**3. Energy supply under fasting conditions**

The liver, muscles, adipose tissues, and brain are all closely involved in energy metabolism and are important organs for understanding whole body metabolism in the fasting state. The brain cannot directly utilize fat because it is not capable of fatty acid oxidation unlike many other organs. Therefore, during prolonged fasting, glucose is supplied to the brain from multiple sources to maintain its functions. In addition, ketone bodies are produced as an energy source for the brain during extremely long fasting. The reason why the brain is unable to oxidize fatty acids remains controversial, but it is thought to be because fatty acids cannot cross the blood–brain barrier and the brain does not have the enzymes necessary for β-oxidation.

In the normal state of energy metabolism under feeding conditions, dietary sugar is commonly used as an energy source in all organs. Glucose is oxidized via glycolysis to eventually yield two molecules of pyruvate. In the presence of oxygen, pyruvate enters the TCA cycle, and it is completely oxidized to produce six molecules of carbon dioxide. At this step, NAD+ and FAD are reduced to produce NADH and FADH2, respectively, and are transported to the respiratory chain of mitochondria to produce energy in the form of ATP.

In the early stage of fasting, stored glycogen, a branched polymer of glucose, is degraded to provide glucose via glucose 6-phosphate (**Figure 2**). Glycogen-derived glucose is used for energy supply by the glycolytic pathway and the TCA cycle in most of organs except the liver, muscles, and adipose tissue. However, the amount of energy stored as glycogen is consumed in a day of fasting, and hepatic glycogen is almost completely depleted after two to three days of starvation. The degradation of glycogen proceeds as phosphorylase sequentially removes glucose monomers by cleaving the α-(1 → 4) bond in a phosphate-dependent manner. At the branched end of glycogen, when degradation proceeds to four glucose residues near the α-(1 → 6) bond, three glucose molecules at the branching end are transferred to the other chain end by a group of glycogen debranching enzymes. The remaining glucose molecules are then hydrolyzed to remove them, and further degradation by phosphorylases continues. The glucose 1-phosphate produced by phosphorylase is converted to glucose 6-phosphate by phosphoglucomutase and enters the glycolytic pathway (**Figure 2**). Glucose 6-phosphate is converted to glucose by the action of glucose 6-phosphatase (G6Pase) in the liver, and then released into the bloodstream via Glut2 to supply glucose to the brain and other organs. On the other hand, G6Pase is not expressed in the muscles, so stored glycogen is used only for local energy production.

**Figure 2.** *Gluconeogenesis under the fasting condition.*

During fasting, TGs in the adipose tissue are also degraded and used for fatty acid oxidation (**Figures 2** and **3**). TGs are broken down into fatty acids and glycerol by hormone-sensitive lipase (HSL) and a rate-limiting enzyme, adipose triglyceride lipase (ATGL). Fatty acids are bound to albumin and transported to the liver and muscles, where they are taken up by facilitated transport for fatty acid oxidation. Long-chain fatty acids taken up by the cells pass through the mitochondrial membrane to the matrix. Fatty acids are converted to acyl-CoA by acyl-CoA synthase and then conjugated with carnitine to form acyl-carnitine by carnitine palmitoyltransferase (CPT1) on mitochondrial outer membrane (CPT1a: mainly in the liver, CPT1b: mainly in the skeletal muscle and brown fat) and pass through mitochondrial inner membrane to the matrix via carnitine-acylcarnitine translocase (CACT). Fatty acids are then released from carnitine through the action of CPT2 on mitochondrial inner membrane (**Figure 3**). In mitochondrial matrix, fatty acids are continuously oxidized via FAD-dependent acyl-CoA dehydrogenase to produce FADH2, NADH+H+ , and acetyl-CoA (**Figure 3**). Although fatty acid-derived acetyl-CoA enters the TCA cycle for energy production, it does not contribute to gluconeogenesis because two carbon atoms derived from the acetyl-CoA are removed in the TCA cycle. In contrast, the glycerol produced by lipolysis can be used as a substrate for gluconeogenesis (**Figure 2**).

When fasting persists, glucose is supplied to multiple organs via gluconeogenesis in the liver. Gluconeogenesis is particularly important to provide glucose as an energy source for the brain, which is unable to oxidize fatty acids. In the liver, gluconeogenesis is carried out from amino acids, pyruvate, lactate, glycerol, and ketone bodies (**Figures 1B** and **2**). During several weeks of starvation, about 80 grams of glucose is produced daily via gluconeogenesis, of which 15–20 grams is derived from amino acids, mainly alanine, 35–40 grams from pyruvate and lactate, 20 grams from glycerol from fat, and 10 grams from ketone bodies [1]. Amino acids are supplied by degradation of proteins in the muscles during fasting. Alanine is transported to the liver via the bloodstream, and is then converted to pyruvate for gluconeogenesis (**Figure 2**). Pyruvate is metabolized to oxaloacetate by pyruvate carboxylase (PC)

#### **Figure 3.**

*Lipid metabolism and ketone body production during fasting.*

and then decarboxylated by phosphoenolpyruvate carboxykinase (PEPCK) to produce phosphoenolpyruvate (PEP) (**Figure 2**). PEP is an intermediate product of the glycolytic pathway, and the following gluconeogenic process utilizes the glycolytic enzymes except for the step of fructose-1,6-bisphosphate to fructose-6-phosphate which requires fructose-1,6-bisphosphatase [4] and the step of glucose-6-phosphate to glucose mediated by G6Pase. Lactic acid is converted to pyruvate by lactate dehydrogenase (LDH) for gluconeogenesis (**Figure 2**). Glycerol is phosphorylated by glycerol kinase in the liver to produce glycerol 3-phosphate, which is then oxidized by glycerol 3-phosphate dehydrogenase (G3PDH) to dihydroxyacetone phosphate, an intermediate of the glycolytic pathway. Dihydroxyacetone phosphate undergoes gluconeogenesis via glyceraldehyde 3-phosphate or fructose 1,6-diphosphate (**Figure 2**). Furthermore, under long-term starvation for several days or a week where water, vitamins, salt, and other minerals are supplied, glucose production occurs not only in the liver but also in the renal cortex, which is responsible for about 40% of total body glucose production [1].

Gluconeogenesis is tightly regulated by the products of fatty acid oxidation such as NADH+H+ , acetyl-CoA, and ATP (**Figure 4**). In normal glucose metabolism, pyruvate enters the TCA cycle via acetyl-CoA by the action of pyruvate dehydrogenase (PDH). However, during fasting, pyruvate is converted to oxaloacetate by PC for gluconeogenesis as described above (**Figure 4**). The activity of PC is stimulated by acetyl-CoA. In addition, NADH+H+ , acetyl-CoA, and ATP inhibit PDH activity (**Figure 4**). Furthermore, NADH+H+ inhibits isocitrate dehydrogenase (IDH), one of the enzymes responsible for the TCA cycle (**Figure 4**). Thus, NADH+H+ ,

**Figure 4.** *Fatty acid oxidation and gluconeogenesis during fasting.*

acetyl-CoA, and ATP, which are generated by fatty acid oxidation, regulate the activities of enzymes in the gluconeogenesis pathway, and these regulatory mechanisms ensure a consistent flow of metabolites for energy supply through gluconeogenesis and fatty acid oxidation during fasting. Since PEP produced by PEPCK is an intermediate product of the glycolytic pathway, it could theoretically be converted to pyruvate and re-enter the TCA cycle. However, because pyruvate kinase (PK) in the liver is inhibited by alanine and inactivated by protein kinase A (PKA), which is activated by glucagon, PEP produced during fasting is used for gluconeogenesis but not glycolysis. In other words, gluconeogenesis and amino acid metabolism are consistently regulated under fasting conditions. In addition, nitrogen sources stored in the muscles are used for gluconeogenesis in the renal cortex under prolonged fasting. Glutamine and alanine are metabolized from branched-chain amino acids such as leucine, isoleucine, and valine in the muscles, and released into the bloodstream. Glutamine is then primarily used for gluconeogenesis via the TCA cycle in the renal cortex. This indicates that diet therapies that focus primarily on fasting result not only in burning of stored fat but also loss of muscle mass.

#### **4. Ketone body as an energy source during long-term fasting**

Under glucose-depleted conditions during fasting, the brain and muscles use ketone bodies and acetate as energy sources other than glucose (**Figure 1B**) [3, 5]. These energy sources are critical to sustain function of the brain because it cannot directly metabolize fatty acids. A human study investigated the energy sources of the brain under long-term starvation for 5–6 weeks. When only water, vitamins, and minerals such as salt are supplied, urinary nitrogen excretion, an indicator of amino acid-derived gluconeogenesis, dropped to about 4–5 grams per day, and two-thirds of the energy source of the brain comes from β-hydroxybutyrate and acetoacetate [1].

The metabolic systems that use ketone bodies as an energy source can be traced back to bacteria, archaea, and protozoa. In humans, most ketone bodies are produced in the liver. During fasting, the most part of acetyl-CoA produced by fatty

#### *Metabolic Responses to Energy-Depleted Conditions DOI: http://dx.doi.org/10.5772/intechopen.100391*

acid oxidation is used for ketone body production, while only the limited amount enters the TCA cycle (**Figure 3**). At the first step of the TCA cycle, acetyl-CoA is conjugated to oxaloacetate through the action of citrate synthase to produce citrate. However, oxaloacetate is relatively scarce during fasting because it is consumed by glucose production. As a result, excess acetyl-CoA produced by fatty acid oxidation is used exclusively for ketone body production. In addition, NADH+H<sup>+</sup> , acetyl-CoA, and ATP, which are produced by fatty acid oxidation, regulate gluconeogenesisrelated enzymes to stimulate gluconeogenesis. In addition, NADH+H<sup>+</sup> suppresses the activity of IDH in the TCA cycle, which in turn suppresses the TCA cycle and directs fatty acid-derived acetyl-CoA toward ketone body production.

Ketone bodies are produced in mitochondria in the following reactions: two molecules of acetyl-CoA are combined by acetoacetyl-CoA thiolase to produce acetoacetyl-CoA, and an additional molecule of acetyl-CoA is conjugated by HMG-CoA synthase 2 (HMGCS2) to produce HMG-CoA, which is then cleaved by lyase to form acetoacetate (**Figures 3** and **5**). Acetoacetate can be converted to acetone through spontaneous non-enzymatic decarboxylation or to β-hydroxybutyrate (D-3-hydroxybutyrate) by 3-hydroxybutyrate dehydrogenase (BDH1) (**Figure 3**). β-Hydroxybutyrate is the most abundant ketone body in the blood. Here, the irreversible reaction by HMGCS2 is a key reaction for ketone body production, and the activity of BDH1 is increased by NADH+H+ .

The basal level of β-hydroxybutyrate in humans is at the level of a few μM under feeding conditions, and the blood concentration increases to 200–300 μM after 12–16 hours of fasting, 1–2 mM after 2 days of fasting, and as high as 6–8 mM after prolonged fasting [6]. Ketone bodies also reach more than 2 mM with a ketogenic diet that excludes most carbohydrates, and intense exercise for about 90 minutes also increases ketone bodies to 1–2 mM. In neonates, the production and utilization of ketone bodies are more efficient than adults. The serum concentration of ketone bodies is as high as 2–3 mM just after birth, and the neonatal brain uses ketone bodies as an important energy source.

Ketone bodies are produced by the liver and supplied to the brain, muscles, and kidneys during fasting, but the liver cannot utilize ketone bodies as an energy source because it does not express 3-keto acid CoA transferase (OXCT1/SCOT). β-Hydroxybutyrate is produced in the liver and released into the bloodstream via

**Figure 5.** *Production and utilization of ketone bodies.*

monocarboxylic acid transporter 7 (MCT7/SLC16A6) (**Figure 5**) [6]. During prolonged fasting, the high concentration of β-hydroxybutyrate in the blood is taken up by the brain through the blood–brain barrier via several monocarboxylic acid transporters, including MCT1 and MCT2 (**Figure 5**). Once taken up into neurons, β-hydroxybutyrate reverses the ketone body production pathway to produce acetoacetate by BDH1 and further converts to acetoacetyl-CoA by OXCT1/SCOT. The activity of BDH1 is promoted by NADH+H+ produced during fatty acid oxidation (**Figures 3** and **5**). In addition, the reaction of OXCT1/SCOT is the rate-limiting step to produce acetoacetyl-CoA, which is accompanied by the production of an intermediate metabolite of the TCA cycle, succinate, in a succinyl CoA-dependent manner (**Figure 5**).

In addition, acetate is also utilized as an energy source during prolonged fasting. In the liver, acetate is produced from acetyl-CoA by the action of acetyl-CoA hydroxylase during fasting, and is released from the liver to other organs. Acetate is utilized as an energy source for acetyl-CoA production via type 2 acetyl-CoA synthase (AceCS2), which is particularly abundant in mitochondria of the muscles [2].

## **5. Regulatory mechanisms of ketone body production**

HMGCS2 is a key enzyme for the regulation of ketone body production. The expression of HMGCS2 is strongly regulated by forkhead box protein A2 (FOXA2), peroxisome proliferators-activated receptor α (PPARα), fibroblast growth factor 21 (FGF21), and mTOR, and its activity is enhanced through deacetylation by sirtuin 3 (SIRT3) [6]. FOXA2 binds directly to the promoter region of HMGCS2 and activates its transcription. The expression of FOXA2 is regulated by insulin and glucagon. Insulin-mediated phosphorylation inactivates FOXA2 by translocating it out of the nucleus, while glucagon activates FOXA2 through p300-mediated acetylation, thereby contributing to ketone body production. FOXA2 deacetylation is also regulated by class I and class II HDACs and SIRT1, a class III HDAC. In addition, mTORC1 complex is known to suppress PPARα, and rapamycin promotes ketone body production by inhibiting mTORC1 complex.
