Textbook - BIOLOGICAL CHEMISTRY - Gubsky Yu.I. - 2000

Chapter III. METABOLISM OF MAJOR CLASSES OF BIOMOLECULES

CHAPTER 12. CARBOHYDRATE METABOLISM. II. ALTERNATIVE PATHWAYS OF MONOSACCHARIDE METABOLISM. REGULATION OF GLUCOSE METABOLISM

12.3. GLUCOSE BIOSYNTHESIS AND ITS REGULATION

The primary source of glucose as metabolic fuel for The Human Body is dietary intake in the form of the polysaccharide starch and sucrose. Excess glucose that is not utilized in the oxidative reactions of aerobic and anaerobic Glycolysis is stored in energy depots as Glycogen and triacylglycerols. However, metabolic pathways exist that supply the body with glucose through its synthesis from non-carbohydrate Biomolecules.

Gluconeogenesis (glyconeogenesis) is the Synthesis of glucose from non-carbohydrate metabolic precursors, which include: Pyruvate (and lactate); Certain Amino Acids (glucogenic amino acids); and a specific amount of glucose can be generated from glycerol and the catabolic products of Fatty acids with an odd number of carbon atoms in their hydrocarbon chain.

Class="center">Physiological Significance of Gluconeogenesis

Gluconeogenesis reactions occur predominantly in The Liver and, to some extent, in the renal cortex, as the Cells of these specific Organs contain the complete set of required Enzymes.

Up to 80 g of glucose is synthesized in the adult human body per day. Glucose Biosynthesis ensures its normal concentration under conditions of reduced monosaccharide intake from the environment and the depletion of the primary stored glucose source—liver and Muscle glycogen. Such a physiological state is observed a few hours after a meal (the postabsorptive state, occurring in the morning on an empty Stomach), during prolonged fasting, and following exhaustive physical exertion. The human Brain is particularly sensitive to a decrease in intracellular glucose concentration, relying on glucose as the primary substrate for METABOLISM/26.html">Energy Metabolism at a rate of about 120 g per day.

Metabolic Pathway of Gluconeogenesis

The metabolic pathway of gluconeogenesis is largely the reverse of glycolysis (which converts glucose into pyruvate and lactate), with the exception of three thermodynamic irreversible glycolytic "kinase" reactions that require bypass (shunt) mechanisms.

The irreversible reactions of glycolysis are:

1) the hexokinase (or glucokinase) reaction:

2) the Phosphofructokinase reaction:

3) the pyruvate kinase reaction:

Given the irreversibility of these reactions, The conversion of pyruvate (or lactate) into glucose requires additional enzymatic reactions that are specific to gluconeogenesis. These reactions are:

- conversion of glucose-6-phosphate to glucose;

- conversion of fructose-1,6-bisphosphate to fructose-6-phosphate;

- conversion of pyruvate to phosphoenolpyruvate.

Reactions and Enzymes of Gluconeogenesis

1. Conversion of pyruvate to phosphoenolpyruvate.

The reaction proceeds in two consecutive stages:

1). Conversion of pyruvate into oxaloacetate catalyzed by the enzyme pyruvate carboxylase:

Pyruvate carboxylase is localized within the Cell/35.html">Mitochondria. The prosthetic group of the enzyme is carboxybiotin, the coenzyme form of vitamin H, which acts as a CO₂ carrier. The macroergic bond of ATP is expended on the carboxylation of the enzyme-bound biotin (see Chapter 15, Section 15.1).

2). Conversion of oxaloacetate into phosphoenolpyruvate (PEP) catalyzed by the enzyme phosphoenolpyruvate carboxykinase (PEP carboxykinase):

In the cells of most animals, PEP carboxykinase is localized in the Cytosol, whereas in humans it is found in the cytosol and, partially, in the mitochondria.

Compartmentation of the Conversion of Pyruvate to PEP

The pyruvate carboxylase reaction is one of the anaplerotic reactions of The Tricarboxylic Acid Cycle, which help maintain high concentrations of certain TCA cycle metabolites—in this case, oxaloacetate (see Chapter 10, Section 10.4). Because the reaction takes place inside the mitochondria, oxaloacetate must be transported across the mitochondrial membranes into the cytosol to participate in gluconeogenesis (driven by cytosolic PEP carboxykinase and other gluconeogenic enzymes).

The transport of oxaloacetate into the cytosol is mediated by one of several shuttle systems. The underlying principle of these shuttles is the conversion of mitochondrial oxaloacetate—to which The inner mitochondrial membrane is impermeable—into an intermediate that can diffuse into the cytosol and be cleaved back into oxaloacetate. Shuttle mechanisms are utilized to export oxaloacetate and acetyl-CoA from the mitochondria into the cytosolic space, and to import glycolytic NADH from the cytosol into the mitochondria. The shuttle systems for oxaloacetate transport include:

1) The malate shuttle system, which involves: the reduction of oxaloacetate to malate catalyzed by the mitochondrial isoenzyme of malate dehydrogenase:

the efflux of malate across the mitochondrial membranes into the cytosol, followed by The oxidation of malate back to oxaloacetate by the cytosolic isoenzyme of malate dehydrogenase:

2) The aspartate shuttle system, which Functions through the intra-mitochondrial conversion of oxaloacetate into aspartate:

followed by the export of aspartate into the cytosol and its reconversion back into oxaloacetate; this mechanism is driven by the sequential action of mitochondrial and cytosolic Isoenzymes of aspartate aminotransferase.

3) The citrate shuttle system, which operates via the intra-mitochondrial synthesis of citrate from oxaloacetate (catalyzed by citrate synthase):

followed by the efflux of citrate into the cytosol and its Cleavage to release oxaloacetate once again, catalyzed by citrate lyase:

The primary mechanism for exporting oxaloacetate to the cytosol to supply cytosolic PEP for subsequent gluconeogenesis reactions is the malate shuttle, the principles of which are illustrated in Scheme 12.5:

Fig. 12.5. Schematic diagram of the malate shuttle.

2. Conversion of fructose-1,6-diphosphate to fructose-6-phosphate:

The reaction is catalyzed by the regulatory enzyme fructose-1,6-diphosphatase (Fru-1,6-Pase), found predominantly in the liver, as well as in the Kidneys and intestinal epithelial cells. The fructose-1,6-diphosphatase reaction bypasses the irreversible phosphofructokinase step of glycolysis.

3. Conversion of glucose-6-phosphate to glucose:

This reaction bypasses the irreversible hexokinase (glucokinase) step of glycolysis and is catalyzed by glucose-6-phosphatase (G-6-Pase), which is most abundant in the membranes of The Endoplasmic reticulum of hepatocytes. Due to the high concentration of glucose-6-phosphatase in the liver, this organ is primarily responsible for releasing free glucose into the bloodstream via the Hydrolysis of glucose-6-phosphate derived from gluconeogenesis or the phosphorolytic breakdown of glycogen. This mechanism plays a crucial role in maintaining normal glycemia, especially during fasting.

Overall Reaction and Energetics of Gluconeogenesis

The remaining reactions required to convert non-carbohydrate substrates into glucose are standard glycolytic reactions that can proceed physiologically in both forward and reverse directions. The overall reaction of gluconeogenesis (accounting for the expenditure of 2 ATP and 2 NADH molecules for the reverse conversion of 2 molecules of 3-phosphoglyceric acid into 2 molecules of 3-phosphoglyceraldehyde) is expressed as:

The equation above demonstrates that gluconeogenesis is a strongly endergonic metabolic pathway: the synthesis of a single glucose molecule from two pyruvate molecules requires the consumption of six high-energy bonds. Consequently, like glycolysis, gluconeogenesis is an irreversible biochemical process.

The General Overview of gluconeogenesis is presented in Fig. 12.6. Solid arrows indicate the reactions of gluconeogenesis, while dashed arrows represent the reactions of glycolysis and the TCA cycle. The diagram also shows the entry points of major glucogenic amino acids into gluconeogenesis (see below).

Fig. 12.6. Metabolic map of gluconeogenesis.

Abbreviations: G-6-P — glucose-6-phosphate; Fru-6-P — fructose-6-phosphate; Fru-1,6-P2 — fructose-1,6-diphosphate; G-3-P — glyceraldehyde-3-phosphate; 3-PGA — 3-phosphoglyceric acid; 2-PGA — 2-phosphoglyceric acid; PEP — phosphoenolpyruvate.

Substrates for Gluconeogenesis

The primary precursors (substrates) for gluconeogenesis are pyruvate (lactate) and amino acids (predominantly Alanine), which are generated mainly in active skeletal Muscles, erythrocytes, and certain other tissue cells.

1. Formation of Pyruvate from Lactate in the Liver

Lactate, produced from pyruvate during anaerobic glycolysis, is a metabolic end product; its re-entry into metabolism (via oxidation or utilization in glucose synthesis, i.e., gluconeogenesis) is only possible through re-oxidation to pyruvate in the Lactate dehydrogenase reaction:

This reaction is catalyzed by lactate dehydrogenase (discussed previously in Chapter 11) and proceeds in the reverse direction relative to the lactate dehydrogenase reaction that produces lactate from pyruvate during anaerobic glycolysis.

The directional drive of the LDH reaction toward pyruvate formation occurs specifically in gluconeogenic Tissues (liver, kidneys), where it is governed by the prevailing NAD⁺/NADH and pyruvate/lactate ratios, as well as the presence of specific LDH isoenzymes. The source of lactate for hepatic gluconeogenesis is the lactate delivered via Blood Plasma from its Sites of production—primarily skeletal muscles.

Glucose-Lactate Cycle

Thus, a cyclic process is established—the glucose-lactate cycle, or Cori cycle—which links lactate production in Muscle tissue cells during anaerobic glycolysis, its release into the blood through the Plasma Membranes of muscle cells, and the utilization of lactate (following oxidation to pyruvate) in hepatocytes for gluconeogenesis (Fig. 12.7). A fraction of the pyruvate (not shown in the diagram) is oxidized via the pyruvate dehydrogenase reaction to acetyl-CoA.

Fig. 12.7. Glucose-lactate cycle (Cori cycle).

In skeletal muscles, the LDH reaction proceeds predominantly toward The formation of lactate from pyruvate (Chapter 11). By reducing pyruvate to lactate and subsequently utilizing it for gluconeogenesis in the liver (the Cori cycle), skeletal muscles not only rid themselves of "excess" lactic acid—which is produced in particularly large amounts during intense physical activity—but also maintain the high NAD⁺/NADH ratio required for glycolysis to function actively (utilizing NAD⁺ in the glycolytic oxidoreduction process).

2. Glucogenic Amino Acids

The direct precursors of glucose during its synthesis (and essentially the metabolites of gluconeogenesis) are pyruvate, oxaloacetate, and phosphoenolpyruvate. Amino acids that are converted into these non-nitrogenous compounds As a result of losing an amino group (via deamination or Transamination reactions) can thus be regarded as substrates for gluconeogenesis; such Amino acids are termed glucogenic amino acids (whereas amino acids whose hydrocarbon Skeleton is converted into acetoacetate or acetyl-CoA, from which glucose synthesis via gluconeogenesis is impossible, are called ketogenic amino acids).

Specific glucogenic Amino Acids and their points of entry into the gluconeogenic metabolic pathway are shown in Fig. 12.6.

Gluconeogenesis involving amino acids is most active during complete starvation, when the energy processes of the body—specifically maintaining normal blood and brain glucose concentrations—are sustained through the Catabolism of endogenous tissue Proteins.

Glucose-Alanine Cycle

An important substrate for hepatic gluconeogenesis is alanine, which can be formed in skeletal muscles via the reverse transamination reaction between pyruvate and glutamate:

Upon release from working muscles into the bloodstream, alanine is taken up by hepatocytes and (after being converted into pyruvate) is utilized in gluconeogenesis (the glucose-alanine cycle):

Regulation of Gluconeogenesis

The rate of gluconeogenesis is controlled through metabolite (allosteric) regulation, as well as Hormonal Regulation of the activity and Synthesis of specific gluconeogenic enzymes.

1. Metabolite Regulation of Gluconeogenesis — is achieved at the level of the following regulatory enzymes:

pyruvate carboxylase — an enzyme whose positive modulator is acetyl-CoA; in the absence of acetyl-CoA, pyruvate carboxylase is practically inactive;

fructose-1,6-bisphosphatase — an enzyme whose activity depends on the concentration ratio between the positive modulator ATP and the negative modulator AMP, which acts as an inhibitor of fructose-1,6-bisphosphatase activity.

Thus, by regulating the catalytic activity levels of these allosteric enzymes, the overall rate of glucose synthesis is controlled in accordance with The Cell's metabolic status:

- gluconeogenesis is activated when intracellular glucose concentration decreases, as indicated by the accumulation of acetyl-CoA (a product of aerobic glycolysis), and under conditions of an adequate supply of chemical energy (in the form of ATP);

- gluconeogenesis is inhibited when the concentration of acetyl-CoA decreases (reflecting a reduced rate of glucose breakdown) and when energy supply is insufficient (an increased AMP/ATP ratio).

2. Hormonal Regulation of Gluconeogenesis — is carried out by Glucagon, adrenaline (epinephrine), glucocorticoid Hormones of the adrenal cortex, and Insulin.

2.1. Glucagon, adrenaline, and glucocorticoids increase the synthesis rates of the bypass enzymes of gluconeogenesis in hepatocytes — PEP carboxykinase, Fru-1,6-bisphosphatase, and G-6-Pase.

2.2. Insulin suppresses the synthesis of these gluconeogenic enzymes, thereby inhibiting the gluconeogenic process.