14.4 Gluconeogenesis in Chapter 14 Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

14.4 Gluconeogenesis

The central role of glucose in metabolism arose early in evolution, and this sugar remains the nearly universal fuel and building block in modern organisms, from microbes to humans. In mammals, some tissues depend almost completely on glucose for their metabolic energy. For the human brain and nervous system, as well as the erythrocytes, testes, renal medulla, and embryonic tissues, glucose from the blood is the sole or major fuel source. The brain alone requires about 120 g of glucose each day — more than half of all the glucose stored as glycogen in muscle and liver. However, the supply of glucose from these stores is not always sufficient; between meals and during longer fasts, or after vigorous exercise, glycogen is depleted. For these times, organisms need a method for synthesizing glucose from noncarbohydrate precursors. This is accomplished by a pathway called gluconeogenesis (“new formation of sugar”), which converts pyruvate and related three- and four-carbon compounds to glucose.

Gluconeogenesis occurs in all animals, plants, fungi, and microorganisms. The reactions are essentially the same in all tissues and all species. The important precursors of glucose in animals are three-carbon compounds such as lactate, pyruvate, and glycerol, as well as certain amino acids (Fig. 14-15). In mammals, gluconeogenesis takes place mainly in the liver, and to a lesser extent in the renal cortex and in the epithelial cells that line the small intestine. The glucose produced passes into the blood to supply other tissues. After vigorous exercise, lactate produced by anaerobic glycolysis in skeletal muscle returns to the liver and is converted to glucose, which moves back to muscle and is converted to glycogen — a circuit called the Cori cycle (see Fig. 23-17). In plant seedlings, stored fats and proteins are converted, via paths that include gluconeogenesis, to the disaccharide sucrose for transport throughout the developing plant. Glucose and its derivatives are precursors for the synthesis of plant cell walls, nucleotides and coenzymes, and a variety of other essential metabolites. In many microorganisms, gluconeogenesis starts from simple organic compounds of two or three carbons, such as acetate, lactate, and propionate, in their growth medium.

A figure shows how carbohydrates can be synthesized from a variety of simple precursors in animals and plants. — FIGURE 14-15 Carbohydrate synthesis from simple precursors. The pathway from phosphoenolpyruvate to glucose 6-phosphate is common to the biosynthetic conversion of many different precursors of carbohydrates in animals and plants. The path from pyruvate to phosphoenolpyruvate leads through oxaloacetate, an intermediate of the citric acid cycle, which we discuss in Chapter 16. Any compound that can be converted to either pyruvate or oxaloacetate can therefore serve as starting material for gluconeogenesis. This includes alanine and aspartate, which are convertible to pyruvate and oxaloacetate, respectively, and other amino acids that can also yield three- or four-carbon fragments, the so-called glucogenic amino acids. Plants and photosynthetic bacteria are uniquely able to convert ${CO}_{2}$ $CO Subscript 2$ to carbohydrates, using the Calvin cycle, as we shall see in Section 20.4.

FIGURE 14-15 Carbohydrate synthesis from simple precursors. The pathway from phosphoenolpyruvate to glucose 6-phosphate is common to the biosynthetic conversion of many different precursors of carbohydrates in animals and plants. The path from pyruvate to phosphoenolpyruvate leads through oxaloacetate, an intermediate of the citric acid cycle, which we discuss in Chapter 16. Any compound that can be converted to either pyruvate or oxaloacetate can therefore serve as starting material for gluconeogenesis. This includes alanine and aspartate, which are convertible to pyruvate and oxaloacetate, respectively, and other amino acids that can also yield three- or four-carbon fragments, the so-called glucogenic amino acids. Plants and photosynthetic bacteria are uniquely able to convert ${CO}_{2}$ $CO Subscript 2$ to carbohydrates, using the Calvin cycle, as we shall see in Section 20.4.

The figure has three rectangular pieces: a left side labeled animals, a middle region representing animals and plants, and a right side labeled plants. At the bottom of the animal piece, lactate has an orange arrow up to pyruvate, from which an orange arrow bends to the right to point to the left side of the citric acid cycle just above the nine o’clock position in the region representing animals and plants. In the center piece for animals and plants, glucogenic amino acids has a short blue arrow that joins with the bottom of the citric acid cycle at the seven o’clock position. The citric acid cycle has a long blue arrow from just past the nine o’clock position to the two o’clock position, then a blue arrow from the 2 o’clock position to the seven o’clock position, then a blue arrow from the seven o’clock position to just past the nine o’clock position. A blue piece branches from the top arrow near the twelve o’clock position to indicate phosphoenolpyruvate, from which a long blue arrow points up to glucose 6-phosphate. At the bottom of the animals and plants piece, triacylglycerols has a blue arrow to glycerol, from which a long blue arrow extends up past the citric acid cycle and bends left to join the arrow from phosphoenolpyruvate to glucose 6-phosphate. At the bottom of the plant piece, C O 2 fixation has a blue arrow to 3-phosphoglycerate, from which a short green arrow points up to a longer green arrow that curves left to join a blue arrow up from phosphoenolpyruvate in the animal and plant piece below the arrow from glycerol. A short purple arrow labeled energy joins the arrow from phosphoenolpyruvate to glucose 6-phosphate above the place that the blue arrow from glycerol joined it. Glucose 6-phosphate has an arrow pointing up that forms a horizontal line that splits into seven pieces. The central pieces are blue and within the animal and plant piece of the illustration. From left to right, these blue arrows point to glycoproteins, disaccharides, and other monosaccharides. The horizontal line becomes orange to the left and forms two branches in the animal piece of the illustration. From left to right, these orange arrows point to blood glucose and glycogen. The horizontal line becomes green to the right and forms two branches in the plant piece of the illustration. From left to right, these green arrows point to starch and sucrose.

Although the reactions of gluconeogenesis are the same in all organisms, the metabolic context and the regulation of the pathway differ from one species to another and from tissue to tissue. In this section we focus on gluconeogenesis as it occurs in the mammalian liver. In Chapter 20 we show how photosynthetic organisms use this pathway to convert the primary products of photosynthesis into glucose, to be stored as sucrose or starch.

Gluconeogenesis and glycolysis are not identical pathways running in opposite directions, although they do share several steps (Fig. 14-16); 7 of the 10 enzymatic reactions of gluconeogenesis are the reverse of glycolytic reactions. However, three reactions of glycolysis are essentially irreversible in vivo and cannot be used in gluconeogenesis: the conversion of glucose to glucose 6-phosphate by hexokinase, the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate by phosphofructokinase-1, and the conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase. In cells, these three reactions are characterized by a large negative free-energy change, whereas other glycolytic reactions have a $Δ G$ $upper Delta upper G$ near 0 (Table 14-2). In gluconeogenesis, the three irreversible steps are bypassed by a separate set of enzymes, catalyzing reactions that are sufficiently exergonic to be effectively irreversible in the direction of glucose synthesis. Thus, both glycolysis and gluconeogenesis are irreversible processes in cells. In animals, both pathways occur largely in the cytosol, necessitating their reciprocal and coordinated regulation, described in Section 14.5.

A figure shows relationships between opposing pathways of glucose and gluconeogenesis in the liver. — FIGURE 14-16 Opposing pathways of glycolysis and gluconeogenesis in liver. The reactions of glycolysis are on the left side, in red; the opposing pathway of gluconeogenesis is on the right, in blue. The major sites of regulation of gluconeogenesis shown here are discussed in Section 14.5.

The figure has an orange heading labeled glycolysis above a downward arrow on the left and a blue heading labeled gluconeogenesis above a blue arrow on the right. Glucose is shown at the top center. A red arrow curves down on the left from glucose to glucose 6-phosphate accompanied by a red arrow showing that A T P is added and A D P is lost. Hexokinase catalyzes the reaction. On the right, a blue arrow points up from glucose 6-phosphate to glucose accompanied by a blue arrow showing that H 2 O is added and P subscript I is lost. Glucose 6-phosphatase catalyzes the reaction. Glucose 6-phosphate has a red arrow pointing down to fructose 6-phosphate and a blue arrow pointing up to show the reverse reaction. A red arrow curves down on the left from fructose 6-phosphate to fructose 1,6-bisphosphate accompanied by a red curved arrow showing that A T P is added and A D P is lost. Phosphofructokinase-1 catalyzes the reaction. On the right, a blue arrow points up from fructose 1,6-bisphosphate to fructose 6-phosphate accompanied by a blue arrow showing that H 2 O is added and P subscript i is lost. Fructose 1,6-bisphosphatase-1 catalyzes the reaction. A red arrow points down from fructose 1,6-bisphosphate on the left and splits to indicate dihydroxyacetone phosphate to the left and (2) glyceraldehyde 3-phosphate below. Diagonal arrows point to the upper left and lower right to show that dihydroxyacetone phosphate can be reversibly converted to glyceraldehyde 3-phosphate. On the right, a blue arrow points up from (2) glyceraldehyde 3-phosphate to fructose 1,6-bisphosphate and is joined by a line from dihydroxyacetone phosphate to the right. Diagonal arrows point to the upper right and lower left to show that dihydroxyacetone phosphate can be reversibly converted to glyceraldehyde 3-phosphate. A red arrow points down on the right from (2) glyceraldehyde 3-phosphate to (2) 1,3-bisphosphoglycerate. A red line shows the addition of 2 P subscript i and a curved arrow shows that 2 N A D plus are added and 2 N A D H plus 2 H plus are produced. On the right, a blue arrow points up from (2) 1,3-bisphosphoglycerate to (2) glyceraldehyde 3-phosphate. A curved arrow shows that 2 N A D H plus 2 H plus are added and that 2 N A D plus are produced and an arrow leaves to show that 2 P i are lost. A red arrow points down from (2) 1,3-bisphosphoglycerate on the left to (2) 3-phosphoglycerate on the left accompanied by a curved arrow showing the addition of 2 A D P and loss of 2 A T P. On the right, a blue arrow shows the reverse reaction accompanied by a curved arrow showing the addition of 2 A T P and loss of 2 A D P. (2) 3-phosphoglycerate has a red arrow pointing down to (2) 2-phosphoglycerate and a blue arrow pointing up on the right to show the reverse reaction. (2) 2-phosphoglycerate has a red arrow pointing down to (2) phosphoenolpyruvate on the left and a blue arrow on the right pointing up to show the reverse reaction. (2) phosphoenolpyruvate has a large arrow that curves left to form a half circle before curving back to end at a box labeled (2) pyruvate at the bottom center. This arrow is accompanied by a curved arrow showing the addition of 2 A D P and loss of 2 A T P and by pyruvate kinase. The half circle back from (2) pyruvate to (2) phosphoenolpyruvate on the right side contains two reactions. The first reaction has a blue arrow from (2) pyruvate to (2) oxaloacetate accompanied by a curved arrow to show the addition of 2 A T P and loss of 2 A D P. Pyruvate carboxylate catalyzes the reaction. The second reaction has a blue arrow from (2) oxaloacetate to (2) phosphoenolpyruvate with a curved arrow showing that 2 G T P is added and 2 G D P is lost. P E P carboxykinase catalyzes the reaction.

upper Delta bold-italic upper G Superscript prime Baseline degree left-parenthesis bold kJ slash mol right-parenthesis

upper Delta bold-italic upper G left-parenthesis bold kJ slash mol right-parenthesis

TABLE 14-2 Free-Energy Changes of Glycolytic Reactions in Erythrocytes
Glycolytic reaction step	$Δ G^{'} ° (kJ/mol)$ $upper Delta bold-italic upper G Superscript prime Baseline degree left-parenthesis bold kJ slash mol right-parenthesis$	$Δ G (kJ/mol)$ $upper Delta bold-italic upper G left-parenthesis bold kJ slash mol right-parenthesis$
$Glucose + ATP \to glucose 6 -phosphate + ADP$ $Glucose plus ATP right-arrow glucose 6 hyphen phosphate plus ADP$	$- 16.7$ $negative 16.7$	$- 33.4$ $negative 33.4$
$Glucose 6 hyphen phosphate right harpoon over left harpoon fructose 6 hyphen phosphate$ $Glucose 6 hyphen phosphate right harpoon over left harpoon fructose 6 hyphen phosphate$	1.7	0 to 25
$Fructose 6 -phosphate + ATP \to fructose 1, 6 -bisphosphate + ADP$ $Fructose 6 hyphen phosphate plus ATP right-arrow fructose 1 comma 6 hyphen bisphosphate plus ADP$	$- 14.2$ $negative 14.2$	$- 22.2$ $negative 22.2$
$\begin{matrix} Fructose 1, 6 -bisphosphate ⇌ \\ dihydroxyacetone phosphate + glyceraldehyde 3 -phosphate \end{matrix}$ $StartLayout 1st Row Fructose 1 comma 6 hyphen bisphosphate right harpoon over left harpoon 2nd Row dihydroxyacetone phosphate plus glyceraldehyde 3 hyphen phosphate EndLayout$	23.8	$- 6 to 0$ $negative 6 to 0$
$Dihydroxyacetone phosphate right harpoon over left harpoon glyceraldehyde 3 hyphen phosphate$ $Dihydroxyacetone phosphate right harpoon over left harpoon glyceraldehyde 3 hyphen phosphate$	7.5	0 to 4
$\begin{matrix} Glyceraldehyde 3 -phosphate + P_{i} + {NAD}^{+} ⇌ \\ 1, 3 -bisphosphoglycerate + NADH + H^{+} \end{matrix}$ $StartLayout 1st Row Glyceraldehyde 3 hyphen phosphate plus upper P Subscript i Baseline plus NAD Superscript plus Baseline right harpoon over left harpoon 2nd Row 1 comma 3 hyphen bisphosphoglycerate plus NADH plus upper H Superscript plus EndLayout$	6.3	$- 2 to 2$ $negative 2 to 2$
$1, 3 -Bisphosphoglycerate + ADP ⇌ 3 -phosphoglycerate + ATP$ $1 comma 3 hyphen Bisphosphoglycerate plus ADP right harpoon over left harpoon 3 hyphen phosphoglycerate plus ATP$	$- 18.8$ $negative 18.8$	0 to 2
$3 hyphen Phosphoglycerate right harpoon over left harpoon 2 hyphen phosphoglycerate$ $3 hyphen Phosphoglycerate right harpoon over left harpoon 2 hyphen phosphoglycerate$	4.4	0 to 0.8
$3 -Phosphoglycerate ⇌ phosphoenolpyruvate + H_{2} O$ $3 hyphen Phosphoglycerate right harpoon over left harpoon phosphoenolpyruvate plus upper H Subscript 2 Baseline upper O$	7.5	0 to 3.3
$Phosphoenolpyruvate + ADP \to pyruvate + ATP$ $Phosphoenolpyruvate plus ADP right-arrow pyruvate plus ATP$	$- 31.4$ $negative 31.4$	$- 16.7$ $negative 16.7$

We begin by considering the three bypass reactions of gluconeogenesis. (Keep in mind that “bypass” refers throughout to the bypass of irreversible glycolytic reactions.)

The First Bypass: Conversion of Pyruvate to Phosphoenolpyruvate Requires Two Exergonic Reactions

The first of the bypass reactions in gluconeogenesis is the conversion of pyruvate to phosphoenolpyruvate (PEP). This reaction cannot occur by simple reversal of the pyruvate kinase reaction of glycolysis (p. 521), which has a large, negative free-energy change and is therefore irreversible under the conditions prevailing in intact cells (Table 14-2, step ). Instead, the phosphorylation of pyruvate is achieved by a roundabout sequence of reactions that in eukaryotes requires enzymes in both the cytosol and mitochondria. As we shall see, the pathway shown in Figure 14-16 and described in detail here is one of two routes from pyruvate to PEP; it is the predominant path when pyruvate or alanine is the glucogenic precursor. A second pathway, described later, predominates when lactate is the glucogenic precursor.

Pyruvate is first transported from the cytosol into mitochondria or is generated from alanine within mitochondria by transamination, in which the α-amino group is transferred from alanine (leaving pyruvate) to an α-keto carboxylic acid (transamination reactions are discussed in detail in Chapter 18). Then pyruvate carboxylase, a mitochondrial enzyme that requires the coenzyme biotin, converts the pyruvate to oxaloacetate:

Pyruvate + {HCO}_{3}^{-} + ATP \to oxaloacetate + ADP + P_{i}

$Pyruvate plus HCO Subscript 3 Superscript minus Baseline plus ATP right-arrow oxaloacetate plus ADP plus upper P Subscript i Baseline$

(14-4)

The carboxylation reaction involves biotin as a carrier of activated bicarbonate, as shown in Figure 14-17; the reaction mechanism is shown in Figure 16-16. (Note that ${HCO}_{3}^{-}$ $HCO Subscript 3 Superscript minus$ is formed by ionization of carbonic acid formed from $CO Subscript 2 Baseline plus upper H Subscript 2 Baseline upper O$ $CO Subscript 2 Baseline plus upper H Subscript 2 Baseline upper O$ .) ${HCO}_{3}^{-}$ $HCO Subscript 3 Superscript minus$ is phosphorylated by ATP to form a mixed anhydride (a carboxyphosphate); then biotin displaces the phosphate in the formation of carboxybiotin.

A figure shows how biotin participates in the pyruvate carboxylase reaction in three steps. — FIGURE 14-17 Role of biotin in the pyruvate carboxylase reaction. The cofactor biotin is covalently attached to pyruvate carboxylase through an amide linkage to the ε-amino group of a Lys residue, forming a biotinyl-enzyme. The reaction takes place in two phases, which occur at two different sites in the enzyme. The long biotinyl-Lys arm carries the substrate from one site to the other.

Red highlighted H C O 3 minus has an arrow pointing right accompanied by a curved arrow showing that A T P is added and that A D P is lost and a second branching arrow showing that P i is lost. This yields a complex molecule above a curved surface of pyruvate carboxylase with crescent-shaped pieces on the left and right ends labeled as site 1 and site 2, respectively. Step 1: In site 1, bicarbonate is converted to C O 2 at the expense of A T P. Biotin picks up C O 2. The atoms from C O 2 are shown just above site 1 as red highlighted C bonded to red highlighted O minus to the upper left, double bonded to red highlighted O below. The red highlighted C is bonded to N above that forms the left side of a five-membered ring that shares its base with the top of a five-membered ring below. This five-membered ring has N substituted for C at the lower left vertex as previously described, C at the upper left vertex double bonded to O, N substituted for C at the adjacent upper right vertex and bonded to H, and the remaining two vertices forming a shared bond with the five-membered ring below. The second five-membered ring shares its upper left bond with the five-membered ring to its left, has S substituted for C at its lower right vertex, and has its upper right vertex bonded to a five-carbon zigzag chain with its terminal C double bonded to O and bonded to N H below that is bonded to L y s in the pyruvate carboxylase molecule. Step 2: Long biotinyl-L y s tether moves C O 2 from site 1 to site 2. A dashed arrow points from N H of the left-hand biotin molecule to N H on the left-side of a second biotin molecule, shown with the red highlighted C O 2 atoms positioned in site 2 and with the zigzag chain bonded to the second five-membered ring showing three carbons and then a break before the remaining part of the tail extending down to L y s. Step 3: Biotin delivers C O 2 to pyruvate in site 2, forming oxaloacetate. An arrow points right to show pyruvate as a blue highlighted molecule with a left-hand C double bonded to O to the upper left, bonded to O minus to the lower right, and bonded to a central C to the right bonded to O minus above and double bonded to C H 2 to the right. Red highlighted C O 2 is shown to the right. Curved red arrows point from the negative sign on O to the bond between the same O and the central C of pyruvate and from the double bond between the central and right-hand C atoms of pyruvate to the red highlighted C of C O 2. An arrow points right to show that this yields oxaloacetate, which is mostly highlighted in blue and has a left-hand C double bonded to O to the upper left, bonded to O minus to the lower left, and bonded to a central C to the right double bonded to O above and bonded to C H 2 to the right connected by a blue bond to red highlighted C double bonded to red highlighted O to the upper right and bonded to red highlighted O minus to the lower right.

Pyruvate carboxylase is the first regulatory enzyme in the gluconeogenic pathway, requiring acetyl-CoA as a positive effector. Acetyl-CoA is produced by fatty acid oxidation (Chapter 17), and its accumulation signals the availability of fatty acids as fuel. As we shall see in Chapter 16, the pyruvate carboxylase reaction can replenish intermediates in another central metabolic pathway, the citric acid cycle.

Because the mitochondrial membrane has no transporter for oxaloacetate, before export to the cytosol the oxaloacetate formed from pyruvate must be reduced to malate by mitochondrial malate dehydrogenase, at the expense of NADH:

Oxaloacetate + NADH + H^{+} ⇌ L -malate + {NAD}^{+}

$Oxaloacetate plus NADH plus upper H Superscript plus Baseline right harpoon over left harpoon upper L hyphen malate plus NAD Superscript plus$

(14-5)

The standard free-energy change for this reaction is quite high, but under physiological conditions (including a very low concentration of oxaloacetate) $Δ G \approx 0$ $upper Delta upper G almost-equals 0$ and the reaction is readily reversible. Mitochondrial malate dehydrogenase functions in both gluconeogenesis and the citric acid cycle, but the overall flow of metabolites in the two processes is in opposite directions.

Malate leaves the mitochondrion through a specific transporter in the inner mitochondrial membrane (see Fig. 19-31), and in the cytosol it is reoxidized to oxaloacetate, with the production of cytosolic NADH:

Malate + {NAD}^{+} \to oxaloacetate + NADH + H^{+}

$Malate plus NAD Superscript plus Baseline right-arrow oxaloacetate plus NADH plus upper H Superscript plus$

(14-6)

The oxaloacetate is then converted to PEP by phosphoenolpyruvate carboxykinase (Fig. 14-18). This ${Mg}^{2 +}$ $Mg Superscript 2 plus$ -dependent reaction requires GTP as the phosphoryl group donor:

Oxaloacetate + GTP ⇌ PEP + {CO}_{2} + GDP

$Oxaloacetate plus GTP right harpoon over left harpoon PEP plus CO Subscript 2 Baseline plus GDP$

(14-7)

A figure shows the reaction of oxaloacetate and G T P to produce phosphoenolpyruvate. — FIGURE 14-18 Synthesis of phosphoenolpyruvate from oxaloacetate. In the cytosol, oxaloacetate is converted to phosphoenolpyruvate by PEP carboxykinase. The ${CO}_{2}$ $CO Subscript 2$ incorporated in the pyruvate carboxylase reaction is lost here as $CO Subscript 2$ $CO Subscript 2$ . The decarboxylation leads to a rearrangement of electrons that facilitates attack of the carbonyl oxygen of the pyruvate moiety on the γ phosphate of GTP.

Oxaloacetate is a four-carbon chain with a red highlighted C double bonded to red highlighted O to the upper left, bonded to red highlighted O minus to the lower left, and bonded to C H 2 to the right further bonded to C double bonded to O below and bonded to C to the right double bonded to O to the upper right and bonded to O minus to the lower right. G T P has a rectangle labeled guanosine bonded to O bonded to P double bonded to O above, bonded to O minute below, and bonded to O to the right further bonded to O bonded to P double bonded to O above, bonded to O minus below, and bonded to O to the right further bonded to P double bonded to O above and bonded to O minus to the right and below. Curved red arrows point from red highlighted O minus at the left side of oxaloacetate to the bond between the same O and the red highlighted C, from the bond between the same red highlighted C and C H 2 to its right to the bond between the same C H 2 and nonhighlighted C double bonded to O to its right, from the double bond between the same nonhighlighted C and O to P at the right end of G T P, and from the bond between the same P and O to its left to the same O. An arrow labeled P E P carboxykinase has two arrows branching off to show the loss of G D P and then the loss of red highlighted C O 2. This yields phosphoenolpyruvate, which has C H 2 double bonded to C bonded to O above bonded to P O subscript 3 superscript 2 minus and to C O O minus to the right.

The reaction is reversible under intracellular conditions; the formation of one high-energy phosphate compound (PEP) is balanced by the hydrolysis of another (GTP). The overall equation for this set of bypass reactions, the sum of Equations 14-4 through 14-7, is

\begin{matrix} Pyruvate + ATP + GTP + {HCO}_{3}^{-} \to PEP + ADP + GDP + P_{i} + {CO}_{2} \\ Δ G^{'} ° = 0.9 kJ/mol \end{matrix}

$StartLayout 1st Row Pyruvate plus ATP plus GTP plus HCO Subscript 3 Superscript minus Baseline right-arrow PEP plus ADP plus GDP plus upper P Subscript i Baseline plus CO Subscript 2 Baseline 2nd Row upper Delta upper G Superscript prime Baseline degree equals 0.9 kJ slash mol EndLayout$

(14-8)

Two high-energy phosphate equivalents (one from ATP and one from GTP), each yielding about $50 kJ/mol$ $50 kJ slash mol$ under cellular conditions, must be expended to phosphorylate one molecule of pyruvate to PEP. In contrast, when PEP is converted to pyruvate during glycolysis, only one ATP is generated from ADP. Although the standard free-energy change $(Δ G^{'} °)$ $left-parenthesis upper Delta upper G prime degree right-parenthesis$ of the two-step path from pyruvate to PEP is 0.9 kJ/mol, the actual free-energy change $left-parenthesis upper Delta upper G right-parenthesis$ $left-parenthesis upper Delta upper G right-parenthesis$ , calculated from measured cellular concentrations of intermediates, is very strongly negative $left-parenthesis negative 25 kJ slash mol right-parenthesis$ $left-parenthesis negative 25 kJ slash mol right-parenthesis$ ; this results from the ready consumption of PEP in other reactions such that its concentration remains relatively low. The reaction is thus effectively irreversible in the cell.

Note that the ${CO}_{2}$ $CO Subscript 2$ added to pyruvate in the pyruvate carboxylase step (Fig. 14-17) is the same molecule that is lost in the PEP carboxykinase reaction (Fig. 14-18). This carboxylation-decarboxylation sequence represents a way of “activating” pyruvate, in that the decarboxylation of oxaloacetate facilitates PEP formation. In Chapter 21 we shall see how a similar carboxylation-decarboxylation sequence is used to activate acetyl-CoA for fatty acid biosynthesis (see Fig. 21-1).

There is a logic to the route of these reactions through the mitochondrion. The $[NADH] / [{NAD}^{+}]$ $left-bracket NADH right-bracket slash left-bracket NAD Superscript plus Baseline right-bracket$ ratio in the cytosol is several orders of magnitude lower than in mitochondria. Because cytosolic NADH is consumed in gluconeogenesis (in the conversion of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate; Fig. 14-16), glucose biosynthesis cannot proceed unless NADH is available. The transport of malate from the mitochondrion to the cytosol and its reconversion there to oxaloacetate effectively moves reducing equivalents to the cytosol, where they are scarce. This path from pyruvate to PEP therefore provides an important balance between NADH produced and consumed in the cytosol during gluconeogenesis.

A second pyruvate → PEP bypass predominates when lactate is the glucogenic precursor (Fig. 14-19). This pathway makes use of lactate produced by glycolysis in erythrocytes or anaerobic muscle, for example, and it is particularly important in large vertebrates after vigorous exercise (Box 14-2). The conversion of lactate to pyruvate in the cytosol of hepatocytes yields NADH, and the export of reducing equivalents (as malate) from mitochondria is therefore unnecessary. After the pyruvate produced by the lactate dehydrogenase reaction is transported into the mitochondrion (by a transporter in the inner mitochondrial membrane specific for pyruvate), it is converted to oxaloacetate by pyruvate carboxylase, as described above. This oxaloacetate, however, is converted directly to PEP by a mitochondrial isozyme of PEP carboxykinase, and the PEP is transported out of the mitochondrion to continue on the gluconeogenic path. The mitochondrial and cytosolic isozymes of PEP carboxykinase are encoded by separate genes in the nuclear chromosomes, providing another example of two distinct enzymes catalyzing the same reaction but having different cellular locations or metabolic roles (recall the isozymes of hexokinase).

A figure shows two alternative pathways to convert pyruvate to phosphoenolpyruvate. — FIGURE 14-19 Alternative paths from pyruvate to phosphoenolpyruvate. The relative importance of the two pathways depends on the availability of lactate or pyruvate and the cytosolic requirements for NADH for gluconeogenesis. The path on the right predominates when lactate is the precursor, because cytosolic NADH is generated in the lactate dehydrogenase reaction and does not have to be shuttled out of the mitochondrion (see text).

Pyruvate is shown in the cytosol at the lower left beneath a mitochondrion with its inner and outer membranes shown. An arrow points to pyruvate in the mitochondrial matrix. An arrow labeled pyruvate carboxylase points from pyruvate to oxaloacetate accompanied by a line showing the addition of C O 2 and a curved arrow showing the addition of an orange oval of A T P and loss of A D P plus P subscript i. An arrow labeled mitochondrial malate dehydrogenase points from oxaloacetate to malate and is accompanied by a curved arrow showing the addition of an orange oval labeled N A D H plus H plus outside of the oval and loss of N A D plus. An arrow points from malate inside of the mitochondrion to malate outside of the mitochondrion. An arrow labeled cytosolic malate dehydrogenase points from malate to oxaloacetate and is accompanied by a curved arrow showing the addition of N A D plus and loss of an orange oval labeled N A D H and H plus outside of the oval. An arrow labeled cytosolic P E P carboxykinase points from oxaloacetate to P E P at the top center of the illustration. The arrow is accompanied by a curved arrow showing the addition of an orange oval labeled A T P and the loss of A D P. Another arrow shows that C O 2 is lost. Lactate at the lower right begins another series of reactions to produce P E P. An arrow labeled lactate dehydrogenase points from lactate to pyruvate and is accompanied by a curved arrow showing the addition of N A D plus and loss of an orange oval labeled N A D H plus H plus outside of the oval. An arrow points from pyruvate in the cytosol to pyruvate in the mitochondrial matrix. An arrow labeled pyruvate carboxylase points from pyruvate to oxaloacetate accompanied by a line showing the addition of C O 2 and a curved arrow showing the addition of an orange oval of A T P and loss of A D P plus P subscript i. An arrow labeled mitochondrial P EP carboxykinase points from oxaloacetate to P E P accompanied by a curved arrow showing the addition of an orange oval labeled G T P and loss of G D P. An additional arrow shows the loss of P E P. An arrow points from P E P in the mitochondrion to P E P outside of the mitochondrion at the top center of the illustration.

The Second and Third Bypasses Are Simple Dephosphorylations by Phosphatases

The second glycolytic reaction that cannot participate in gluconeogenesis is the phosphorylation of fructose 6-phosphate by PFK-1 (Table 14-2, step ). Because this reaction is highly exergonic and therefore irreversible in intact cells, the generation of fructose 6-phosphate from fructose 1,6-bisphosphate (Fig. 14-16) is catalyzed by a different enzyme, ${Mg}^{2 +}$ $Mg Superscript 2 plus$ -dependent fructose 1,6-bisphosphatase (FBPase-1), which promotes the essentially irreversible hydrolysis of the C-1 phosphate (not phosphoryl group transfer to ADP):

\begin{matrix} Fructose 1, 6 -phosphate + H_{2} O \to fructose 6 -phosphate + P_{i} \\ Δ G^{'} ° = - 16.3 kJ/mol \end{matrix}

$StartLayout 1st Row Fructose 1 comma 6 hyphen phosphate plus upper H Subscript 2 Baseline upper O right-arrow fructose 6 hyphen phosphate plus upper P Subscript i Baseline 2nd Row upper Delta upper G Superscript prime Baseline degree equals negative 16.3 kJ slash mol EndLayout$

FBPase-1 is so named to distinguish it from another, similar enzyme (FBPase-2) with a regulatory role, which we discuss in Section 14.5.

The third bypass is the final reaction of gluconeogenesis, the dephosphorylation of glucose 6-phosphate to yield glucose (Fig. 14-16). Reversal of the hexokinase reaction (p. 514) would require phosphoryl group transfer from glucose 6-phosphate to ADP, forming ATP, an energetically unfavorable reaction (Table 14-2, step ). The reaction catalyzed by glucose 6-phosphatase does not require synthesis of ATP; it is a simple hydrolysis of a phosphate ester:

\begin{matrix} Glucose 6 -phosphate + H_{2} O \to & glucose + P_{i} \\ Δ G^{'} ° = - 13.8 kJ/mol \end{matrix}

$StartLayout 1st Row 1st Column Glucose 6 hyphen phosphate plus upper H Subscript 2 Baseline upper O right-arrow 2nd Column glucose plus upper P Subscript i Baseline 2nd Row 1st Column Blank 2nd Column upper Delta upper G Superscript prime Baseline degree equals negative 13.8 kJ slash mol EndLayout$

This ${Mg}^{2 +}$ $Mg Superscript 2 plus$ -activated enzyme is a membrane protein in the lumen of the endoplasmic reticulum of hepatocytes, renal cells, and epithelial cells of the small intestine (see Fig. 15-6), but not in other tissues, which are therefore unable to supply glucose to the blood. If other tissues had glucose 6-phosphatase, this enzyme’s activity would hydrolyze the glucose 6-phosphate needed within those tissues for glycolysis. Glucose produced by gluconeogenesis in the liver or kidney or ingested in the diet is delivered to these other tissues, including brain and muscle, through the bloodstream.

Gluconeogenesis Is Energetically Expensive, But Essential

The sum of the biosynthetic reactions leading from pyruvate to free blood glucose (Table 14-3) is

\begin{matrix} 2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H^{+} + 4 H_{2} O \to \\ glucose + 4 ADP + 2 GDP + 6 P_{i} + 2 {NAD}^{+} \end{matrix}

$StartLayout 1st Row 2 Pyruvate plus 4 ATP plus 2 GTP plus 2 NADH plus 2 upper H Superscript plus Baseline plus 4 upper H Subscript 2 Baseline upper O right-arrow 2nd Row glucose plus 4 ADP plus 2 GDP plus 6 upper P Subscript i Baseline plus 2 NAD Superscript plus EndLayout$

(14-9)

Pyruvate plus HCO Subscript 3 Superscript minus Baseline plus ATP right-arrow oxaloacetate plus ADP plus upper P Subscript i Baseline

TABLE 14-3 Sequential Reactions in Gluconeogenesis Starting from Pyruvate
$Pyruvate + {HCO}_{3}^{-} + ATP \to oxaloacetate + ADP + P_{i}$ $Pyruvate plus HCO Subscript 3 Superscript minus Baseline plus ATP right-arrow oxaloacetate plus ADP plus upper P Subscript i Baseline$	$\times 2$ $times 2$
$Oxaloacetate + GTP ⇌ phosphoenolpyruvate + {CO}_{2} + GDP$ $Oxaloacetate plus GTP right harpoon over left harpoon phosphoenolpyruvate plus CO Subscript 2 Baseline plus GDP$	$\times 2$ $times 2$
$Phosphoenolpyruvate + H_{2} O ⇌ 2-phosphoglycerate$ $Phosphoenolpyruvate plus upper H Subscript 2 Baseline upper O right harpoon over left harpoon 2 hyphen phosphoglycerate$	$\times 2$ $times 2$
$2-Phosphoglycerate ⇌ 3 -phosphoglycerate$ $2 hyphen Phosphoglycerate right harpoon over left harpoon 3 hyphen phosphoglycerate$	$\times 2$ $times 2$
$3 -Phosphoglycerate + ATP ⇌ 1, 3 -bisphosphoglycerate + ADP$ $3 hyphen Phosphoglycerate plus ATP right harpoon over left harpoon 1 comma 3 hyphen bisphosphoglycerate plus ADP$	$\times 2$ $times 2$
$\begin{matrix} 1, 3 -Bisphosphoglycerate + NADH + H^{+} ⇌ \\ glyceraldehyde 3 -phosphate + {NAD}^{+} + P_{i} \end{matrix}$ $StartLayout 1st Row 1 comma 3 hyphen Bisphosphoglycerate plus NADH plus upper H Superscript plus Baseline right harpoon over left harpoon 2nd Row glyceraldehyde 3 hyphen phosphate plus NAD Superscript plus Baseline plus upper P Subscript i Baseline EndLayout$	$\times 2$ $times 2$
$Glyceraldehyde 3 -phosphate ⇌ dihydroxyacetone phosphate$ $Glyceraldehyde 3 hyphen phosphate right harpoon over left harpoon dihydroxyacetone phosphate$
$\begin{matrix} Glyceraldehyde 3 -phosphate + dihydroxyacetone phosphate ⇌ \\ fructose 1, 6 -bisphosphate \end{matrix}$ $StartLayout 1st Row Glyceraldehyde 3 hyphen phosphate plus dihydroxyacetone phosphate right harpoon over left harpoon 2nd Row fructose 1 comma 6 hyphen bisphosphate EndLayout$
$Fructose 1, 6 -bisphosphate \to fructose 6 -phosphate + P_{i}$ $Fructose 1 comma 6 hyphen bisphosphate right-arrow fructose 6 hyphen phosphate plus upper P Subscript i Baseline$
$Fructose 6 -phosphate ⇌ glucose 6 -phosphate$ $Fructose 6 hyphen phosphate right harpoon over left harpoon glucose 6 hyphen phosphate$
$Glucose 6 -phosphate + H_{2} O \to glucose + P_{i}$ $Glucose 6 hyphen phosphate plus upper H Subscript 2 Baseline upper O right-arrow glucose plus upper P Subscript i Baseline$
$\begin{matrix} S u m : 2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H^{+} + 4 H_{2} O \to \\ glucose + 4 ADP + 2 GDP + 6 P_{i} + 2 {NAD}^{+} \end{matrix}$ $StartLayout 1st Row upper S u m colon 2 Pyruvate plus 4 ATP plus 2 GTP plus 2 NADH plus 2 upper H Superscript plus Baseline plus 4 upper H Subscript 2 Baseline upper O right-arrow 2nd Row glucose plus 4 ADP plus 2 GDP plus 6 upper P Subscript i Baseline plus 2 NAD Superscript plus EndLayout$

For each molecule of glucose formed from pyruvate, six high-energy phosphate groups are required: four from ATP and two from GTP. In addition, two molecules of NADH are required for the reduction of two molecules of 1,3-bisphosphoglycerate. Clearly, Equation 14-9 is not simply the reverse of the equation for conversion of glucose to pyruvate by glycolysis, which would require only two molecules of ATP:

Glucose + 2 ADP + 2 P_{i} + {NAD}^{+} \to 2 pyruvate + 2 ATP + 2 NADH + 2 H^{+} + 2 H_{2} O

$Glucose plus 2 ADP plus 2 upper P Subscript i Baseline plus NAD Superscript plus Baseline right-arrow 2 pyruvate plus 2 ATP plus 2 NADH plus 2 upper H Superscript plus Baseline plus 2 upper H Subscript 2 Baseline upper O$

This makes the synthesis of glucose from pyruvate a relatively expensive process. Much of this high energy cost is necessary to ensure the irreversibility of gluconeogenesis. Under intracellular conditions, the overall free-energy change of glycolysis is at least $negative 63 kJ slash mol$ $negative 63 kJ slash mol$ . Under the same conditions the overall $Δ G$ $upper Delta upper G$ of gluconeogenesis is $negative 16 kJ slash mol$ $negative 16 kJ slash mol$ . Thus both glycolysis and gluconeogenesis are essentially irreversible processes in cells. A second advantage to investing energy to convert pyruvate to glucose is that if pyruvate were instead excreted, its considerable potential for ATP production by complete, aerobic oxidation would be lost (more than 10 ATP are produced per pyruvate, as we shall see in Chapter 16).

The biosynthetic pathway to glucose described above allows the net synthesis of glucose not only from pyruvate but also from the four-, five-, and six-carbon intermediates of the citric acid cycle (Chapter 16). The citric acid cycle intermediates can undergo oxidation to oxaloacetate (see Fig. 16-7). Some or all of the carbon atoms of most amino acids derived from proteins are ultimately catabolized to pyruvate or to intermediates of the citric acid cycle. Such amino acids can therefore undergo net conversion to glucose and are said to be glucogenic (Table 14-4). Alanine and glutamine, the principal molecules that transport amino groups from extrahepatic tissues to the liver (see Fig. 18-9), are particularly important glucogenic amino acids in mammals. After removal of their amino groups in liver mitochondria, the carbon skeletons remaining (pyruvate and α-ketoglutarate, respectively) are readily funneled into gluconeogenesis.

TABLE 14-4 Glucogenic Amino Acids, Grouped by Site of Entry
Pyruvate	Succinyl-CoA
Alanine	Isoleucine^a
Cysteine	Methionine
Glycine	Threonine
Serine	Valine
Threonine	Fumarate
Tryptophan^a	Phenylalanine^a
α-Ketoglutarate	Tyrosine^a
Arginine	Oxaloacetate
Glutamate	Asparagine
Glutamine	Aspartate
Histidine
Proline

Mammals Cannot Convert Fatty Acids to Glucose; Plants and Microorganisms Can

No net conversion of fatty acids to glucose occurs in mammals. As we shall see in Chapter 17, the catabolism of most fatty acids yields only acetyl-CoA. Mammals cannot use acetyl-CoA as a precursor of glucose, because the pyruvate dehydrogenase reaction is irreversible and cells have no other pathway to convert acetyl-CoA to pyruvate. Plants, yeast, and many bacteria do have a pathway (the glyoxylate cycle; see Fig. 20-45) for converting acetyl-CoA to oxaloacetate, so these organisms can use fatty acids as the starting material for gluconeogenesis. This is important during the germination of seedlings, for example; before leaves develop and photosynthesis can provide energy and carbohydrates, the seedling relies on stored seed oils for energy production and cell wall biosynthesis.

Although mammals cannot convert fatty acids to carbohydrate, they can use the small amount of glycerol produced from the breakdown of fats (triacylglycerols) for gluconeogenesis. Phosphorylation of glycerol by glycerol kinase, followed by oxidation of the central carbon, yields dihydroxyacetone phosphate, an intermediate in gluconeogenesis in liver.

As we shall see in Chapter 21, glycerol phosphate is an essential intermediate in triacylglycerol synthesis in adipocytes, but these cells lack glycerol kinase and so cannot simply phosphorylate glycerol. Instead, adipocytes carry out a truncated version of gluconeogenesis, known as glyceroneogenesis: the conversion of pyruvate to dihydroxyacetone phosphate via the early reactions of gluconeogenesis, followed by reduction of the dihydroxyacetone phosphate to glycerol 3-phosphate (see Fig. 21-21).

SUMMARY 14.4 Gluconeogenesis

Gluconeogenesis is a multistep process in which glucose is produced from lactate, pyruvate, or oxaloacetate, or any compound (including citric acid cycle intermediates) that can be converted to one of these intermediates. Seven steps are the reversal of glycolytic reactions; three differ and must be bypassed with exergonic reactions.
In the first bypass, pyruvate is converted to PEP via oxaloacetate in two steps catalyzed by pyruvate carboxylase (which uses ATP) and PEP carboxykinase (which uses GTP).
In the second bypass, FBPase-1 removes a phosphate group from fructose 1,6-bisphosphate, producing fructose 6-phosphate. In the third bypass, glucose 6-phosphatase converts glucose 6-phosphate to glucose.
In mammals, gluconeogenesis in the liver, kidney, and small intestine provides glucose for use by the brain, muscles, and erythrocytes. Formation of one molecule of glucose from pyruvate requires four ATP, two GTP, and two NADH; it is expensive.
Animals cannot convert acetyl-CoA derived from fatty acids into glucose; they lack the enzymatic machinery to convert acetyl-CoA to pyruvate. Plants and microorganisms have the glyoxylate pathway, which allows them to make glucose from fatty acids.