrane free fatty acid transporter, (ii) plasma membrane carnitine transporter, (iii) CPT system (CPT I, translocase, CPT II), and (iv) β-oxidation spiral.

3.1.1 Disorders of Plasma Membrane Functions

Disorders of plasma membrane functions include defects in cellular uptake of fatty acids and primary carnitine deficiency.

Al Qdaib et al. (178) reported that two white boys presenting a defect in the transport of long-chain fatty acids at the plasma membrane level, resulting in

36 liver failure at the young age. Tanaka et al. (179) reported that the defect in uptake of radioactive labeled long-chain fatty acids by the heart is probably related to mutations of FAT/CD36 in fatty acid transport deficient patients.

Nezu et al. (180) first reported that primary carnitine deficiency is due to loss of

OCTN2 function, which is a sodium ion-dependent, high affinity human carnitine transporter. Stanley et al. (181) reported that cardiomyopathy is the most common presentation in primary carnitine deficiency.

3.1.2 Disorders of the Carnitine Palmitoyltransferase System

Disorders of the CPT system include defects in CPT I, translocase, and CPT II.

To date, there is only L-CPT I deficiency has been reported (182). Also, the molecular basis of CPT I deficiency is not very clear. In 1998, Ijlst et al. (183) provided evidence that a missense mutation in D454G causes hepatic CPT I deficiency. The Hutterite communities have a high carrier frequency for CPT I deficiency (176; 184).

Stanley et al. (185) first reported that there is a translocase deficiency. Huizing et al. (186) reported that the molecular basis of translocase deficiency is a homozygous cytosine nucleotide insertion in patient’s translocase gene.

CPT II deficiency has been described as the most common defect in disorders of the CPT cycle (176). It is also reported that CPT II deficiency is the most common metabolic cause of rhabdomyolysis in adults (182). Taroni et al. (187)

37 reported that in an early-onset patient, three missense mutations in CPT II allele are the molecular basis of CPT II deficiency: (i) V368I, a substitution of valine by isoleucine, (ii) R631C, a substitution of arginine by cysteine, and (iii)

M647V, a substitution of methionine by valine. Later, Verderio et al. (188) reported that a proline to histidine substitution (C665A) and an aspartate to asparagine substitution (G2173A) cause CPT II deficiency. Taggart et al. (189) reported that patients who have two novel S113L and R503C mutations are susceptible of CPT II deficiency.

3.1.3 Long-chain Fatty Acid Oxidation Disorders

Typically, long-chain FODs can affect translocase, CPT II, very-long-chain acyl-CoA dehydrogenase (VLCAD), isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), and mitochondrial trifunctional protein (MTP) complex.

In 1992, from the rat liver mitochondria extract Izai et al. (190) first purified and identified a novel VLCAD, which has different properties and chain-length specificity from other acyl-CoA dehydrogenase. Since then, Yamaguchi et al.

(191) reported the inability to dehydrogenate palmitoyl-CoA in patients. This was thought to result from a deficiency of long-chain acyl-CoA dehydrogenase, but is actually caused by a deficiency of VLCAD. Mathur et al. (192) reported that 80% of mutations on VLCAD gene are associated with cardiomyopathy.

38

The conclusion was drawn from 18 defective infants (192).

In 1990, LCHAD deficiency was first identified and reported by Wanders et al.

(193) in a 5 months old infant. In isolated LCHAD deficiency there are almost normal activities of long-chain 2-enoyl-CoA hydratase and long-chain

3-ketoacyl-CoA thiolase in the MTP complex (194). Ijlst et al. (195) reported that the G1528C mutation on α-subunit of MTP complex is directly responsible for the loss of LCHAD activity. A specific feature of LCHAD deficiency is peripheral neuropathy (196). It has also been reported that female LCHAD deficient patients are highly susceptible to have AFLP (acute fatty liver of pregnancy) and HELLP (hemolysis, elevated liver function tests, low platelets) syndrome (197-199). There is also a high frequency to get a LCHAD deficient fetus in female patients with LCHAD deficiency (198).

MTP deficiency is the defect with lost activities of all three enzymes. Isolated

LCHAD deficiency is much more common than MTP deficiency (182) and peripheral neuropathy is also detected in MTP deficiency (196). It seems that

MTP deficient patients are prone to have more severe clinical presentations than LCHAD patients (182; 200).

3.1.4 Disorders of Medium-chain Fatty Acid Oxidation

Disorders of medium-chain fatty acid oxidation involve medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, medium- and short-chain

39

3-hydroxyacyl-CoA dehydrogenase (M/SCHAD) deficiencies, and medium-chain 3-ketoacyl-CoA thiolase (MCKAT) deficiency.

MCAD deficiency was first reported by Stanley et al. (201) in 1983 in three children who had less than 2.5% normal MCAD activity against octanoyl-CoA.

MCAD deficiency is the most common FOD with a high incidence in newborns in Europe (182; 184). Gregersen et al. (202) and Matsubara et al. (203) reported that an adenine to guanine transition at position 985 of MCAD cDNA

(G985) results in a lysine to glutamate substitution, which is related to up to 90% of the disease in Caucasians.

SCHAD was shown to have activity against medium-chain length acyl-CoA substrates (204), so the lack of SCHAD would also cause medium-chain fatty acid oxidation disorder.

MCKAT deficiency was first reported in 1997 in a Japanese male neonate who had significantly reduced octanoate and palmitate oxidation (205).

3.1.5 Disorders of Short-chain Fatty Acid Oxidation

Disorders of short-chain fatty acid oxidation can affect short-chain acyl-CoA dehydrogenase (SCAD).

Corydon et al. (206) reported that 625G>A and 511C>T are two susceptible mutations on the SCAD gene which are probably responsible for SCAD deficiency. Tein et al. (207) reported that SCAD deficient patients specifically

40 present ophthalmoplegia and multicore myopathy.

3.2 Pathogenesis of Fatty Acid Oxidation Disorders

All kinds of FODs share some common clinical symptoms. For example, FOD patients generally present hypoketotic hypoglycemia, rhabdomyolysis, muscle weakness, cardiomyopathy, liver disease, and congenital abnormalities.

These symptoms can be ascribed to several mechanisms. First of all, due to the fact that the body cannot mobilize fat stores and due to the impaired ketogenesis, there is no glucose sparing effect from ketone bodies. Thus, FOD patients exhaust glucose quickly, leading to hypoketotic hypoglycemia.

Second, inadequate ATP production results from the defective fatty acid oxidation. Third, due to the inability to breakdown long-chain fatty acids, toxic metabolites such as long-chain fatty acyl-CoAs accumulate in the cell.

Accumulation of large amount of long-chain fatty acyl-CoA results in sequestration of CoASH, thus decreasing acetyl-CoA and further inhibiting energy production from CAC cycle. Moreover, long-chain fatty acyl-CoAs or long-chain fatty acylcarnitines are detergents which cause the leakage of mitochondrial and cell membranes. As a result, creatine kinase and CAC intermediates easily leak through the permeable mitochondrial membranes.

Thus, a massive cataplerosis occurs, which is not compensated by physiological anaplerosis. Furthermore, increased concentration of circulating

41 free fatty acids that resulted from reduced fatty acid oxidation induce insulin resistance (208).

3.3 Diagnosis of Fatty Acid Oxidation Disorders

The biochemical diagnosis of FODs involves three components (209): (i) routine testing, (ii) specialized testing, and (iii) in vitro testing.

Routine laboratory investigations include plasma or serum testing for electrolytes, glucose, ammonia, lactate, uric acid, ketone bodies, creatine kinase and so on (177). Routine laboratory investigations also include urine testing for ketone bodies (177).

Specialized laboratory investigations include plasma or serum testing for carnitine, acylcarnitines, free fatty acids, 3-hydroxy fatty acids and so on (177).

Specialized laboratory investigations also include urine testing for organic acids and acylglycines (177).

Although abnormal concentrations of plasma or urine metabolites provide hints for recognizing FODs, the final confirmation requires in vitro investigations.

Confirmatory laboratory investigations are just such common testing usually using skin fibroblasts for enzymatic and functional assays (209). Confirmatory testing is necessary to be followed by (209): (i) metabolic flux studies which measure the end products of labeled fatty acid oxidation in intact cells, (ii) direct enzyme and transport analysis which assays all the enzymes and

42 transporters involved in fatty acid oxidation in cultured cells, and (iii) molecular analysis which directly screens mutations in specific genes in biological samples.

3.4 Treatment of Fatty Acid Oxidation Disorders

3.4.1 Carnitine Supplementation

Carnitine has two important functions: (i) to help transport of long-chain fatty acids into mitochondria for oxidation to make energy, and (ii) to help removal of toxic accumulation of long-chain acyl-CoAs from the cell. Primary carnitine deficiency can be effectively treated by L-carnitine supplementation (182; 210).

Recommended doses of carnitine supplementation were reported to range from 50 mg/kg/day in children to 150 mg/kg/day in adults (211). L-carnitine can convert toxic long-chain acyl-CoAs to acylcarnitines, which are excreted into the urine, thus recycling CoA. Thus, it seems that carnitine is useful for the treatment of other FODs. However, Treem et al. (212) reported that in a patient who had MCAD deficiency, after 3 months oral L-carnitine treatment, the treatment failed to prevent accumulation of medium-chain fatty acids in plasma, lethargy, vomiting and hypoglycemia. In another patient who has multiple acyl-CoA dehydrogenation deficiency, the patient was pale, drowsy, vomiting, and had a throat infection and very low blood glucose during carnitine treatment. However, after the stop of carnitine supplementation, such 43 symptoms disappeared for 4 years (213). That is why Green et al. (213) reported that the carnitine supplementation may be even harmful in disorders of fatty acid oxidation. In addition, Bonnet et al. (214) reported that the accumulation of long-chain acylcarnitines is probably responsible for arrhythmias. Thus, it is still controversial to use carnitine supplementation for the treatment of FODs except in primary carnitine deficiency.

3.4.2 Decreased Dietary Fat intake

Since FOD patients cannot oxidize long-chain fat, they should be fed a long-chain fat restricted diet. Generally, long-chain fat intake in health human ranges from 30 to 40% of total energy. With a long-chain fat restricted diet for

FOD patients, long-chain fat intake should be adjusted to about 15% of total calories. However, Piper et al. (215) reported that consumption of a very-low-fat diet may lead to essential fatty acid deficiency. Thus, the fat restricted diet must include essential fatty acids, which should reach to about 1

– 2% of total calories (216).

3.4.3 Increased Dietary Carbohydrate

For FOD patients, the decreased energy from long-chain fat can be compensated by a high carbohydrate diet. The high carbohydrate intake can

44 also prevent hypoglycemia which is often met by FOD patients. FOD patients should be fed by raw cornstarch at bed time or before intense activity as a good long-lasting energy source (211). Raw cornstarch provides a slow glucose release, thus preventing both hypoglycemia and lipolysis (211; 216).

3.4.4 Minimizing Fat Mobilization

Lipolysis is positively correlated to plasma long-chain fatty acid levels.

Lipolysis and fatty acid oxidation occurs during times of increased energy demand. Thus, in order to minimize fat mobilization from adipose tissue in

FOD patients, exertion must be strictly avoided. Also, due to the inability to metabolize fat, FOD patients cannot undergo fasting. Thus, increased meal frequency is necessary for the treatment of FOD patients (211).

3.4.5 Infusion of Glucose and Insulin as Acute Treatment

During acute FOD decompensation episodes induced by fasting, fever, infection, or trauma, nasogastric tube administration of carbohydrates is a good treatment. However, nasogastric tube feeding cannot be used under conditions of recurrent vomiting or severe gastroenteritis. Instead, the usual treatment of acute FOD decompensation involves intravenous infusion of 10% glucose solution (10 – 12 mg/kg/min or greater) and insulin, in order to

45 promptly block lipolysis, overcome hypoglycemia, and provide energy (217).

3.4.6 Medium-chain Triglycerides (MCT)

The decreased energy from long-chain fat not only can be compensated by a high carbohydrate diet, but also can be compensated by MCT. Brown-Harrison et al. (218) reported that the dietary therapy with MCT can rapidly reverse cardiomyopathy and other severe clinical symptoms in VLCAD deficient patients. In the case of long-chain FOD, the current treatment suggests to provide up to 30% of total calories as MCT (182). The mostly predominant

MCT is trioctanoin, which is an even-carbon (C8) medium-chain triglyceride.

The recommended use of trioctanoin for the treatment of long-chain FOD is based on the notion that medium-chain fatty acids can directly enter mitochondria without the help of the carnitine shuttle system, and also bypass the membrane bound long-chain β-oxidative specific enzymes (VLCAD,

LCHAD, and MTP) (148; 148; 182). Medium-chain fatty acids can enter mitochondria as carboxylates and require only shorter chain-length specific enzymes (182). Another advantage of trioctanoin for the treatment of long-chain FOD is its C4-ketogenic capacity. The formed C4-ketone bodies are good energy source for extrahepatic tissues and can spare glucose utilization.

However, trioctanoin is not gluconeogenic because octanoate is an even-chain fatty acid. In addition, MCT cannot be used in patients with disorders of short-

46 and medium-chain fatty acid oxidation.

3.4.7 Other Strategies

Recently, Djouadi et al. (219; 220) reported that FOD may be corrected by drugs. In cells from VLCAD deficient patients, bezafibrate can normalize fatty acid oxidation capacity, increase VLCAD mRNA, protein as well as enzyme activity, decrease the toxic accumulation of long-chain acylcarnitines, and also stimulate gene expression of other enzymes involved in the β-oxidation pathway (220). Glycine administration at a rate of 300 mg/kg/day was reported to be good for FOD patients because glycine can conjugate with toxic long-chain acyl-CoAs which are not metabolized and these conjugants will be excreted into the urine (176).

3.5 Triheptanoin as the Treatment for Long-chain Fatty Acid Oxidation

Disorders

3.5.1 Introduction of Triheptanoin

Triheptanoin is an odd-carbon medium-chain triglyceride. Triheptanoin is esterified with one glycerol backbone and 3 molecules of heptanoate (C7). The formula of triheptanoin is shown in Fig 8. Since 2002, triheptanoin has been considered as a promising treatment of long-chain FOD (148).

47

Figure 8. Formula of triheptanoin

48

3.5.2 Metabolism of Triheptanoin

When it is infused into the blood or ingested, one mole of triheptanoin is easily hydrolyzed to one mole of glycerol and 3 moles of heptanoate (221). Similar to other medium-chain fatty acids, heptanoate can enter mitochondria as carboxylate independent of CPT system. The mitochondrial oxidation of heptanoate requires only medium- and short-chain specific β-oxidative enzymes. After one and half cycles of β-oxidation, heptanoate is converted to

BKP-CoA, which is an intermediate in the C5-ketogenesis pathway. BKP-CoA can either go through HMG-CoA cycle for C5-ketone body production or be cleaved to provide one acetyl-CoA and one propionyl-CoA for fueling the CAC cycle. The hepatic oxidation of heptanoate is illustrated in Fig 9.

49

Figure 9. Hepatic oxidation of heptanoate

Modified from (221)

50

3.5.3 Triheptanoin versus Trioctanoin

In 2002, Roe et al. (148) reported that three VLCAD deficient patients significantly improved clinical status including muscle weakness, cardiomyopathy, and rhabdomyolysis with triheptanoin diet. However, when these patients were treated with trioctanoin diet, there was no change in such clinical status (148). Why is triheptanoin more effective than trioctanoin for the treatment of long-chain FOD? One has to consider differences in the metabolism between odd-chain heptanoate and even-chain octanoate.

Even-chain fatty acids like octanoate can only be oxidized to acetyl-CoA and

C4-ketone bodies (Fig 10). However, odd-chain heptanoate is not only the precursor for acetyl-CoA but also the precursor for propionyl-CoA (Fig 10). In addition, odd-chain heptanoate can be converted to C5-ketone bodies (Fig 10).

Peripheral utilization of C5-ketone bodies also leads to production of acetyl-CoA and propionyl-CoA (Fig 10). As mentioned earlier, long-chain FOD patients experience hypoglycemia and have increased permeability of CAC intermediates from leaky mitochondrial and cell membranes. The provision of propionyl-CoA plays two important metabolic roles in the case of long-chain

FOD (148): (i) propionyl-CoA is a gluconeogenic substrate which elevates blood glucose level, and (ii) propionyl-CoA is an anaplerotic substrate which replenishes the pool of CAC intermediates, thus compensating massive cataplerosis. In contrast, the provision of acetyl-CoA alone from trioctanoin and high-carbohydrate diet is not always sufficient to improve the clinical status of 51 long-chain FOD patients (148).

52

Figure 10. Metabolism of heptanoate versus even-chain fatty acids

1: 3-ketoacyl-CoA thiolase, 2: HMG-CoA synthase, 3: HMG-CoA lyase, 4: BHB dehydrogenase

Modified from (132)

53

3.5.4 Previous Studies of Triheptanoin

After triheptanoin was reported to improve the clinical conditions and quality of life in VLCAD deficient patients (148), in 2005, Mochel et al. (222) reported that administration of triheptanoin at 35% of total calories can serve as the therapy for a patient with pyruvate carboxylase deficiency. They also reported that

C5-ketone bodies derived from triheptanoin metabolism can pass across the blood brain barrier, serving as anaplerotic substrates for CNS, and increasing energy fuel, glutamine and GABA levels in brain fluid of the patient (222).

In 2006, Kinman et al. (133) reported that intravenous infusion of triheptanoin at 40% of calorie requirements in normal rats resulted in a significant increase in blood glycerol turnover, indicating that activated lipolysis occurred. We know that activation of lipolysis would be deleterious for long-chain FOD patients, so this observation put triheptanoin as the acute treatment for long-chain FOD decompensation in a dilemma. Thus, the lipolytic process of the intravenous triheptanoin administration would be intensively investigated in my present study.

In 2008, Roe et al. (223) reported that diet treatment with triheptanoin at 30 –

35% of total caloric intake impressively improved exercise tolerance in seven

CPT II deficient patients.

In the same year, Oliveira et al. (224) reported that short-term rather than long-term treatment of triheptanoin in young rats significantly reduced brain cerebral excitability compared to rats on either control diet or long-chain 54 triglyceride diet. They reported that their study was the first time to demonstrate that triheptanoin can directly effect on central nervous system

(224).

4. ANAPLEROSIS

4.1 Introduction of Anaplerosis

The CAC cycle has two most important functions: (i) oxidize acetyl groups to generate CO2 and reducing equivalents which are used for ATP synthesis

(225), and (ii) regenerate the acceptor of the acetyl groups (149). There are eight reactions and eight intermediates involved in the CAC cycle. The total pool size of the eight CAC intermediates was reported to be small (1 - 2 μmol/g tissue) (149). Since the throughput of the cycle is 1 – 2 μmol acetyl/g per min

(149), the turnover of these intermediates is really rapid. Normally, there is no big change in the pool size of CAC intermediates (226). However, there is a physiological cataplerosis, which means the leakage of CAC intermediates through the mitochondrial and cell membranes (149). The rate of physiological cataplerosis usually accounts for 1 – 2% of the total pool per min (149). In order to operate the CAC cycle properly and regenerate ATP, it requires an influx of intermediates to compensate for the physiological cataplerosis. The re-filling of the pool of the CAC cycle is called anaplerosis. When massive

55 cataplerosis occurs, for example, in the case of long-chain FOD, physiological anaplerosis from endogenous substrates cannot compensate for the increased leakage of the CAC intermediates. Thus, carbon flux through the CAC cycle, energy production, and cellular homoeostasis would be impaired.

There are six major anaplerotic reactions in mammalian cells (149): (i) pyruvate enters the CAC cycle to generate oxaloacetate via pyruvate carboxylase, (ii) pyruvate is anaplerotic via malic enzyme to form malate, (iii) glutamate is converted to α-ketoglutarate catalyzed by glutamate dehydrogenase, (iv) aspartate forms oxaloacetate via aspartate transaminase,

(v) aspartate replenishes the CAC cycle as fumarate by purine nucleotide cycle and urea cycle (149; 227), and (vi) precursors of propionyl-CoA generates succinyl-CoA catalyzed by propionyl-CoA carboxylase, methylmalonyl-CoA racemase, and methylmalonyl-CoA mutase.

Castillo et al. (228) reported that a prolonged 72-h fasting inhibited anaplerosis indicated by decreased citrate concentration in human skeletal muscle whereas a 10-h starvation did not. Gibala et al. (229) reported that the concentration of CAC intermediates significantly increased after 5 min and 15 min exercise compared to that of at rest in human muscle. Even though the concentration of CAC intermediates was still higher at exhaustion than at rest, anaplerosis after prolonged exercise became lower compared with that during the initial minutes of exercise (229). The initial rapid expansion of the pool of

CAC intermediates was ascribed to an increased flux through the alanine

56 aminotransferase reaction, which is the most important anaplerotic process during the initial period of exercise (225; 230). Bruce et al. (231) reported that subjects consuming glutamine had greater total pool size of CAC intermediates in skeletal muscle after 10 min exercise compared to subjects who ingested placebo or ornithine α-ketoglutarate. Taegtmeyer et al. (232) reported that in isolated rat working hearts perfused with AcAc, which only leads to acetyl-CoA, the contractile function decreased rapidly. The damaged work output can be reversed by the addition of pyruvate (233) or propionyl-L-carnitine (234) to the perfusate, thus demonstrating the importance of anaplerosis in the maintenance of normal heart function.

Fransson et al. (235) reported that perifusion of rat pancreatic islet with phenylacetic acid, which is an inhibitor of pyruvate carboxylase, significantly decreased insulin secretion. Hasan et al. (236) reported that the transfection of small interfering RNA generated pyruvate carboxylase knockdown insulinoma cell lines, in which glucose-induced insulin release was impaired. These studies demonstrate the importance of anaplerosis on insulin secretion in

β-cells. Brain also needs anaplerosis because neurons keep losing CAC intermediates, especially α-ketoglutarate, resulting from the release of glutamate and GABA as neurotransmitter (237). Sibson et al. (238) reported that anaplerosis accounted for 19 – 26% of the total glutamine synthesis in rat brain under normoammonemic conditions, and this anaplerotic contribution was significantly increased to 32% during hyperammonemia conditions. It

57 demonstrates that anaplerosis in the brain plays an important role in ammonia detoxification (238).

4.2 Anaplerotic Substrates

4.2.1 Pyruvate

Pyruvate serves as one of anaplerotic substrates mainly via pyruvate carboxylase. Pyruvate carboxylase is positively regulated by acetyl-CoA, glucagon and glucocorticoids while negatively regulated by insulin (239). Large et al. (240) reported that increased anaplerotic pyruvate flux can be used for gluconeogenesis in rat livers. In adipose tissue, anaplerotic pyruvate is used for both de novo fatty acid synthesis and glyceroneogenesis (239). Reshef et al. (241) reported that in rat epididymal fat pad slices incubated with 1 – 2.5 mM pyruvate, glyceride-glycerol synthesis was largely increased. In pancreatic

β-cells, anaplerotic pyruvate is associated to glucose-induced insulin secretion

(239). Xu et al. (242) reported that the increased anaplerotic pyruvate flux resulted from overexpression of pyruvate carboxylase in INS-1 cells led to elevated insulin release and cell proliferation. In the heart, anaplerotic pyruvate is related to maintain cardiac function (149). Bunger et al. (243) reported that in isolated guinea-pigs heart, 2 – 20 mM pyruvate markedly increased ventricular work output under normoxia conditions. Even only physiological concentration of pyruvate already significantly improved ventricular performance during 58 reperfusion. In addition, cardiac reperfusion function could be almost fully restored in the presence of 5 – 10 mM pyruvate (243). Similarly, Tejero-Taldo et al. (244) reported that 5 mM pyruvate largely restored contractile performance in isolated stunned guinea-pig hearts. The mechanism is based on the hypothesis that anaplerotic pyruvate can potentiate β-adrenergic responsiveness in ischemic-reperfused hearts (244). Stanley et al. (245) reported that intravenous administration of dipyruvyl-acetyl-glycerol, a precursor of pyruvate, significantly reduced myocardial infarct size in pigs with ischemia-reperfusion injury. Dipyruvyl-acetyl-glycerol is reported to be a novel and efficient anaplerotic pyruvate precursor, which would not result in toxic sodium overload as with sodium pyruvate (245). In the nervous system, anaplerotic pyruvate contributes to de novo synthesis of glutamate (239).

Gamberino et al. (246) reported that increased anaplerotic pyruvate flux is required for enhanced glutamate production in cultured rat astrocytes.

4.2.2 Glutamate/Glutamine

Glutamate can be derived from either glutamine via glutaminase or

α-ketoglutarate via aminotransferase. Primarily, glutamate is anaplerotic via the reaction catalyzed by glutamate dehydrogenase to form back to

α-ketoglutarate, thus entering the CAC cycle. In the kidney, anaplerotic glutamate/glutamine is the donor of NH3, which can be used to titrate the

59 acidity of the urine (247). Moreover, in the kidney, anaplerotic glutamate/glutamine is used for gluconeogenesis. Stumvoll et al. (248) reported that renal gluconeogenesis derived from anaplerotic glutamate/glutamine is responsible for 20 – 25% of total body glucose production. After prolonged starvation or under metabolic acidosis, anaplerotic glutamate/glutamine in the kidney was reported to be essential, which contributes to up to 50% of whole body glucose production (249). In human small intestine, about 73% of anaplerotic [13C]glutamine derived from nasogastric infusion was reported to be oxidized to CO2 (250). In skeletal muscle, during initial minutes of exercise, glutamate concentration was reported to be largely decreased whereas alanine concentration was increased, indicating that glutamate is a very important anaplerotic substrate

(230; 249). In the heart, anaplerotic glutamate/glutamine is believed to improve post-ischemic cardiac functions (251). Khogali et al. (252) reported that in isolated perfused rat heart, addition of 1.25 mM glutamine to perfusate improved the impaired post-ischemic cardiac output and prevented the harmful changes in myocardial metabolites. Moreover, addition of 2.5 mM glutamine is responsible for the complete recovery to normal cardiac functions (252).

4.2.3 Precursors of Propionyl-CoA

Anaplerotic propionyl-CoA can be derived from: (i) the activation of propionate,

60

(ii) the catabolism of branched-chain amino acids, (iii) the degradation of odd-chain fatty acids, and (iv) the utilization of C5-ketone bodies.

Propionyl-CoA is converted to succinyl-CoA and enters the CAC cycle via sequential reactions catalyzed by propionyl-CoA carboxylase, methylmalonyl-CoA racemase and methylmalonyl-CoA mutase. See Fig 11.

(253).

61

Figure 11. Sources and anaplerotic pathway of propionyl-CoA

The enzymes involved are: 1, propionyl-CoA carboxylase, 2, methylmalonyl-CoA racemase, 3, methylmalonyl-CoA mutase

(253)

62

Based on 13C NMR technology, Sherry et al. (254) reported that the total anaplerosis flux was increased from 18% to 29% when rat hearts were perfused with 2.5 mM [3-13C]pyruvate plus 2 mM unlabeled propionate. In the heart, anaplerotic propionyl moiety seems to improve cardiac functions.

Russell et al. (234) reported that in isolated working rat hearts, addition of 2 mM propionyl-L-carnitine to 7.5 mM AcAc markedly increased cardiac power and also the utilization of AcAc. Anaplerosis from propionyl moiety is also related to a possible improvement in peripheral muscle metabolism. Anand et al. (255) reported that chronic treatment of propionyl-L-carnitine significantly increased exercise duration in patients with congestive heart failure. In the liver, anaplerosis from propionyl moiety can be used for gluconeogenesis. Deng et

13 al. (132) reported that rat liver perfused with [ C3]propionate and

13 [5,6,7- C3]heptanoate resulted in labeled glucose in the effluent perfusate, although the labeling was low (0.8 – 1%). In adipose tissue, anaplerotic propionyl moiety probably has an effect on glyceroneogenesis. Reshef et al.

(241) reported that addition of 0.25 mM propionate as a second substrate to

0.25 mM [2-14C]pyruvate markedly stimulated glyceride-glycerol formation in incubated epididymal fat pad slices from fasted rats.

4.3 Measurement of Anaplerosis

Anaplerosis can be measured based on 13C labeling pattern of CAC

63 intermediates by either mass isotopomer distribution (MID) using mass spectrometry or positional isotopomer distribution using NMR. In the following, several studies that calculate anaplerosis by MID analysis will be introduced.

Comte et al. (256) reported that the relative contribution of anaplerotic pyruvate can be calculated by: (i) MIDs of the oxaloacetate and acetyl moieties of citrate, and (ii) MIDs of pyruvate. In isolated rat hearts perfused with

13 [U- C3](lactate + pyruvate), M3 pyruvate would only be decarboxylated to M2 acetyl-CoA, while M3 pyruvate would enter the CAC cycle through

13 carboxylation to [1,2,3- C3]OAA (M3) (256). In addition, M3 pyruvate would

13 also enter the CAC cycle as [1,2,3- C3]malate via malic enzyme, which would

13 be further converted to [2,3,4- C3]OAA (M3) due to the reversibility of the fumarase reaction (256). Through the condensation catalyzed by citrate synthase, there are many kinds of possible citrate isotopomers formed from incoming labeled pyruvate (256). Rates of anaplerosis from pyruvate can thus be calculated based on the labeling patterns of OAA and the acetyl moiety of citrate. Briefly, the relative rate of pyruvate carboxylation is measured as the enrichment ratio (M3 OAA moiety of citrate)/(M3 pyruvate), whereas the relative rate of pyruvate oxidation is measured as the enrichment ratio (M2 acetyl-CoA moiety of citrate)/(M3 pyruvate) (257). Comte et al. (258) reported

13 that anaplerosis flux from 0.2 mM [U- C3]pyruvate accounted for 6.3% of the

13 supply of citrate synthesis in the presence of 1 mM [U- C3]lactate and 0.2 mM

13 [1- C3]octanoate.

64

Martini et al. (259) reported that anaplerosis from propionate can be quantitatively assessed from the MIDs of succinate in pig heart in vivo. During

13 infusion of [U- C3]propionate, the only pathway for M3 propionate entering the

CAC cycle is to form M3 succinate. Thus, the anaplerosis flux from propionate is calculated as the enrichment ratio (M3 succinate in tissue)/(M3 propionate in coronary vein) (259). Martini et al. demonstrated that 0.25 mM propionate accounts for about 8.9% of the CAC flux, indicating that propionate is an effective anaplerotic precursor even at a low concentration (149; 259).

Kasumov et al. (253) reported that propionate was a pure anaplerotic substrate in the heart since it did not label mitochondrial acetyl-CoA. In perfused rat

13 heart with [U- C3]propionate, the relative rate of anaplerosis from exogenous

M3 propionate was calculated as the enrichment ratio (M3 succinyl-CoA in heart)/(M3 propionate in perfusate) (253). Note that the real anaplerosis starts from propionyl-CoA, while it ends at succinyl-CoA. There should be some differences between the labeling of propionate and propionyl-CoA.

Endogenous unlabeled substrates would dilute the enrichment of M3 propionyl-CoA from M3 propionate. Actually, the enrichment ratio (M3 succinyl-CoA in heart)/(M3 propionyl-CoA in heart) represents the contribution from all sources to the pool of CAC intermediates (253). This demonstrates

13 that in rat hearts perfused with 0 – 2 mM [U- C3]propionate, anaplerosis from exogenous M3 propionate increased to a maximum of 17% of the turnover of

CAC intermediates (253). Furthermore, this relative contribution from

65 exogenous M3 propionate to the pool of CAC intermediates almost equals to total anaplerosis from all the sources (253). In contrast, relative anaplerosis from endogenous substrates decreased to nearly zero, resulting from the

13 inhibition by increased [U- C3]propionate concentration in the perfusate (253).

5. GLYCERONEOGENESIS

5.1 Introduction of Glyceroneogenesis

Glyceroneogenesis was first pointed out in the late 1960s (260; 261).

Glyceroneogenesis is a truncated version of gluconeogenesis pathway (262).

The glyceroneogenic pathway converts intermediates other than glucose or glycerol, such as pyruvate, lactate, and some gluconeogenic amino acids to glycerol-3-phosphate, which is esterified with three molecules of fatty acyl-CoA to synthesize triglyceride. The most important enzyme involved in glyceroneogenesis is the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C). Glyceroneogenesis occurs in the white adipose tissue (241; 260; 263), brown adipose tissue (264-266), and liver (267; 268).

Glyceroneogenesis occurs during fasting, or after having a diet without carbohydrates (269). However, recently Nye et al. (270) has reported rats fed with sucrose-supplementation and received glucose infusion during the study have significant increases in glyceroneogenesis in both adipose tissues and

66 gastrocnemius. Hanson et al. (262) explained glyceroneogenesis occurs probably because extra fuel released from lipolysis exceeds the energy demands of peripheral tissues, also triglyceride/fatty acid cycling has an advantage of heat generation, as well as the potential of re-deposit triglyceride as an energy reserve.

The glyceroneogenesis pathway during fasting is illustrated in Fig 12. (271).

67

Figure 12. The glyceroneogenesis pathway

In the fed state, glycerol-3-phosphate used for fatty acid re-esterification is primarily derived from glycolysis via glucose. During fasting in the adipose tissue, glucose availability is limited and glycerol kinase is absent.

Glycerol-3-phosphate used for re-esterification of fatty acids released from lipolysis can thus be derived from glyceroneogenesis.

68

5.2 Regulation of Glyceroneogenesis

Since PEPCK catalyzes the rate-limiting step in glyceroneogenesis, the regulation of the pathway should be reviewed at the level for this key enzyme

PEPCK. PEPCK is believed to be regulated at transcriptional level. The expression of the gene for PEPCK is specifically inhibited by insulin within the physiological range of concentration (10-12 - 10-9 M) (272). Hormones that upregulate PEPCK include glucagon (273-275) and epinephrine (275), both acting via cAMP. Glucocorticoids positively regulate PEPCK gene expression in the liver and kidney (262; 276; 277), and negatively regulate it in the adipose tissue (262; 275; 278). Other factors such as thyroid hormone (279) and retinoic acid (280) are also transcriptional inducers of the PEPCK gene.

Recent interest has demonstrated that the transcription and activity of PEPCK is sensitive to pharmacological manipulation by thiazolidinediones (TZDs), the latest antidiabetic drugs (281-283). Among the TZDs class, rosiglitazone, the

PPARγ agonist, stimulates glyceroneogenesis probably via increased PEPCK gene expression (284; 285).

5.3 Measurement of Glyceroneogenesis

Nye et al. (270) investigated glyceroneogenesis in rats in vivo in response to a control diet, 48 h fast, and high sucrose diet. The relative contribution of glyceroneogenesis and glycolysis was quantified using the rate of 69

3 3 14 14 incorporation of [ H] from [ H2]O and [ C] from [U- C]glucose into glycerol-3-phosphate in adipose tissue, skeletal muscle and liver. In this study, it was assumed that only two of five hydrogens in glycerol-3-phosphate are derived from pyruvate (glyceroneogenesis), and only two of three hydrogens on C3 of pyruvate are incorporated into glycerol-3-phosphate. According to this assumption, the rate of glyceroneogenesis can thus be calculated using the measured specific activity of pyruvate. It was shown that the contribution of glyceroneogenesis is predominant as compared with the contribution of glucose for triglyceride glycerol synthesis in white adipose tissue, skeletal muscle, and liver in control and extended fasting animals. Surprisingly, the highest rate of glyceroneogenesis was obtained in the adipose tissue of high sucrose supplemented animals and the glyceroneogenesis remained higher for the contribution to triglyceride synthesis than glucose via glycolysis.

However, adipose tissue PEPCK activity in high sucrose supplemented animals was lowest. The conflicts between high glyceroneogenesis flux and low PEPCK activity might be due to overestimation of the rate of glyceroneogenesis from calculation, which will be discussed later.

Botion et al. (286) determined glyceroneogenesis in rats fed a high-protein, carbohydrate-free diet by subtracting the rate of glycerol-3-phosphate synthesis via glucose from the rate of glycerol-3-phosphate synthesis via all carbon sources in the carcass, liver, retroperitoneal and epididymal adipose tissue. Triglyceride glycerol synthesis from all carbon sources was evaluated

70

3 3 from the assumption that 3.3 atoms of [ H] from [ H2]O were incorporated into the glycerol moiety of triglyceride. While triglyceride glycerol synthesis from glucose carbon was evaluated from the rate of incorporation of [14C] from

[U-14C]glucose into the glycerol moiety of triglyceride. It was shown glyceroneogenesis was significantly increased in both adipose tissues from rats adapted to a high-protein, carbohydrate-free diet compared to the control.

Chen et al. (285) investigated glyceroneogenesis in low-carbohydrate, high-fat diet fed mice, rosiglitazone treated mice, and fructose infused rats. The relative contribution of glyceroneogenesis to glycerol-3-phosphate was estimated by

2 application of H2O labeling combined with mass isotopomer distribution analysis. They assume the glycerol-3-phosphate generated via glyceroneogenesis has all five hydrogens exchangeable with body water, thus resulting in an n of 5. The glycerol-3-phosphate generated via glycolysis has an n of 3.5. In the liver, the glycerol-3-phosphate generated via glycerol kinase results in an n of 3. Thus, based on the measured n of triglyceride glycerol moiety, one can obtain the relative contribution of glyceroneogenesis to triglyceride synthesis. It was shown that glyceroneogenesis was significantly higher in mice fed a low-carbohydrate, high fat diet compared to mice fed a high-carbohydrate diet. The administration of rosiglitazone to mice also significantly increased glyceroneogenesis. However, fructose infused rats decreased glyceroneogenesis from 66 to 34% because of increased glycolytic input. These results are opposite with what Nye et al. (270) observed in high

71 sucrose fed glucose infused rats.

Recently, Bederman et al. (287) determined the contribution of glucose versus non-glucose carbon sources to triglyceride glycerol synthesis in C57BL/6J

2 2 mice in vivo using [6,6- H2]glucose and H2O. Mice were fed either a high-carbohydrate, low-fat (HC) diet or a high-fat, carbohydrate-free (CF) diet.

Surprisingly, they found mice fed the CF diet have a much higher triglyceride turnover as compared with mice fed the HC diet even though the caloric intakes were similar between groups. In HC-fed mice, glucose is the predominant carbon source for making triglyceride glycerol. In CF-fed mice, the absolute rate of glyceroneogenesis significant increased compared to that of HC-fed mice. The observation that glucose makes a substantial contribution to triglyceride glycerol when glucose availability is high whereas glyceroneogenesis makes a substantial contribution when glucose availability becomes limiting is consistent with the reports by Chen et al. (285) and Botion et al. (286) but opposite with the finding by Nye et al. (270).

5.4 Problems in Measuring Glyceroneogenesis in vivo

In the study of Nye et al. (270), the glyceroneogenesis flux is quantified by comparing the 3H labeling of triglyceride glycerol moiety versus pyruvate.

However, glucose can also be labeled by tritium. Tritium labeled glucose can subsequently label glycerol-3-phosphate. The ignorance of this labeling

72 recycling overestimates the relative contribution of glyceroneogenesis to triglyceride synthesis. Moreover, when calculating the indirect rate of the glycolytic contribution to triglyceride glycerol via lactate, 14C specific activity of lactate was multiplied by the estimated glyceroneogenesis flux from pyruvate corrected with a dilution factor of 2.2 (270). The dilution factor used is problematic because the loss of 14C in the CAC cycle is uncertain (287).

In the study of Botion et al. (286), the rate of lipid synthesis from all carbon sources was calculated assuming that each glycerol incorporated into triglyceride contained 3.3 atoms of 3H. Based on the knowledge from Chen et al.’s study (285), we know that glycerol-3-phosphate derived from glyceroneogenesis can result in all five hydrogens to be labeled, whereas glycerol-3-phosphate generated from glycolysis or glycerol kinase will have three and a half or three 3H. Thus, the average factor of 5, 3.5, and 3 will be absolutely greater than 3.3 which was used in Botion et al.’s study (286). The underestimation of the exchange factor leads to an overestimation of total triglyceride glycerol synthesis.

In the study of Chen et al. (285), they assumed in adipose tissue glucose is unlabeled and glycerol-3-phosphate produced from glycolysis has an n of 3.5.

However, glucose will be rapid and greatly labeled by deuterium as shown by

Bederman et al. (287). It is recognized that at most seven hydrogens in C-H bonds of glucose could be labeled by 2H. Therefore, n would increase up to 5 instead of 3.5 via glycolysis. As a result, the relative contribution of

73 glyceroneogenesis to adipose tissue triglyceride glycerol synthesis would be overestimated if the equation of n = 5x + 3.5(1-x) is still used.

In the study of Bederman et al. (287), they noted that the labeling of triglyceride glycerol should be multiplied by the factor of 2 because the precursor glucose can convert to 2 triose phosphates. This point is ignored by

Botion et al. (286). Furthermore, in order to avoid problems which were not prevented in both studies of Nye et al. (270) and Chen et al. (285), Bederman et al. (287) measured the incorporation of glucose into triglyceride glycerol by

2 14 2 from [6,6- H2]glucose, instead of [U- C]glucose and H2O in the previous studies. Thus, the problem of tracer recycling can be solved.

6. STATEMENT OF PURPOSES

The initial clinical investigations reported that patients with long-chain FOD greatly improved their clinical status when they were switched from chronic dietary treatment with high carbohydrates and trioctanoin to the treatment with triheptanoin (219). However, there is no effective acute treatment for long-chain FOD decompensation currently. It is very necessary to develop a treatment that suppresses lipolysis, prevents hypoglycemia, and replenishes the leakage of the CAC intermediates during acute long-chain FOD episodes.

Otherwise, the lives of long-chain FOD patients would be threatened.

Because triheptanoin has been proposed to be a promising treatment for 74 long-chain FOD, the goal of this research is to characterize the metabolism and metabolic effects of triheptanoin in vivo and to test the potential of triheptanoin as the acute treatment of long-chain FOD decompensation. More specifically, we have investigated lipolysis, gluconeogenesis, and anaplerosis during triheptanoin administrations via parenteral and enteral routes. We have also determined the fatty acid re-esterification during triheptanoin infusions.

The studies provide us evidences that the triheptanoin administration is able to be considered as the acute treatment of long-chain FOD patients.

7. HYPOTHESES

The hypothesis that triheptanoin is a potential acute treatment of long-chain

FOD is based on the metabolism of heptanoate. As mentioned earlier, the oxidation of heptanoate leads to acetyl-CoA and propionyl-CoA. The propionyl-CoA is not only an anaplerotic substrate for the CAC cycle, but also a gluconeogenic substrate in the liver and kidney cortex.

However, in a previous study (150), we observed an apparent increase of

13 lipolysis, measured from the dilution of [ C3]glycerol tracer during intravenous infusion of triheptanoin. We did not know whether this elevated glycerol turnover was accompanied with the increased long-chain fatty acid release from adipocytes. In the case of acute long-chain FOD decompensation, the increased long-chain fatty acid flux always leads to life-threatening symptoms. 75

Thus, activation of lipolysis is extremely harmful for long-chain FOD patients.

We further hypothesized that the triheptanoin-mediated lipolysis could be blunted if a large amount of glucose and insulin was given. Moreover, we hypothesized that long-chain fatty acids released from adipocyte lipolysis during intravenous infusion of triheptanoin could be re-esterified within the same cells. The increased fatty acid re-esterification in adipose tissue is due to a large supply of glycerol-3-phosphate during intravenous triheptanoin infusion, which can be either derived from hyperglycemia via glycolysis, or from propionyl-CoA, pyruvate/lactate via glyceroneogenesis.

We lastly hypothesized that the enteral administration of triheptanoin is another option for the acute treatment of long-chain FOD decompensation because it does not activate lipoprotein lipase in the vascular bed, thus does not activate lipolysis.

8. EXPERIMENTAL PROCEDURES

8.1 Animals

Male Sprague-Dawley rats were purchased from Harland Industries

(Indianapolis, IN). The animals were housed in the Animal Resource Center

(ARC) of Case Western Reserve University under controlled temperature (22 ±

1 ºC) and light (on at 6 am, off at 6 pm). All procedures of the animal

76 experiments in this study were reviewed and approved by the Institutional

Animal Care and Utilization Committee (IUCAC) of the School of Medicine of

Case Western Reserve University and conformed to American Association for

Accreditation of Laboratory Animal Care Guidelines.

Rats weighing 250 - 310 g were fed on Harlan Teklad rat chow (Madison, WI) ad libitum and given free access to water at all times. The animals were also provided with the environmental enrichment (chewing toys and nestlets). After entry into the ARC, the rats were allowed one week to acclimate prior to the study. Food was removed at 4 pm the day before the study and water was provided ad libitum. On the following experimental day, surgery was started at

9 am. There were 2 different types of catheter placement: (i) intravenous infusion: catheters were inserted into jugular vein and carotid artery; and (ii) intraduodenal infusion: catheters were inserted into duodenum, jugular vein and carotid artery. Followed by the successful surgery, isotonic saline solution

(58.3 µl/min) was infused via the jugular vein catheter for 20 min. An equilibration period was allowed for animals to reach the normal body temperature (37.5 - 38.2 ºC) and the normal breathing rate (75 - 115/min).

8.2 Protocol

The protocol is illustrated in Fig 13.

77

Figure 13. Experimental protocol

78

A basal blood sample (150 µL) was obtained via the carotid artery catheter before the start of each protocol. There were 6 groups of animals (6 - 7 rats/group) used. The duration of the study was 125 min. The animals were anesthetized with 2 - 2.5% isoflurane in pure oxygen during the experiment.

Group 1 was the control group and the rats were infused isotonic saline solution throughout the experiment. Groups 2 - 5 received intravenously heptanoate-Na solution (150mM) or 10% triheptanoin emulsion in amounts that provided 40% of the caloric requirement from the heptanoate moiety.

2 Group 6 received triheptanoin intraduodenally. Tracers of [6,6- H2]glucose,

13 13 [ C3]glycerol, and [ C18]oleate were administrated at constant rates via the jugular vein catheter for 125 min in groups 1-6. In group 6, an additional tracer

2 of [1,1,2,3,3- H5]glycerol was infused into the rats via the duodenum catheter.

The substrates and isotopic tracers infused, as well as the routes of administration are indicated in Table 1.

Blood samples (150 µL) were drawn from the carotid artery catheter at 20, 40,

60, 75, 90, 105, 120, 125 min and plasma was separated. At 125 min, after the last blood sampling, the tissues of interest (liver and epididymal adipose tissue) were harvested while the infusion of substrates and tracers was continued.

The tissues were clamped immediately and quick-frozen with liquid nitrogen.

Plasma was stored at -20 ºC and tissues were stored at -80 ºC until further analyses.

79

µmol·kg Substrates and tracers were infused intravenously (IV) intraduodenallyor (ID) atthe indicated rates expressed in Table 1. Rates of administration of substrates and isotopic tracers Group 6 (**), [1,1,2,3,3,- -1 ·min -1 . In Group . In Group 5 (*), and glucose insulin were infused IV at 145 µmol·kg 2 H 5 ]glycerol was infused µmol·kg ID (3.7 infused was ]glycerol -1 ·min -1 ) in addition to [ in additionto ) -1 ·min 13 -1 and 4 mU·kg 4 and C 3 ]glycerol IV.]glycerol -1

·min -1 , respectively. In

80

8.3 Tracer Studies

2 13 13 We selected [6,6- H2]glucose, [U- C3]glycerol, and [U- C18]oleate for the measurements of glucose Ra, glycerol Ra and oleate Ra. Tracers were

2 administrated as a constant infusion in the V - A mode. [6,6- H2]glucose was infused at 1.2 µmol·kg-1·min-1 via the jugular vein catheter for 125 min in all

13 -1 -1 groups. [U- C3]glycerol was infused at 1.5 µmol·kg ·min in Group 1 and 3.7

µmol·kg-1·min-1 in Groups 2 - 6 via the jugular vein catheter for 125 min.

2 -1 -1 [1,1,2,3,3- H5]glycerol was infused at 3.7 µmol·kg ·min via the duodenal

13 catheter for 125 min in Group 6. [U- C18]oleate was infused at 0.3

µmol·kg-1·min-1 in Group 1 and 0.6 µmol·kg-1·min-1 in Groups 2 - 6 via the jugular vein catheter for 125 min.

8.4 Rationale for Selection of Infusion Groups

In a previous study, we observed an apparent increase in lipolysis, as shown by elevated endogenous glycerol Ra, during intravenous infusion of triheptanoin (133). The lipolysis probably results from the infusion of triglyceride emulsion, which activates plasma lipoprotein lipase. We would like to compare the lipolysis induced by the administration of heptanoate moiety as either carboxylate or triglyceride. Thus, we first selected the groups of heptanoate-Na I.V. infusion and heptanoate-Na + glycerol I.V. infusion. In

81 these two groups, heptanoate-Na was infused at the same rate as the heptanoate equivalent in triheptanoin, and free glycerol was infused at the same rate as the total glycerol equivalent in triheptanoin. Furthermore, activation of lipolysis induced by triheptanoin I.V. infusion is not desirable for the acute treatment of decompensated long-chain FOD patients. We want to investigate whether the lipolysis can be blunted by intravenous infusion of glucose and insulin, which is currently the commonest acute treatment of FOD.

Accordingly, we selected the group of hyperglycemia hyperinsulinemia clamp.

In addition, we want to test the new avenue for the acute treatment of long-chain FOD via intraduodenal infusion. It is assumed that triheptanoin I.D. administration can mimic the conditions in unconscious patients who receive triheptanoin through the nasogastric tube.

8.5 Assay of Glucose and Glycerol in the Plasma

Arterial blood samples (150 µL) were immediately centrifuged to obtain plasma.

For the assays of glucose and glycerol, 20 µL plasma were pipetted into glass tubes containing a 275 µL of an aqueous solution of internal standards:

13 2 [U- C6]glucose (50 nmol), [1,1,2,3,3- H5]glycerol (5 nmol, in Groups 1 to 5) or

[2-13C]glycerol (0.16 nmol, in Group 6). After quick mixing, the solutions were treated with 100 µL of 1 M sodium borodeuteride in 0.1 M NaOH. The treatment with sodium borodeuteride converts glucose to M1 sorbitol.

82

The borodeuteride-treated plasma samples were acidified with 100 µL of 12 N

HCl (to destroy excess borodeuteride), and evaporated under nitrogen. The residue was reacted with 150 µL of acetic anhydride and 300 µL of pyridine, heated for 1 hr at 100 ºC, and then left overnight at room temperature. The next day, samples were extracted 3 times with diethyl ether, and the combined extract was dried over Na2SO4 before evaporation. The residue was dissolved in 50 µl of ethyl acetate.

Either 1 µL (glycerol assay) or 2 µL (sorbitol derived from glucose) was injected into an Agilent 6890 gas chromatograph linked to a 5973 MSD mass spectrometer. The chromatograph was equipped a 30 m OV-225 capillary column (Quadrex). The carrier gas was helium, and the injection mode was either split (glycerol) or splitless (sorbitol derived from glucose). It was necessary to use the splitless mode for the sorbitol derivative because it does not extract well with diethyl ether. The GC injector temperature was set at either 190 ºC (glycerol) or 235 ºC (glucose), and the transfer line was held at

240 ºC. For glycerol, the column temperature was increased from 80 ºC by 4

ºC/min to 190 ºC, and then by 50 ºC/min from 190 ºC to 220 ºC, where it was held for 10 min. For glucose, the column temperature was increased from 100

ºC by 20 ºC/min to 190 ºC, by 5 ºC/min from 190 ºC to 220 ºC, and then by 20

ºC/min from 220 ºC to 235 ºC, where it was held for 45 min. The mass spectrometer was operated under ammonia positive chemical ionization with the source pressure adjusted to obtain the maximal signal. The retention times

83 and ions monitored were as follows: glycerol (6.9 min, m/z 236, 237, 238, 239,

241); glucose converted to sorbitol (25.6 min, m/z 453, 454, 455, 459).

8.6 Assay of Heptanoate, C4-ketone Bodies, C5-ketone Bodies, and

Oleate in the Plasma

Plasma (40 µL) were pipetted into glass tubes containing a 660 µL solution of

2 2 internal standards: [ H13]heptanoate (20 nmol), R,S-ß-hydroxy-[ H6]butyrate

2 (40 nmol), R,S-ß-hydroxy-[ H5]pentanoate (20 nmol), and heptadecanoic acid

(16 nmol). After quick mixing, the solutions were treated with 100 µL of 1 M sodium borodeuteride in 0.1 M NaOH. The treatment with sodium borodeuteride converts unstable AcAc and BKP to the stable M1 BHB and M1

BHP, respectively, which can be distinguished by GC-MS from the unlabeled

BHB and BHP (134).

Plasma samples were deproteinized with 3 ml acetonitrile:methanol (v:v 7:3).

After centrifugation, the supernatant was transferred to new tubes and evaporated. The residue was reacted with 100 µL TMS and heated for 1 h at

90 ºC and 2 µL were injected into the same gas chromatograph- mass spectrometer as above. The chromatograph was equipped with a 60m Varian

CP 9017 VF-5 capillary column. The carrier gas was helium (10.3 ml/min) and the injection mode was splitless. The injector temperature was set at 290 ºC, and the transfer line was held at 290 ºC. The column temperature was

84 increased from 100 ºC by 2 ºC/min to 135 ºC, by 10 ºC/min from 135 ºC to 200

ºC, by 4 ºC/min from 200 ºC to 300 ºC, where it was held for 5 min, and then by

50 ºC/min from 300 ºC to 310 ºC, where it was held for 10 min. The mass spectrometer was operated under ammonia positive chemical ionization with the source pressure adjusted to obtain the maximal signal. The retention times and ions monitored were as follows: heptanoate (13.8 min, m/z 220, 233); BHB

(13.2 min, m/z 249, 250, 255); BHP (16.4 min, m/z 263, 264, 268); oleate (38.3 min, m/z 372, 390).

8.7 Assay of Short-chain and Medium-chain Acyl-CoAs in the Liver

Frozen, powdered liver samples (~ 200 mg) were homogenized with a Polytron homogenizer in a 50 mL screw-cap tube containing 4 mL methanol:H2O (v:v

2 1:1) with 2% acetic acid and 1 nmol [ H5]propionyl-CoA as internal standard.

The centrifuged extract was loaded onto a Supelco solid phase extraction cartridge (2-(pyridyl)-ethyl functionalized silica gel) pre-conditioned with 3 mL methanol, then 3 mL buffer A (1:1 methanol:H2O with 2% acetic acid). The cartridge was then washed with 3mL buffer A to elute impurities, followed sequentially by 3 mL buffer B (1:1 methanol:H2O with 50 mM ammonium formate), 3 mL buffer C (3:1 methanol:H2O with 50 mM ammonium formate), and 3 mL methanol to elute the acyl-CoAs. The eluent was evaporated under nitrogen.

85

After dissolving the residue in 100 µL of mobile phase A (100 mM ammonium formate in 5% acetonitrile, pH 5.0), 15 µL of sample was injected on a Hypersil

Gold C18 column (150 x 2.1 mm, 5 μm particle size, Thermo Electron) protected by a guard column (Hypersil Gold 10 x 2.1 mm, 5 μm), in a Dionex

Ultimate 3000 liquid chromatograph. The flow rate was constant at 0.2 mL/min.

For elution: (i) for the first 7 min, mobile phase A was 98%; (ii) from 7 to 25 min,

2 to 60% mobile phase B (5 mM ammonium formate in 95% acetonitrile); (iii) from 25 to 26 min, to 90% B; (iv) from 26 to 31 min, 90% B; and (v) for re-equilibration, the mobile phase was brought back to 98% A within one minute and held for 10 min.

The order of acyl-CoA elution (min) was malonyl-CoA (4.8), methylmalonyl-CoA (9.7), succinyl-CoA (12.4), HMG-CoA (13.1), acetyl-CoA

(13.9), BHB-CoA, AcAc-CoA (14.2), HEG-CoA (14.7), BHP-CoA, BKP-CoA

(15.4), propionyl-CoA (15.4), pentanoyl-CoA (18.5), and heptanoyl-CoA (21.7).

In the absence of standards, HEG-CoA, BHP-CoA, and BKP-CoA were identified from (i) mother/daughter ion pairs; (ii) comparison with the spectra of analogs (HMG-CoA, BHB-CoA, AcAc-CoA); and (iii) labeling in rat livers

13 perfused with [5,6,7- C3]heptanoate (132).

The liquid chromatograph was coupled to an API 4000 QTrap mass spectrometer (Applied Biosystems, Foster City, CA) operated under positive ionization mode with the following source settings: turbo-ion-spray source at

500C under N2 nebulization at 60 psi, N2 heater gas at 60 psi, curtain gas at

86

30 psi, collision-activated dissociation gas pressure was held at high, turbo ion-spray voltage at 4,500 V, declustering potential at 90 V, entrance potential at 10 V, collision cell exit potential at 50 V. The Analyst software (version 1.4.2;

Applied Biology) was used for data analysis.

8.8 Calculations

The rates of appearance (Ra) of glucose, glycerol and oleate were calculated according to the steady-state equation:

Total Ra = [(IEinfusate/IEplasma) - 1] x (INF) where IEinfusate is the isotopic enrichment of the infused tracer, IEplasma is the isotopic enrichment of the tracee in plasma, and INF is the rate of the tracer infusion (µmolkg-1min-1).

Whenever exogenous unlabeled glucose (Group 5) or glycerol (Groups 3-6) was infused, the corresponding total Ra were corrected for the amounts of exogenous substrates infused:

Endogenous Ra = [(IEinfusate/IEplasma) - 1] x (INF) - Ginf where Ginf is the total exogenous glycerol or glucose infusion rate

(µmolkg-1min-1). Note that the glycerol content of the triheptanoin emulsion

(467 µmol/ml) has two almost equal components: the glycerol moiety of triheptanoin, and the free glycerol which keeps the emulsion stable.

In Group 6, rats were infused intraduodenally with triheptanoin and

87

2 13 [1,1,2,3,3- H5]glycerol (M5 glycerol). The other tracers, including [ C3]glycerol

(M3 glycerol), were infused intravenously. The two glycerol tracers were infused at the same rate (3.7 µmolkg-1min-1). The total glycerol Ra

13 (endogenous + exogenous) was calculated from the arterial [ C3]glycerol (M3 glycerol) enrichment. We calculated the endogenous glycerol Ra according to the equation:

Endogenous glycerol Ra = total glycerol Ra - Ginf x [(M5/M3)arterial] where (M5/M3)arterial is the labeling ratio of glycerol in arterial plasma. This ratio represents the fraction of the intraduodenally infused glycerol (as triheptanoin

+ free glycerol) that escaped uptake by the liver. The identical rates of infusion of the M3 and M5 glycerol tracers are not included in equation because they cancel out.

Assuming that oleate accounts for 1/4 of plasma long-chain fatty acids, the percentage of the long-chain fatty acids released by lipolysis that was re-esterified in the same cells was calculated as:

100 x [(3 x glycerol Ra) - (4 x oleate Ra)] / (3 x glycerol Ra)

8.9 Statistics

Data are reported as mean ± SE. One-way ANOVA followed by Tukey post-hoc comparisons was used to identify significant differences across six experimental groups (Graphpad Prism Software, version 3.03, La Jolla, CA).

88

The significant level was set at p < 0.05. Statistical analysis was performed on either the plateau period of the concentration or enrichment of metabolites, or the last time-point.

9. RESULTS

9.1 Plasma Heptanoate Concentrations

The profile of plasma heptanoate concentrations in the six groups of rats was shown in Fig 14. Baseline heptanoate was undetectable in all groups and in the saline-infused control group. The infusion of Na-heptanoate at 40% of the caloric requirement resulted in stable heptanoate concentration of 0.34 ± 0.016 mM between 90 and 125 min (p < 0.001 compared to control). Addition of glycerol to the Na-heptanoate infusion did not appreciably increase plasma heptanoate concentration. However, the infusion of triheptanoin markedly increased heptanoate concentration to 1.58 ± 0.11 mM compared to controls

(p < 0.001). This, in spite of the fact that the infusions of triheptanoin and of

(Na-heptanoate + glycerol) supplied the same amounts of heptanoate and of glycerol equivalents. Addition of glucose + insulin to the triheptanoin infusion further increased heptanoate concentration to 1.78 ± 0.11 mM compared to controls (p < 0.001). In contrast, as shown previously (133), the intraduodenal infusion of triheptanoin led to very low arterial plasma concentrations of

89 heptanoate, about 0.069 ± 0.021 mM (not significantly different from control), which indicated that all the heptanoate derived from enteral triheptanoin hydrolysis was taken up by the liver, and little escaped the utilization of the liver.

90

Figure 14. Profile of heptanoate concentrations in rat plasma

Data are presented as Mean ± SE (n = 6 or 7)

91

9.2 Plasma C5-ketone Bodies Concentrations

The basal concentration of C5-ketone bodies (Fig 15) was undetectable in all groups and did not change during saline infusion. Similar concentrations of

C5-ketone bodies were achieved at the end of infusions either of

Na-heptanoate (0.66 ± 0.076 mM), Na-heptanoate + glycerol (0.63 ± 0.036 mM), or triheptanoin (0.59 ± 0.070 mM). The accumulation of C5-ketone bodies was markedly blunted to 0.22 ± 0.013 mM (p < 0.05) when glucose + insulin were added to the triheptanoin infusion. It is possible that insulin inhibits

C5-ketogenesis by inhibiting HMG-CoA synthase. And it is also possible that the high glucose load decreases the production of C5-ketone bodies. As previously shown (133), intraduodenal infusion of triheptanoin led to the highest concentrations of C5-ketone bodies to 0.76 ± 0.041 mM (Fig 15) and the lowest concentrations of heptanoate, except for the controls (Fig 14).

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Figure 15. Profile of total C5-ketone body concentrations in rat plasma

Data are reported as Mean ± SE (n = 6 or 7)

93

9.3 Plasma C4-ketone Bodies Concentrations

The basal concentration of C4-ketone bodies (Fig 16) was high in all groups

(1.37 ± 0.10 to 2.08 ± 0.17 mM). This is the result of anesthesia that decreases the uptake of C4-ketone bodies by the brain (288). This is also due to basal lipolysis increases after overnight fasting and surgery. In the saline infused rats, the concentration of C4-ketone bodies increased steadily to 2.48 ± 0.28 mM, presumably as a result of continuously increased fasting and experimental duration. In the rats infused intravenously with Na-heptanoate, the concentration of C4-ketone bodies did not change. However, intravenous infusion of Na-heptanoate + glycerol or triheptanoin slowly decreased the concentration of C4-ketone bodies. The same profile was observed during intraduodenal infusion of triheptanoin. Since heptanoate is C4-ketogenic, it was assumed that plasma concentrations of C4-ketone bodies would somewhat increase after heptanoate and triheptanoin infusions. However, C4-ketone bodies concentrations did not change or slightly decreased in groups 2, 3, 4 or

6. It is likely that the unchanged or even decreased long-chain fatty acid flux from adipocyte lipolysis results in the inhibition of C4-ketogenesis. The addition of glucose + insulin to the intravenous infusion of triheptanoin led to a 95% decrease in C4-ketone bodies concentration (p < 0.001 compared to controls) over 2 hr.

94

Figure 16. Profile of total C4-ketone body concentrations in rat plasma

Data are reported as Mean ± SE (n = 6 or 7)

95

9.4 Mid-potential of the BHP/BKP System

In all groups, the [BHB] / [AcAc] and [BHP] / [BKP] ratios, measured in plasma, were stable from 60 to 125 min in all groups. The [BHB] / [AcAc] ratio was 1.49

± 0.19 (SD, n = 216, all groups). The [BHP] / [BKP] ratio was 1.09 ± 0.18 (SD, n = 180, groups 2 to 6). Since both ratios are in equilibrium with the [NADH] /

[NAD+] ratio in liver mitochondria via BHB dehydrogenase, we calculated the mid-potential (in mV) of the BHP/BKP system using the equation:

0 E’ BHP/BKP = -297 - 13.5log ([BHB]/[AcAc] / [BHP]/[BKP]) where -297 mV is the mid-potential of the BHB / AcAc couple (289). Using all the ratios measured between 60 and 125 min, the calculated mid-potential of the BHP / BKP couple is - 298.8 ± 0.6 mV (SD, n = 180). It is the first time the mid-potential of the BHP / BKP system is reported.

9.5 Plasma Glycerol Concentrations

The plasma glycerol concentrations were 0.21 ± 0.011 mM in the controls (Fig

17), and were not affected by the intravenous infusion of Na-heptanoate (0.26

± 0.019 mM) or by the intraduodenal infusion of triheptanoin (0.29 ± 0.011 mM).

Addition of glycerol to the intravenous Na-heptanoate infusion significantly increased glycerol concentration from 0.21 ± 0.011 mM to 0.57 ± 0.017 mM (p

< 0.001). Obviously, the increase in glycerol concentration in group 3 is the result of exogenous unlabeled glycerol infusion. Intravenous infusions of 96 triheptanoin lead to very high glycerol concentration (1.71 ± 0.11 mM, p <

0.001 compared to controls); this again in spite of the fact that the infusions of triheptanoin and of (Na-heptanoate + glycerol) supplied the same amounts of heptanoate and glycerol equivalents. Addition of glucose + insulin to the triheptanoin infusion blunted by about 1/3 the glycerol concentration, compared to the infusion of triheptanoin alone (p < 0.001), but the glycerol concentration was still significantly higher compared to the control (p < 0.001).

The very high glycerol concentration is contradicted with the decreased concentration of C4-ketone bodies in the intravenous infusion of triheptanoin

(Fig 16). The difference in glycerol concentrations between the infusion of triheptanoin and heptanoate + glycerol, is supposed to be resulted from the activation of triglyceride hydrolysis in the adipose tissue and plasma during triheptanoin infusion. However, the arterial concentration of C4-ketone bodies decreased in triheptanoin infusion. If there is an apparent lipolysis as indicated by the very high glycerol concentration, an increase in the supply of long-chain fatty acids to the liver would be expected to result in enhanced C4-ketogenesis.

The fact in this study is that only glycerol was released into the circulation during lipolysis when triheptanoin was infused intravenously, but long-chain fatty acids seemed to be re-esterified in the adipocytes, whereas heptanoate was preferentially oxidized. Preferential oxidation of heptanoate would be favored because it does not accumulate in adipose tissue and its oxidation bypasses the CPT system.

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Figure 17. Profile of glycerol concentrations in rat plasma

Data are reported as Mean ± SE (n = 6 or 7)

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9.6 Plasma Glucose Concentrations

The basal glucose concentration (6.6 to 7.4 mM (Fig 18)) was slightly above physiological, presumably because of the surgical stress and isoflurane anesthesia. During saline infusion, glucose concentration decreased to almost normal values (5.75 ± 0.33 mM). Intravenous infusion of Na-heptanoate or

Na-heptanoate + glycerol significantly increased glucose concentration to 7.88

± 0.16 mM (p < 0.05) or 7.98 ± 0.15 mM (p < 0.01) compared to controls, respectively. Infusion of triheptanoin, whether intravenous or intraduodenal, significantly increased glucose concentration to 9.12 ± 0.089 mM (p < 0.001) or 10.3 ± 0.29 mM (p < 0.001) compared to controls, respectively. Lastly, addition of glucose + insulin to the intravenous triheptanoin infusion brought glucose concentration up to 12.4 ± 0.26 mM (p < 0.001 compared to controls).

9.7 Liver Glycogen Contents

The liver glycogen content (Table 2) was very low in the control group, 0.76 mg glucose equivalent/g, as expected for rats deprived of food for about 18 hr. In groups 2 to 6 which were infused for 2 hr with gluconeogenic substrate(s) (and glucose + insulin in group 5), the liver glycogen content increased to a maximum of 6.9 mg glucose equivalents/g in group 5. However, we did not assay the glycogen contents in the skeletal muscle.

99

Figure 18. Profile of glucose concentrations in rat plasma

Data are reported as Mean ± SE (n = 6 or 7)

100

μ Liverglycogen contents expressedare are rates glucose equivalents/g expressed All as mg in = 6-8). (MEAN ±SE, n Table 2. glycogenLiver contents and endogenous Ra of glucose, andglycerol oleate substrate. The two values two substrate. The in compared between Group 4 and 5 or between 4 Group 6.and mol  kg -1  min -1 (MEAN ±SE, n = 6-8). 6-8). of appearance (MEANRa, rate corrected ±SE, n = infusion for of exogenous tracer, + unlabeled or tracer italics are not considered are precise values. compared< 0.05, (*) controlp to 0.05, group; (‡) p <

101

9.8 Plasma Oleate Concentrations and Enrichments

The concentration of plasma oleate (Fig 19) remained stable from the start in all groups, except in the group infused intravenously with triheptanoin.

However, in the latter group, the oleate concentration stabilized after 1 hr. The

M18 enrichment of plasma oleate remained stable in all groups (Fig 20). The concentrations of plasma palmitate and stearate remained stable in all groups

(not shown). Since steady state concentration and enrichment of oleate were achieved in all groups during the last 30 min of the experiments, we calculated the turnover of oleate using the standard steady state equation (Table 2). The endogenous Ra of oleate was not affected by the intravenous infusion of

Na-heptanoate or of triheptanoin. The lack of increase in the endogenous Ra of oleate by triheptanoin contrasts with the 10.8-fold increase in glycerol endogenous Ra induced by the intravenous infusion of triheptanoin. When glucose + insulin was added to the intravenous infusion of triheptanoin, the endogenous Ra of oleate was halved while the endogenous Ra of glycerol was

5.5-times that of controls. Lastly, the intraduodenal infusion of triheptanoin did not change the endogenous Ra of oleate.

102

Figure 19. Profile of oleate concentrations in rat plasma

Data are reported as Mean ± SE (n = 6 or 7)

103

Figure 20. Profile of M18 oleate enrichments in rat plasma

Data are reported as Mean ± SE (n = 6 or 7)

104

9.9 Plasma Insulin Levels

The plasma insulin concentration remained between 0.3 and 0.4 ng/ml in groups 1, 2, 3, 4, and 6 (not shown). It rose to 4.4 ng/ml in group 5 (infused with triheptanoin + glucose + insulin; not shown). We had expected some increase in insulin concentration in groups 4 and 6 where triheptanoin was infused alone intravenously or intraduodenally, respectively, and where plasma glucose concentration went up to 9 - 10 mM. The assays were repeated with the same results. We ascribe the absence of glucose-induced insulin release to isoflurane anesthesia (290; 291).

9.10 Liver Acyl-CoAs Concentrations

The concentrations of medium- and short-chain acyl-CoAs, as well as free

CoA, in the liver of the rats were shown in Table 3. In all rats infused with heptanoate or triheptanoin, the concentration of free CoA decreased as the concentrations of acyl-CoAs derived from heptanoate (heptanoyl-CoA, pentanoyl-CoA, propionyl-CoA) increased. The sum of assayed concentrations of acyl-CoA + free CoA increased by up to one-half in livers of rats infused with triheptanoin + glucose + insulin intravenously, or triheptanoin intraduodenally.

The concentrations of the intermediates of C4-ketogenesis (HMG-CoA, acetoacetyl-CoA) and C5-ketogenesis (HEG-CoA, BKP-CoA) remained fairly constant, in spite of vastly different rates of C4- and C5-ketogenesis. The 105 concentrations of BHB-CoA (not shown) appear to be overestimated because the liver [BHB-CoA] / [AcAc-CoA] ratio is much larger than the liver [BHP-CoA]

/ [BKP-CoA] ratio, while the plasma [BHB] / [AcAc] ratio is close to plasma

[BHP] / [BKP] ratio. We suspect that the peak of BHB-CoA eluting from the

HPLC column is contaminated by its isomer 3-hydroxyisobutyryl-CoA derived from the catabolism of valine. However, we could not find a standard of

3-hydroxyisobutyryl-CoA to correct the results of BHB-CoA. Thus, we deleted the BHB-CoA concentrations from Table 3.

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All concentrations are expressed nmol·g in Table 3. Liver acyl-CoAconcentrations compared between Group 4 and 5 or between 4 Group 6.and -1 (MEAN ± SE, = 6-8).n * p < 0.05, compared to control Group 1. ‡, p < 0.05,

107

10. DISCUSSION

10.1 Heptanoate Metabolism and C4- and C5-Ketogenesis

The intravenous infusion of equimolar amounts of heptanoate (alone, mixed with glycerol, as triheptanoin, or as triheptanoin + glucose + insulin, groups 2 -

5, Table 1) resulted in different plasma concentrations of heptanoate (Fig 14).

While adding free glycerol to the free heptanoate infusion did not affect heptanoate concentration, infusion of triheptanoin resulted in much higher heptanoate concentrations. Since the heptanoate concentrations reached plateaus during the 2nd hour of the experiments, the rates of heptanoate metabolism were identical in all rats of groups 2 to 5, albeit under very different plasma heptanoate concentrations. Therefore, some factors were inhibiting the metabolism of heptanoate derived from triheptanoin, until the influence of such factors was balanced by higher heptanoate concentrations. In group 5 (infused with triheptanoin + glucose + insulin), the marked hyperglycemia (Fig 18) and massive exogenous hyperinsulinemia favored glucose over heptanoate utilization. Similarly, in group 4, infused with triheptanoin alone, the 4 mM increase in glucose concentration compared to controls (Fig 18) interfered with heptanoate utilization.

As shown in our previous study (133), the intraduodenal infusion of triheptanoin (group 6) resulted in very low arterial heptanoate concentrations

108

(Fig 14) because the whole heptanoate load passed first through the liver. This explains also why the concentration of C5-ketone bodies was the highest in group 6 (Fig 15). In rats infused intravenously with heptanoate, heptanoate + glycerol, or triheptanoin (groups 2 to 4), the profiles of C5-ketone body concentrations were similar (Fig 15). Also, in the same groups, the profiles of

C4-ketone body concentrations were similar (Fig 16). However, the accumulations of C4 and C5-ketone bodies were inhibited in rats infused with triheptanoin + glucose + insulin (Figs 15 and 16, open circles). The rapid inhibition of C5-ketogenesis by supra-physiological concentrations of insulin, in the presence of the highest heptanoate concentrations (Fig 14) shows that insulin can interfere with mitochondrial C5-ketogenesis in a time frame shorter than that needed for modulation of gene expression. This is confirmed by the very strong inhibition of C4-ketogenesis in the same rats (Fig 16).

10.2 Activation of Lipolysis in Triheptanoin Infusions

Lipolysis was assessed by plasma glycerol concentrations and glycerol Ra.

The arterial concentration of glycerol (Fig 17) was not affected by either the intravenous infusion of heptanoate or the intraduodenal infusion of triheptanoin.

This confirms that the glycerol resulting from the enteral hydrolysis of triheptanoin and the free glycerol contained in the triheptanoin emulsion are entirely taken up by the liver in a single pass of portal vein blood. In contrast,

109 the intravenous infusion of equimolar amounts of glycerol (mixed with heptanoate, as triheptanoin, or as triheptanoin + glucose + insulin, groups 2 - 5,

Table 1) resulted in different plasma concentrations of glycerol (Fig 17). This results from the fact that the intravenous administration of triheptanoin led to a major increase in the production of endogenous glycerol from lipolysis (Table 2, groups 4 and 5). In these groups, the load of lipolytic glycerol was equivalent to

10 and 5 times the load of glycerol infused as part of the triheptanoin emulsion

(28.2 μmolmin-1kg-1).

The intravenous administration of heptanoate did not significantly increase endogenous lipolysis (Table 2, groups 1 - 3). The intravenous infusion of triheptanoin (without or with glucose + insulin, groups 4 and 5) resulted in large increases in glycerol Ra, as shown by others with different triglycerides

(292-294). However, the 10.8 fold increase in endogenous glycerol Ra we measured in the present study is much larger than the 2 fold increase we measured in our previous study (133). Actually in group 5 of the present study

(triheptanoin + glucose + insulin IV) the endogenous glycerol Ra was also much higher that any value in our previous study. The only difference between the two studies is the mode of anesthesia. In the previous study, the rats were anesthetized with sodium pentobarbital intraperitoneally followed by a continuous IV infusion. In the present study, the rats were anesthetized with isoflurane. In reviewing the metabolic effects of isoflurane anesthesia, we found reports showing that isoflurane induces hyperglycemia, inhibits

110 glucose-induced insulin secretion and stimulates lipolysis (33; 290; 291).

These effects, ascribed to the stimulation of growth hormone by isoflurane (33) were not observed during anesthesia with pentobarbital (291). The inhibition of insulin secretion by isoflurane probably explains why, in the present experiments, plasma insulin concentration increased only in group 5, when exogenous insulin was infused. In groups 2, 3, 4 and 6, insulin concentration did not increase in spite of hyperglycemia. This may be related to differences in the effect of triheptanoin of the Ra of glucose between our previous study

(Ra increased by half with ID triheptanoin but was not significantly increased by IV infusion) and the present study (opposite findings).

10.3 Fatty Acids Released from Lipolysis Are Re-esterified in

Triheptanoin Infusions

The flux of long-chain fatty acids rather than glycerol is most deleterious for long-chain FOD patients. Thus, endogenous Ra of long-chain fatty acids was measured. Based on endogenous glycerol Ra and long-chain fatty acid Ra, the percentage of fatty acid re-esterification in adipose tissue can be calculated. In rats infused with triheptanoin, the 10.8-fold increase in glycerol endogenous

Ra (Table 2) was not accompanied by any significant increase in the

13 endogenous Ra of plasma oleate traced with [ C18]oleate. The latter tracer was chosen instead of the more commonly used [13C]palmitate and

111

[13C]stearate because the unavoidable contamination of glassware and reagents by ubiquitous palmitate and stearate makes the assay of the enrichment of palmitate and stearate in small samples of rat plasma very imprecise. Fig 19 and Fig 20 show that the enrichment and concentration of plasma oleate were stable at the end of the experiments. From the endogenous Ra of glycerol and oleate, we estimated the percentage of the lipolytic long-chain fatty acids that was re-esterified in adipose tissue. This estimation assumes that oleate accounts for 1/4 of the turnover of plasma long-chain fatty acids in all groups (295). Based on this assumption, the percent of re-esterification is 22% in the control rats and about 90% in rats infused intravenously with triheptanoin (± (glucose + insulin), groups 4 and 5).

We acknowledge that there is some uncertainty in the estimated percent re-esterification, however the comparison of the glycerol and oleate endogenous Ra in groups 4 and 5 supports our conclusion that, in rats infused intravenously with triheptanoin, most of the long-chain fatty acids derived from adipose tissue lipolysis are re-esterified in the same cells.

In rats infused with triheptanoin intraduodenally (group 6), the above calculation yields an impossible -55% re-esterification. Because of the low glycerol endogenous Ra in group 6 was calculated using two glycerol tracers, the calculation of fatty acid reesterification is admittedly imprecise. The

2 [1,1,2,3,3- H5]glycerol infused intraduodenally with triheptanoin would probably has different kinetics from the endogenous glycerol.

112

10.4 Sources of Glycerol-3-Phosphate for Fatty Acid Re-esterification

The percentage of fatty acid re-esterification is very high in rats infused with triheptanoin intravenously. Such a strong re-esterification requires a sufficient supply of glycerol-3-phosphate in adipose tissue. The glycerol-3-phosphate used for fatty acid re-esterification in adipose tissue can be derived from glucose catabolism and, possibly, from glyceroneogenesis via PEPCK (270;

285). One can wonder whether the propionyl moiety of heptanoate could be glyceroneogenic in adipose tissue. However, the supply of propionyl-CoA equivalents during intravenous infusion of triheptanoin (42.3 μmolmin-1kg-1) is much smaller than the amount of fatty acid released by lipolysis that needs to be re-esterified. The latter is 3 times the glycerol Ra, i.e., 3 x 282 = 846

μmolmin-1kg-1) (Table 2). Thus, when triheptanoin was infused, the re-esterification of long-chain fatty acid derived from lipolysis could not be supported by glyceroneogenesis from the propionyl moiety of heptanoate.

Thus, the supply of glycerol-3-phosphate for fatty acid re-esterification must be derived from glycolysis stimulated by hyperglycemia (Fig 18) and possibly, by glyceroneogenesis from pyruvate derived from glycolysis (270).

10.5 Fluxes of Glucose Production

The plasma glucose concentrations (Fig 18) and the endogenous Ra of glucose (Table 2) must be interpreted taking into account (i) the concentrations 113 of gluconeogenic precursors (heptanoate, glycerol, Fig 14, 17), (ii) the concentrations of competing energy fuels (heptanoate, C5- and C4-ketone bodies, Fig 14, 15, 16), and (iii) the metabolic loads corresponding to the supply of exogenous and endogenous gluconeogenic precursors (exogenous glycerol, propionyl moiety of exogenous heptanoate, endogenous glycerol). In

Fig 21, these metabolic loads are compared to the endogenous glucose Ra and to the increase in the concentration of liver glycogen in groups 2 to 6, compared to group 1. Note that all rates presented in Fig 21 are expressed in

(μmol glucose equivalents)min-1kg-1. These comparisons are somewhat difficult because our measurements of the glucose endogenous Ra do not distinguish (i) the two components of the glucose Ra, glycogenolysis and gluconeogenesis, and (ii) the contributions of gluconeogenic precursors, amino acids derived from proteolysis, glycerol, and propionyl-CoA derived from heptanoate.

In rats infused intravenously with gluconeogenic heptanoate (alone, + glycerol, or as triheptanoin, groups 2 to 4), the endogenous Ra of glucose was about

1.5 times that of the control group (Table 2). It is not clear why the glucose endogenous Ra was the same in these 3 groups given that groups 3 and 4

(unlike group 2) were given exogenous glycerol in addition to the same load of propionyl precursor. Also, group 4 (triheptanoin intravenous) and group 5

(triheptanoin + glucose + insulin intravenous) had to dispose of a large load of gluconeogenic glycerol derived from lipolysis. Clearly, this large load of

114 glycerol was not converted to free glucose. In groups 3 to 6, some of the exogenous + endogenous glycerol ended up stored as liver glycogen (Fig 21).

However the increase in liver glycogen content would account for only a small fraction of the metabolized glycerol. Also, the small increase in glycogen content has a component of gluconeogenesis from the propionyl moiety of heptanoate (groups 3 to 6). Lastly, a small fraction of the glucose infused in group 5 probably contributed to liver glycogen synthesis. Overall, the observed increases in liver glycogen contents represent only a small fraction of the potential glycogen precursors available. It is likely that there was some accumulation in muscle glycogen in groups 4 and 5, but we did not assay muscle glycogen content.

115

heptanoate or triheptanoin. All rates are expressed in ( exogenousblack bars: potential glycerol; glucose the production from exogenous propionyl equivalentsderived from potentialthe glucose production from endogenous glycerol Ra; obliquely bars:hatched the potential glucose production from open bars: endogenous glucose Ra; checkered bars:rate the of liver glycogen synthesis control; over vertically hatched bars: potential glucose productions Figure 21. Comparison between the endogenous glucose Ra, the μ mol glucose equivalents) rate of liver glycogen synthesis over control and the controlover synthesisliver glycogen and of rate  min -1  kg

-1

116

10.6 Profiles of Medium-chain and Short-chain Acyl-CoAs in the Liver

Administration of heptanoate or triheptanoin led to substantial changes in the concentrations of some acyl-CoA and of free CoA in the liver (Table 3). The sum of the concentrations of the assayed short- and medium-chain acyl-CoAs increased by up to 50% compared to saline-infused controls. This reflects a redistribution of CoA from long-chain to short- and medium-chain esters.

Concern has been expressed that the administration of some substrates or drugs could lead to “CoA sequestration, toxicity or redistribution” (CASTOR)

(118). In particular, the trapping of CoA into unusually large pools of some acyl-CoAs would decrease the availability of free CoA and impact on the rate of CoA-dependent reactions. In our experiments, the trapping of CoA in the intermedates of heptanoate β-oxidation (heptanoyl-CoA, pentanoyl-CoA,

BKP-CoA, BHP-CoA and propionyl-CoA) resulted in up to ten-fold and one-third decreases in the concentrations of free CoA and acetyl-CoA, respectively. However, the plateauing of the plasma heptanoate concentrations (Fig 14), albeit at different levels, indicated that all the infused heptanoate equivalents were being metabolized. Also, in spite of up to one-third decrease in liver acetyl-CoA concentration, there was a large flow of acetyl groups into C4- and C5-ketogenesis, except when insulin was infused

(Fig 15, 16). Ketogenesis removes from the liver acetyl groups which cannot be oxidized in the citric acid cycle, the rate of which is linked to the rate of ATP

117 regeneration (296). Therefore, the trapping of free CoA by heptanoate metabolism did not impair the energy metabolism of the liver.

In spite of very large differences in rates of C4- and C5-ketogenesis between rats infused with saline and rats infused with heptanoate or triheptanoin (Fig 15,

16), there were little variations in the concentrations of HMG-CoA and

HEG-CoA. This reflects the high activities of HMG-CoA synthase and

HMG-CoA lyase. Thus, under the conditions of the experiments, the rates of

C4- and C5-ketogenesis depend on the supply of acetyl and propionyl groups to the HMG-CoA cycle.

11. CONCLUSIONS AND CLINICAL IMPLICATIONS

The metabolism and metabolic effects of an odd-medium-chain triglyceride, triheptanoin, were thoroughly investigated in the present study. First, in the liver, odd-chain heptanoate is partially converted to C4- and C5-ketone bodies.

Second, odd-chain heptanoate is a substrate of gluconeogenesis. Third, odd-chain heptanoate is a substrate of anaplerosis in all tissues that can use heptanoate and C5-ketone bodies. It is well known that the intravenous infusion of a long-chain triglyceride emulsion induces the activation of plasma lipoprotein lipase which hydrolyzes the infused triglyceride and results in an increase in lipolysis. Our study found that intravenous infusion of odd-medium-chain triheptanoin results in the same problem. Intravenous 118 administration of triheptanoin leads to massive lipolysis. However, although the intravenous administration of triheptanoin stimulates lipolysis in adipose tissue, there is no increase in endogenous fatty acid Ra. This is because of intense re-esterification of fatty acids from adipocyte lipolysis in the same cells. The glycerol-3-phosphate required for fatty acid re-esterification is possibly derived from glycolysis (increased glucose concentrations and glucose Ra) and glyceroneogenesis (propionyl-CoA and pyruvate/lactate) in the adipose tissue.

Thus, the intravenous administration of triheptanoin should be considered for the acute treatment of long-chain FOD patients. Moreover, enteral treatment of triheptanoin (by nasogastric tube) in long-chain FOD patients remains an option because this treatment does not lead to increased lipolysis whereas it actually decreases basal lipolysis (Table 2). Intraduodenal infusion of triheptanoin also increases plasma glucose concentrations. Heptanoate is most easily utilized and converted to C5-ketone bodies when triheptanoin is infused intraduodenally. Last of all, intravenous infusion of triheptanoin does not need to be supplemented with glucose co-infusion because it already leads to moderate hyperglycemia.

12. FURTHER DIRECTIONS

The present study provided new insights on the metabolism and metabolic effects of the odd-chain triheptanoin in vivo. The study clearly demonstrated 119 that (i) the harmful activation of lipolysis induced by intravenous triheptanoin infusion is fully balanced by the fatty acid re-esterification; (ii) intraduodenal triheptanoin infusion does not activate lipolysis, and (iii) triheptanoin infusions generate anaplerotic and gluconeogenic propionyl-CoA. Thus, both parenteral and enteral administrations of odd-chain anaplerotic triheptanoin have the potential to serve as the new avenues for the acute treatment of long-chain

FOD. However, a number of new questions rose up. To address these questions, the following studies and the corresponding strategies are suggested.

1. The high percentage of fatty acid re-esterification in the current study may

not be the case in decompensated long-chain FOD patients. In the present

study, after overnight starvation and the surgery, the basal lipolysis in the

normal rats remained low. The rats in our study were not in a severely

stressed condition comparable to decompensated long-chain FOD

patients whose symptoms are really dangerous. Generally, long-chain

FOD patients suffering from an acute attack have extremely high level of

basal lipolysis. In these patients, the massive basal lipolysis and the

lipolysis induced by intravenous triheptanoin infusion may not be fully

balanced by the re-esterification as demonstrated in our current study.

These patients may benefit from the administration of insulin. This

question should be investigated in animals subjected to lipolytic conditions

120

such as the infusion of a catecholamine. Alternatively, the study should be

performed on knock-out animals under stress.

2. Glyceroneogenesis from propionyl-CoA may contribute to the formation of

glycerol-3-phosphate for fatty acid re-esterification in the current study. It is

interesting to investigate anaplerosis and glyceroneogenesis of

propionyl-CoA in adipose tissue. The epididymal fat pad isolated from rats

can be perfused in situ with labeled propionyl-CoA precursors, such as

13 [5,6,7- C3]heptanoate. Labeled M3 succinyl-CoA and M2 or M3

glycerol-3-phosphate allow one to calculate the relative anaplerosis and

the relative contribution of glyceroneogenesis from propionyl-CoA in

adipose tissue. Alternatively, anaplerosis and glyceroneogenesis from

propionyl-CoA can be studied in 3T3-L1 cells incubated with labeled

odd-chain compounds and glucose.

3. Results from triheptanoin I.D. infusion in the present study support chronic

dietary treatment of triheptanoin on knockout mice. I hypothesize that

knockout mice treated the triheptanoin diet will thrive better than knockout

mice on the trioctanoin diet or high carbohydrate diet.

121

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