Chapter 3

I. INTRODUCTION

II. DIGESTION

III. ABSORPTION

IV. METABOLISM OF ABSORBED CARBOHYDRATES

A. General

B. Storage as Glycogen

C. Glycogen Metabolism

D. Catabolism of Glucose

V. INTERRELATIONSHIPS OF CARBOHYDRATE, LIPID, AND PROTEIN METABOLISM

A. Lipid Metabolism

B. Influence of Glucose Oxidation on Lipid Metabolism

VI. INSULIN AND CARBOHYDRATE METABOLISM

A. Proinsulin and Insulin

B. Insulin Transport

C. Glucose Transport

D. Insulin Action on Biochemical Systems

E. Physiological Effects of Insulin

F. Other Pancreatic Islet Hormones

VII. BLOOD GLUCOSE AND ITS REGULATION

A. General

B. Glucose Supply and Removal

C. Role of the Liver

D. Glucose Tolerance

VIII. METHODOLOGY

A. Blood Glucose

B. Indirect Monitoring of Blood Glucose

C. Tolerance Tests

D. Ketone Bodies

IX. DISORDERS OF CARBOHYDRATE METABOLISM

A. Diabetes Mellitus

B. Hyperinsulinism

C. Hypoglycemia of Baby Pigs

D. Glycogen Storage Diseases

X. DISORDERS OF RUMINANTS ASSOCIATED WITH HYPOGLYCEMIA

A. General

B. Carbohydrate Balance

C. Biochemical Alterations in Body Fluids

REFERENCES

I. INTRODUCTION

The biochemical mechanisms by which the chemical energy contained in foodstuffs are made available to the animal are collectively described as metabolism. Thus, the description of the metabolism of a foodstuff encompasses the biochemical events that occur from the moment of ingestion to its final breakdown and excretion. Classically, these biochemical events have been divided into the metabolism of the three major constituents of food; carbohydrates, proteins, and lipids. The metabolism of the lipids and proteins is discussed in their individual chapters.

The major function of ingested carbohydrates is to serve as energy sources, and their storage function is relatively minor. Carbohydrates are also precursors of essential intermediates for use in synthetic processes. When the metabolic machinery of an animal is disrupted, a disease state prevails (e.g., diabetes). The literature of the biochemistry of metabolism and disease continues to expand as the minute details of individual and overall reaction mechanisms are continually clarified. Additionally, modern molecular approaches have significantly increased our understanding of disease mechanisms and remain fertile fields for investigations into the disease processes. This chapter presents a basis for the better understanding of the biochemical mechanisms underlying those diseases associated with carbohydrate metabolism. The intricate details of carbohydrate metabolic reactions may be found in the many books on fundamental biochemistry.

II. DIGESTION

The digestion of carbohydrates in the animal begins with the initial contact of the carbohydrates in their foodstuffs with the enzymes of salivary juice. Starch of plant foods and glycogen of meat are split into their constituent monosaccharides by the action of amylase and maltase. This activity ceases as the food matter passes into the stomach, where the enzymatic action is destroyed by the hydrochloric acid. Within the stomach, acid hydrolysis may occur, but the stomach empties too rapidly for complete hydrolysis to take place. Thus, only a small portion of the ingested carbohydrate is hydrolyzed before passing into the small intestine. In the small intestine, digestion of carbohydrate takes place quickly by the carbohydrate splitting enzymes contained in the pancreatic juice and in the succus entericus. Starch and glycogen are hydrolyzed to glucose by amylase and maltase; lactose to glucose and galactose by lactase; and sucrose to glucose and fructose by sucrase. The monosaccharide products of enzymatic hydrolysis of carbohydrates, glucose, fructose, and galactose, are the principal forms in which absorption occurs in the monogastric animal.

Liver cells are readily permeable to glucose. This process is facilitated by glucose transporter (GLUT) proteins within the plasma membrane, in particular, GLUT-2 is the transporter in the liver cell plasma membrane (Thorens et al., 1988). Within the liver, there are several pathways by which the immediate fate of the hexoses is determined. Glucose, fructose, and galactose first enter the general metabolic scheme through a series of complex reactions to form glucose phosphates (Fig. 3-1). The enzyme, galactose-1-P uridyl transferase, which catalyzes the reaction

Glycogen is the chief storage form of carbohydrate in animals and is analogous to the storage of starch by plants. It is found primarily in liver and in muscle, where it occurs at about 3% to 6% and about 0.5%, respectively (Table 3-1). Glycogen is composed solely of α-D-glucose units linked together through carbon atoms 1 and 4 or 1 and 6. Straight chains of glucose units are formed by the 1–4 links and these are cross-linked by the 1–6 links. The result is a complex ramification of chains of glucosyl units with branch points at the site of the 1–6 links (Fig. 3-2). The internal chains of the glycogen molecule have an average length of four glucosyl units. The external chains beyond the last 1–6 link are longer and contain between 7 and 10 glucose units. The molecular weight of glycogen may be as high as 4 × 10⁶ Mr and contain about 20,000 glucosyl units.

In Table 3-2, the amount of carbohydrate available to meet the theoretical requirements of a hypothetical dog is shown. The amount present is sufficient for about half a day. It is apparent that the needs of the body that must be met continually are satisfied by alternate means and not solely dependent on continuous ingestion of carbohydrates. During and after feeding (postprandial), absorbed hexoses are converted to glucose by the liver and enter the general circulation. Excesses are stored as glycogen or as fat. During the fasting or postabsorptive state, glucose is supplied by the conversion of protein (gluconeogenesis) and by the breakdown of glycogen (glycogenolysis). The continued rapid synthesis and breakdown of glycogen, (i.e., turnover) is well illustrated by the biological half time of glycogen, which is about a day.

C. Glycogen Metabolism

1. Glycogenesis

The initial reaction required for the entrance of glucose into the series of metabolic reactions, which culminate in the synthesis of glycogen, is the phosphorylation of glucose at the C-6 position. Glucose is phosphorylated with adenosine triphosphate (ATP) in liver by an irreversible enzymatic reaction catalyzed by a specific glucokinase (GK):

Glucokinase (GK) (also called hexokinase-IV [HK-IV]) is one of the four hexokinase isoenzymes that occurs in all tissues. Glucokinase (GK) or HK-IV, which is glucose specific, is the predominant isoenzyme found in liver. The nonspecific hexokinase-I (HK-I) is the isoenzyme found in red cells, brain, and nerve tissue.

Liver contains both GK (HK-IV) and HK-I, but GK is the predominant isoenzyme. GK has a high Michaelis constant (Km = 2 × 10^–2 mol G/l) indicating a low affinity for glucose. The rate of the phosphorylation reaction catalyzed by GK is therefore controlled by the glucose concentration. The activity of GK is increased by glucose feeding and by insulin and is decreased during fasting and in insulin lack (i.e., diabetes). In this regard, GK is an inducible enzyme whose activity is increased by glucose or by insulin. The nonspecific HK-I is found in all tissues including liver, brain, and erythrocytes and has a low Michaelis constant (Km = 5 × 10^–5 mol G/l), indicating a high affinity for glucose. HK-I catalyzes phosphorylation in all tissues, therefore, it is not controlled by glucose concentration. The activity of HK-I is not affected by fasting or by carbohydrate feeding, diabetes, or by insulin. Therefore, in contrast to GK, HK-I is not an inducible enzyme.

The initial unidirectional phosphorylation reaction permits the accumulation of glucose in the liver cells because phosphorylated sugars do not pass freely into and out of the cell in contrast to the readily transported free sugars. The glucose-6-phosphate (G-6-P) accumulated in the cell next undergoes a mutation in which the phosphate group is transferred to the C-1 position of the glucose molecule. This reaction is catalyzed by the enzyme, phosphoglucomutase (PGM) and involves glucose-1–6-diphosphate as an intermediate:

G-1-P is converted to G-6-P by the reversible reaction catalyzed by phosphoglucomutase (PGM, Section IV.C.1, Reaction II). The G-6-P is then irreversibly cleaved to free glucose and phosphate by the enzyme G-6-Pase, which is found in liver and kidney. The free glucose formed can, unlike its phosphorylated intermediates, be transported out of the hepatic cell and enter the general circulation, thereby contributing directly to the blood glucose pool. In muscle tissue, there is no G-6-Pase, and muscle glycogen cannot supply glucose directly to the circulation by glycogenolysis. Muscle glycogen contributes to blood glucose indirectly via the lactate or Cori cycle (Section IV.D). The series of reactions described are illustrated schematically in Figure 3-3.

The fundamental conversion required to initiate the oxidation of glucose by a cell is its phosphorylation to form G-6-P. This reaction has been described in Section IV.C.1. The G-6-P formed as a result of the GK (HK-IV) catalyzed reaction is central to glucose catabolism. There are at least five different pathways that G-6-P can follow: free glucose, glycogenesis, glycolysis, hexose monophosphate, and glucuronate pathway.

Muscle G-6-P, however, because of the absence of G-6-Pase, does not contribute glucose from its glycogen to blood directly. Muscle G-6-P does, however, contribute glucose to blood indirectly via the lactate or Cori cycle. Lactate formed in muscle by glycolysis is transported to the liver, where it is resynthesized to glucose and its precursors as outlined in Figure 3-4.

b. Glycogenesis

This pathway for G-6-Pase leading to the synthesis of glycogen is discussed in Section IV.C.1.

c. Anaerobic Glycolysis

One of the three oxidative pathways of G-6-P is the classic anaerobic glycolytic or Embden-Meyerhof pathway (EMP). The intermediate steps involved in this pathway of breakdown of G-6-P into two three-carbon compounds are summarized in Figure 3-5. A mole of ATP is used to phosphorylate fructose-6-phosphate (F-6-P) to form fructose-1,6-diphosphate (F-1,6-P). This phosphorylation reaction is also irreversible and catalyzed by a specific kinase, phosphofructokinase (PFK). The opposing unidirectional reaction is catalyzed by a specific phosphatase, fructose-1,6-diphosphatase (F-1,6-Pase). These opposing PFK and F-1,6-Pase catalyzed reactions are a second site of metabolic control regulated by the activities of these two highly specific enzymes. At this point in the process, starting from glucose, a total of two high-energy phosphates from ATP have been donated to form a mole of F-1,6-P.

F-1,6-P is next cleaved to form two three carbon compounds as shown in Figure 3-5. The next step is an oxidative step catalyzed by the enzyme glyceraldehyde-3-phosphate dehydrogenase (GA-3-PD) with oxidized nicotinamide adenine dinucleotide (NAD⁺) as the hydrogen acceptor. During the process, the molecule is phosphorylated. In the succeeding steps, the molecule is dephosphorylated at the points indicated, and a mole of ATP is generated at each point.

A third site of control of glycolysis is the irreversible formation of pyruvate catalyzed by the enzyme pyruvate kinase (PK). In the reverse direction, two enzymatic reactions operate. Pyruvate carboxylase (PC) first catalyzes the carboxylation of pyruvate to oxaloacetate (OAA), and the OAA is then converted to phospho-enol-pyruvate (PEP) by the enzyme PEP carboxykinase (PEP-CK) (Figs. 3-5 and 3-8).

Thus, the overall conversion of a mole of glucose to 2 moles of pyruvate requires 2 moles of ATP for the initial phosphorylations and a total of 4 moles of ATP are generated in the subsequent dephosphorylations. This net gain of 2 moles of ATP represents the useful energy of anaerobic glycolysis.

For repeated function of the glycolytic pathway, a supply of NAD⁺ must be available for use in the oxidative (GA-3-PD) step. Normally in the presence of molecular O₂ (i.e., aerobic glycolysis), reduced NADH is reoxidized via the cytochrome system:

This alternate route of G-6-P oxidation has been variously referred to as the pentose phosphate pathway (PPP), direct oxidative pathway, Warburg-Dickens scheme, the hexose monophosphate pathway (HMP), or the hexose monophosphate shunt. The initial step of the shunt pathway involves the oxidation of G-6-P at the C-1 position to form 6-phosphogluconate (6-PG) as summarized in Figure 3-6. The reaction is catalyzed by glucose-6-phosphate dehydrogenase (G-6-PD) and in this pathway, oxidized nicotinamide adenine dinucleotide phosphate (NADP⁺) serves as the hydrogen acceptor. In the second oxidative step, 6-P-G is oxidatively decarboxylated by 6-phosphogluconate dehydrogenase (6-P-GD) to yield a pentose phosphate, ribulose-5-phosphate (Rib-5-P), again in the presence of NADP⁺. Thus, in the initial reactions, which are essentially irreversible, 2 moles of NADPH are formed. In this pathway, only the C-1 carbon atom of the glucose molecule is evolved as CO₂. By contrast, glucose catabolism via the glycolytic scheme results in the loss of both the C-1 and C-6 carbon atoms as CO₂ when pyruvate is oxidatively decarboxylated to form acetyl-CoA. This difference in CO₂ evolution is used to study partitioning of glucose metabolism through the glycolytic (EMP) pathway and the HMP shunt pathway in domestic animals. The subsequent metabolism of the Rib-5-P in the HMP shunt is also shown in Figure 3-6. As a result of the series of transformations, F-6-P and GA-3-P are formed, which serve as recycling links into the glycolytic pathway.

For continued functioning of the HMP shunt pathway, a supply of NADP⁺ must be available to act as the hydrogen acceptor. Oxidized NADP⁺ is regenerated from NADPH via the cytochrome system in the presence of O₂ so the HMP pathway is an aerobic pathway of glucose oxidation. Reduced NADPH is also required as a hydrogen donor in the synthesis of fatty acids. Through generation of NADPH, the HMP shunt route of carbohydrate metabolism is linked to that of fat synthesis. Accordingly, glucose oxidation through the HMP shunt pathway is essential for the synthesis of fat. In general, the HMP pathway is the major source of the NADPH, which maintains the reductive environment for all biosynthetic processes using NADPH as a cofactor.

This is an alternate pathway of G-6-P oxidation, which has been named the uronate pathway, the glucuronate pathway, or the C₆ oxidative pathway. This pathway is shown in Figure 3-7. The initial steps of this pathway involve the formation of uridine diphosphoglucose (UDPG), which, as noted earlier, is an intermediate in glycogen synthesis. G-6-P is first converted to G-1-P, which then reacts with uridine triphosphate (UTP) to form UDPG. This product is then oxidized at the C₆ position of the glucose moiety in contrast to the C₁ position, which is oxidized in the HMP shunt pathway. This reaction requires NAD⁺ as a cofactor and the products of the reaction are uridine diphosphoglucuronic acid (UDPGA) and NADH. This UDPGA is involved in a large number of important conjugation reactions in animals (e.g., bilirubin glucuronide formation, synthesis of mucopolysaccharides [chondroitin sulfate], which contain glucuronic acid, and generally in detoxification reactions). UDPGA is cleaved to release D-glucuronate and UDP.

D-glucuronate is next reduced to L-gulonate in a reaction catalyzed by the enzyme gulonate dehydrogenase (GUD), with NADPH as the hydrogen donor. The L-gulonate may be converted to a pentose, L-xylulose, or to vitamin C. When converted to L-xylulose, the C-6 carbon of L-gulonate is oxidatively decarboxylated and evolved as CO₂. The L-xylulose is then reduced to xylitol, catalyzed by the enzyme L-xylulose reductase. This is the enzyme that is deficient in pentosuria of humans. As shown in Figure 3-7, xylitol is converted to D-xylulose, which is then phosphorylated to D-xylulose-5-P and a cyclical pathway involving the HMP shunt pathway may occur. L-gulonate is also converted by enzyme-catalyzed reactions to L-ascorbate in those species that can synthesize their own vitamin C (i.e., all domestic animals). The enzyme, L-gulonolactone oxidase (GLO), is lacking in humans, nonhuman primates, and guinea pigs, and therefore vitamin C must be supplied in their diets. The enzyme is present only in the liver of the mouse, rat, pig, cow, and dog. In the dog, the liver GLO activity is low and the ascorbate hydrolytic activity is high so dogs may have additional needs for vitamin C during stress (e.g., wound healing, postsurgical stress). For vitamin C synthesis, D-galactose may be an even better precursor than D-glucose. This pathway is also included in Figure 3-7.

2. Terminal Oxidation: Aerobic Glycolysis

The metabolic pathways described thus far are those of the carbohydrates. In analogous fashion, the breakdown of fats and of proteins also follows independent pathways leading to the formation of organic acids. Among the organic acids formed from lipids are acetyl-CoA (AcCoA), acetoacetate (AcAc), and 3-OH-butyrate (3-OH-B) from the β-oxidation of fatty acids. From proteins, pyruvate, oxaloacetate (OAA), and α-ketoglutarate (α-KG) are formed from transamination of their corresponding α-amino acids. Direct deamination of amino acids is also a route of formation of organic acids. These organic acid intermediate metabolites are indistinguishable in their subsequent interconversions. Thus, the breakdown of the three major dietary constituents converges into a final common pathway, which also serves as a pathway for the interconversions between them.

a. Pyruvate Metabolism

The pathway for breakdown of glucose to pyruvate has been described in Section IV.D.1. Pyruvate, if it is not reduced to lactate, is oxidatively decarboxylated in a complex enzymatic system requiring the presence of lipoic acid, thiamine pyrophosphate (TPP), coenzyme A (CoA), NAD⁺, and pyruvate dehydrogenase (PD) to form AcCoA and NADH. Pyruvate may follow a number of pathways as outlined in Figure 3-8. The conversion of pyruvate to lactate was described in Section IV.D.1. By the mechanism of transamination or amino group transfer, pyruvate may be reversibly converted to alanine. The general reaction for an amino group transfer is

The energetics of the reaction from phosphoenolpyruvate (PEP) to form pyruvate and catalyzed by pyruvate kinase (PK) are such that this is an irreversible reaction, as is the PD catalyzed conversion of pyruvate to AcCoA. A two-step separate pathway to reverse this process is present at this step so this is a fourth site of directional metabolic control. Through a CO₂ fixation reaction in the presence of NADP⁺-linked malate dehydrogenase (MD), malate is formed from pyruvate. Malate is then oxidized to OAA in the presence of NAD⁺-linked MD. OAA may also be formed directly from pyruvate by the reaction catalyzed by pyruvate carboxylase (PC). OAA formed by either route may then be phosphorylated and decarboxylated to form PEP in a reaction catalyzed by PEP carboxykinase (PEP-CK). Thus, a pathway in the reverse direction of the PK reaction is present for gluconeogenesis from lower intermediates. These pathways for pyruvate metabolism are outlined in Figure 3-8, which includes the dicarboxylic acid cycle.

AcCoA formed by the oxidative decarboxylation of pyruvate also has a number of metabolic routes available. AcCoA occupies a central position in synthetic and in oxidative pathways as shown in Figure 3-8. The oxidative pathway leading to the breakdown of AcCoA to CO₂ and H₂O follows a cyclical pathway that is the tricarboxylic acid (TCA) cycle, citric acid cycle, or the Kreb’s cycle. The major steps involved are given in Figure 3-9. In a single turn of the cycle, a mole of AcCoA enters, 2 moles of CO₂ are evolved, and a mole of OAA is regenerated. The regenerated OAA may then condense with another mole of AcCoA, and the cycle continues. Citric acid is a symmetrical molecule that behaves asymmetrically as shown in Figure 3-9. Also, the CO₂ that is evolved is derived from that portion of the molecule contributed by OAA during each turn of the cycle. The expected distribution of carbon atoms from AcCoA in one turn of the cycle is also given in Figure 3-9. During one turn of the cycle, a randomization of carbon atoms occurs at the succinate level such that CO₂ derived from the carboxyl group of acetate will be evolved during the next turn of the cycle.

In the process, 3 moles of NAD⁺ and a mole of a flavin nucleotide (FAD) are reduced, and a mole of ATP is generated as noted in Figure 3-9. In animal tissues, there is a cytoplasmic NADP⁺-linked isocitric dehydrogenase (ICD), which is not associated with the mitochondrial NAD⁺-linked ICD or other enzymes of the TCA cycle. The NADP⁺-ICD is another enzyme used as an aid to diagnose liver disease.

3. Carbon Dioxide Fixation in Animals

According to Figure 3-9, the TCA cycle is a repetitive process based on the regeneration of OAA at each turn. In addition, other metabolic pathways are available for intermediates in the cycle. Reversal of the transamination reactions previously described to form aspartate or glutamate would result in a withdrawal of OAA and α-KG, respectively, from the cycle. By decarboxylation, OAA may also be withdrawn to form PEP, and malate may form pyruvate and thence other glycolytic intermediates as shown in Figure 3-8. Continued losses of these intermediates into other metabolic pathways would theoretically result in a decrease in the rate of operation of the cycle. A number of metabolic pathways are known whereby the losses of cycle intermediates may be balanced by replacement from other sources and are shown in Figure 3-8. The amino acids, aspartate and glutamate, may function as sources of supply as well as routes for withdrawal. The CO₂ fixation reactions, which are the reversal of the reactions previously described,

may also function as important sources of supply. A fourth CO2-fixing reaction

is especially important in ruminants because propionate is a major product of rumen fermentation and is a major supplier of intermediates for the TCA cycle. Propionate is one of the three major fatty acids, with acetate and butyrate, involved in ruminant metabolism.

4. Energy Relationships in Carbohydrate Metabolism

The energy of carbohydrate breakdown must be converted to high-energy phosphate compounds to be useful to the organism; otherwise the energy is dissipated as heat. The total available chemical energy in the reaction

is about 50 kcal/mole or about 7% of the 690 kcal/mole, which is available from the complete oxidation of glucose to CO₂ and water. The useful energy of anaerobic glycolysis is represented by the net gain of 2 moles of ATP and the available energy of each is about 7 kcal. Thus, the efficiency of glycolytic breakdown of glucose to pyruvate is 14 kcal or 28% of the available 50 kcal or only 2% of the total available 690 kcal in glucose.

The major portion of the energy of glucose is generated in the further aerobic oxidation of pyruvate to CO₂ and H₂O. In the oxidative or dehydrogenation steps, NADH or NADPH (FAD in the succinate step) is formed. In the presence of molecular O₂, these compounds are reoxidized to NAD⁺ or NADP⁺ in the cytochrome system. In the sequence of reactions of this system, 3 moles of ATP are formed per mole of NADH or NADPH oxidized to NAD⁺ or NADP⁺. This transfer of energy to ATP is known as oxidative phosphorylation, or ox-phos. The yield of high-energy phosphate bonds in the form of ATP in the system per atom of oxygen consumed (½ O₂) is conventionally referred to as the P:O ratio, which in this case is 3.

Table 3-3 presents a balance sheet of the ATPs formed in the various steps, and 36 of the total 38 ATPs are generated in aerobic glycolysis. The complete oxidation of a mole of glucose to CO₂ and water yields 690 kcal, and therefore the net gain of 38 ATPs in anaerobic plus aerobic glycolysis represents 266 kcal for an overall efficiency of 38%. In comparison, the efficiency of the modern internal combustion engine is about 20%.

V. INTERRELATIONSHIPS OF CARBOHYDRATE, LIPID, AND PROTEIN METABOLISM

The pathways by which the breakdown products of lipids and proteins enter the common metabolic pathway have been described in previous sections. The principal points at which carbohydrate carbon may be interconverted between amino acids and fatty acids are outlined in Figure 3-10. Thus, certain amino acids (glycogenic) can serve as precursors of carbohydrate through the transamination reactions, and by reversal of these transaminations, carbohydrates can serve as precursors of amino acids.

AcCoA is also the precursor of cholesterol and the ketone bodies: AcAc, 3-OH-B, and acetone. The synthesis of cholesterol proceeds through a series of reactions beginning with the stepwise condensation of 3 moles of AcCoA to form β-hydroxy-β-methyl glutaryl-CoA (HMG-CoA). As shown in Figure 3-10, HMG-CoA is a common intermediate for the synthesis of cholesterol and ketone bodies in the liver cell. In liver, a deacylating enzyme is present, which cleaves HMG-CoA to yield AcCoA and free AcAc. This is the HMG-CoA cycle. The free AcAc then diffuses out of the cell and enters the general circulation. For further oxidations to occur, AcAc is “reactivated” with CoA in extrahepatic tissues (muscle) by the transfer of CoA from succinyl-CoA to form AcAcCoA. Increased ketogenesis and ketonemia are the net result of alterations in metabolic pathways or enzymes that favor the accumulation of AcAcCoA. Prime examples are diabetes mellitus and bovine ketosis.

The increased mobilization and utilization of fatty acids are a well-known requisite for ketogenesis under conditions of starvation and diabetes. Under these same conditions, lipid synthesis from AcCoA is also depressed. The net effect of either or both of these alterations favors the accumulation of AcCoA and thus ketogenesis.

Increased ketogenesis is always associated with an increased rate of gluconeogenesis in association with an increased activity of the key gluconeogenic enzyme, PEP-carboxykinase (PEP-CK). The increased rate of gluconeogenesis in turn depletes OAA. There is an increase in the NADH/NAD ratio, which would promote the conversion of OAA to malate, thereby depleting OAA. With the depletion of OAA and subsequent OAA deficiency, there is an insufficient condensing partner for AcCoA for the Kreb’s cycle. The AcCoA is then readily diverted to ketone bodies.

Hepatic ketogenesis is regulated by the rate limiting transfer of FFA across the mitochondrial membrane. Carnitine acyl transferase, the enzyme system responsible for the mitochondrial uptake of FFA, is increased in diabetes and contributes to the ketogenesis.

B. Influence of Glucose Oxidation on Lipid Metabolism

In addition to the separation of the biochemical pathways for lipid oxidation and lipid synthesis, an anatomical separation of lipid metabolism is also present. The liver is the major site of fatty acid oxidation and the adipose tissue is the major site of lipid synthesis. Adipose tissue, in vitro, converts glucose carbons to fatty acids faster than does liver tissue.

It is well known that, with excessive carbohydrate feeding, fat depots in the body increase. Fasting, on the other hand, depresses the respiratory quotient (R.Q.) indicating that the animal is now using body fat as the energy source. During fasting, plasma FFA also increase, and when carbohydrate is supplied, they decrease. The presence of glucoseboth stimulates lipogenesis and spares fatty acid from oxidation. In diseases with an inability to utilize glucose (e.g., diabetes), depression of lipogenesis is a characteristic finding. When there is adequate glucose oxidation (e.g., successful insulin therapy in diabetes), lipid synthesis is restored and the animal regains its weight.

In those conditions with decreased glucose use or availability (e.g., diabetes, starvation, ruminant ketosis), there is an increased release of glucose precursors (amino acids) from muscle and FFA from adipose tissues mediated by activated hormone-sensitive lipases (HSL) (Khoo et al., 1973). The amino acids and FFA are transported to the liver where the amino acids follow gluconeogenic pathways. Fatty acids follow pathways toward oxidation and ketogenesis and, additionally, glucagon promotes hepatic ketogenesis. There is also an underutilization of ketones in the peripheral tissues of dogs (Balasse and Havel, 1971; Foster and McGarry, 1982). The net result is an overproduction of glucose and ketones in liver and an underutilization of both in the peripheral tissues.

VI. INSULIN AND CARBOHYDRATE METABOLISM

The internal secretions of the anterior pituitary, adrenal cortex and medulla, and the pancreas are closely associated with carbohydrate metabolism. The pituitary and adrenal factors were discussed in Section IV.C, together with glucagon. More detailed information is available in the chapters on pituitary and adrenal function. Since the successful extraction of insulin by Banting and Best in 1921, a vast amount of literature has accumulated on its role in carbohydrate metabolism and continues to this day. The fine details of insulin action are still being studied, and a basic understanding of the major biochemical events that occur in animals with and without insulin has evolved.

A. Proinsulin and Insulin

The elucidation of the insulin structure by Sanger in 1959 was soon followed by the discovery of its precursor, proinsulin, and its structure was quickly known. It has been the subject of many reviews (Raptis and Dimitriadis, 1985; Steiner, 2004). Proinsulin is a single-chain looped polypeptide linked by disulfide bridges (Fig. 3-11). It varies in length from 78 amino acid residues in the dog to 86 for the human, horse, and rat. Its m.w. is near 9000 daltons. Proinsulin is synthesized in the pancreatic β-cells on the rough endoplasmic reticulum (RER) and transported and stored in the secretory granules on the Golgi apparatus. There, the central connecting polypeptide or C-peptide is cleaved from the chain by proteolytic enzymes, and the two linked end fragments are the monomeric insulin molecule. C-peptide has an m.w. of 3600 daltons and is devoid of biological activity.

Insulin and C-peptide are released into the circulation in equimolar amounts but C-peptide’s circulatory concentration is higher than that of insulin because of its slower clearance half-time of 20 min as compared to 5 to 10 min for insulin. C-peptide is primarily degraded by the kidney and a portion is excreted in the urine (Duckworth and Kitabchi, 1981).

The A chain of insulin consists of 21 amino acids and the B chain of 30 amino acid residues (Fig. 3-11). The m.w. of the insulin monomer is 6000 daltons and is the smallest unit possessing biological activity. Under physiological conditions, 4 molecules of insulin are linked together to form a tetramer, the active molecule. Insulin obtained from various species differs in amino acid composition in Chain A or Chain B or both (Table 3-4). Differences occur within species also as rats and mice (Markussen, 1971) have two nonallelic insulins. These structural differences among the various species of animals are not located at critical sites, however, because they do not affect their biological activity. They do, however, affect their immunological behavior.

The amount of insulin stored in the pancreata of various species also differs. The dog stores about 3.3 units per gram of pancreas, which amounts to about 75 I.U. in a 10-kg dog. This amount, if suddenly released, would be fatal.

Insulin release is affected by glucose, mannose, leucine, other amino acids, ketone bodies, and fatty acids. This release is mediated by glucagon, a hormone, which increases cAMP and potentiates the insulin response. The sulfonylureas are effective as pharmacological agents to release insulin, the basis for their therapeutic use.

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