BIOCHEMISTRY AND MOLECULAR BIOLOGY - W. ELLIOTT - 2002

CHAPTER 11. SYNTHESIS OF GLUCOSE IN THE BODY (GLUCONEOGENESIS)

The body can synthesize glucose from compounds that can be preliminarily converted into Pyruvate, i.e., from Most Amino Acids and lactate delivered to the Blood from working Muscles. The totality of such transformations is called Gluconeogenesis. Glucose cannot be synthesized from acetyl-CoA and Fatty acids. Gluconeogenesis essentially allows the energy of transformations to be stored as Glycogen. However, beyond this, gluconeogenesis in A number of cases saves the Organism from death.

The Brain requires a continuous supply of glucose. This means that to avoid coma or death, blood glucose levels must be maintained within the normal range. Meanwhile, glycogen reserves in the Liver are small and become completely depleted after a day of fasting.

Humans, however, remain alive even during longer periods without food. In this case, the supply of glucose to the blood is provided by the liver. No other organ (except the Kidneys, whose contribution is small) is capable of performing this task. After a day of fasting, the liver must meet The Human Body's demand for glucose (about 100 g per day).

To meet the basic energy needs of the body, fat reserves are sufficient for weeks, but fatty acids do not cross the blood-brain barrier and therefore cannot be used by the brain. Fat mobilization leads to the Formation of Ketone bodies, the presence of which in the blood somewhat reduces The Need for glucose (see p. 84); however, the necessity for its synthesis does not disappear. The brain's demand for glucose during starvation remains the same. Moreover, the brain is only one of its main consumers. Glucose is required by the Cells of the retina, the renal medulla, and erythrocytes—i.e., all Tissues and cells whose vital activity is largely or entirely (erythrocytes lack Cell/35.html">Mitochondria altogether!) supported by Anaerobic METABOLISM.

Gluconeogenesis in the liver begins with pyruvate, which also serves as the starting compound in fatty acid synthesis. It would seem that it could link these two metabolic branches. However, The conversion of pyruvate to acetyl-CoA in animals is irreversible, so they cannot process fatty acids into glucose.

Mechanism of Glucose Synthesis from Pyruvate

Among the reactions of Glycolysis, three are thermodynamically irreversible (see Fig. 8.7):

1) ATP-dependent phosphorylation of glucose by hexokinase (or glucokinase);

2) phosphorylation of fructose-6-phosphate by Phosphofructokinase;

3) conversion of phosphoenolpyruvate to pyruvate. Glucose is synthesized from pyruvate via the same intermediates as in glycolysis, but to bypass the irreversible reactions, alternative pathways must be taken.

The first thermodynamic barrier must be overcome during the conversion of pyruvate to phosphoenolpyruvate. Since the spontaneous Conversion of the enol form of pyruvate to the keto form is strongly exergonic (large negative value of ΔG°′), the reaction Phosphoenolpyruvate -> Pyruvate is irreversible. Therefore, in animals, the conversion of phosphoenolpyruvate from pyruvate occurs via a detour, in two stages using two high-energy phosphates, which makes this conversion thermodynamically favorable:

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The scheme of these transformations is presented in Fig. 11.1.

Fig. 11.1. Gluconeogenesis in the liver: formation of phosphoenolpyruvate from pyruvate (PEP - phosphoenolpyruvate). Note that these reactions form a futile cycle unless the reverse conversion of PEP to pyruvate is blocked. For how this is achieved, see Chapter 12

Why GTP rather than ATP is used In the second stage is unclear. Energetically, both substances are completely equivalent. Note that reaction (1)—the synthesis of oxaloacetate catalyzed by pyruvate carboxylase—plays an important role in regulating The Citric Acid Cycle. However, this is unrelated to gluconeogenesis.

The second reaction is carried out by phosphoenolpyruvate carboxykinase. This enzyme name is explained by the fact that the reverse reaction can be viewed as the carboxylation of phosphoenolpyruvate coupled with The transfer of a phosphate group.

After The formation of phosphoenolpyruvate, all glycolytic reactions are reversible up to the Formation of fructose-1,6-bisphosphate, the conversion of which from fructose-6-phosphate during glycolysis is irreversible. This obstacle is easily overcome, since the removal of the phosphate group via simple Hydrolysis requires no Energy Expenditure.

A similar hydrolysis reaction during gluconeogenesis occurs in the conversion of glucose-6-phosphate to glucose (during glycolysis, the formation of glucose-6-phosphate catalyzed by hexokinase or glucokinase is irreversible).

Glucose-6-phosphatase is present in the liver, which hydrolyzes glucose-6-phosphate, after which free glucose leaves The Cell. The set of gluconeogenesis reactions is presented in Fig. 11.2. The Regulation of Blood glucose levels is discussed in Chapter 12.

Thus, there are four Enzymes responsible for gluconeogenesis that do not participate in glycolysis: pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase. Naturally, they are concentrated predominantly in the liver. In rats, the concentration of these enzymes in the liver is 20-50 times greater than in skeletal muscles.

Where does the liver get pyruvate for gluconeogenesis?

When glycogen stores are depleted during starvation, the primary source of pyruvate becomes the hydrolytic breakdown of Muscle Proteins, generating all 20 amino acids. Although Alanine is just one of them, it accounts for more than 30% of all amino acids delivered to the liver. Alanine is one of the glucogenic amino acids; its carbon Skeleton is utilized by the liver to build glucose molecules (Amino acid metabolism will be discussed in Chapter 15).

Why does The breakdown of muscle proteins produce so much alanine? The metabolism of many Amino Acids and The Citric Acid cycle lead to the accumulation of oxaloacetate, which is converted into pyruvate (see Fig. 11.1). The latter is then transformed into alanine, receiving its amino group from Other Amino Acids.

It is worth noting that during starvation, muscles rely mainly on Fatty Acids and Ketone Bodies for energy production; consequently, they maintain a sufficient supply of acetyl-CoA and do not require pyruvate oxidation to generate it. As will be shown in Chapter 12, a high acetyl-CoA/CoA concentration ratio leads to the inactivation of pyruvate dehydrogenase. As a result, the pyruvate derived from amino acids is channeled into alanine synthesis. Protein degradation in muscles yields various amino acids, many of which are converted into alanine, and this alanine is transported via the bloodstream to the liver. (In addition to alanine, glutamine is also synthesized in muscles and delivered to the liver for glucose synthesis, following the same conversion pathway.) In the liver, alanine is converted back into pyruvate and subsequently into glucose. The scheme of alanine's role in gluconeogenesis is illustrated in Fig. 11.3.

Fig. 11.2. Hepatic gluconeogenesis: Synthesis of glucose from pyruvate. Reactions differing from the corresponding glycolytic steps are highlighted in color

Fig. 11.3. Mechanism by which muscle proteins supply the liver with pyruvate for gluconeogenesis during starvation. The scheme assumes that pyruvate is not converted into acetyl-CoA via the pyruvate dehydrogenase reaction. Alongside alanine, another gluconeogenic substrate, glutamine, is also exported from muscles

As ketone body levels rise in the blood during prolonged starvation, the brain increasingly utilizes them instead of glucose for energy production. This diminishes (though does not eliminate) the demand for gluconeogenesis. Since producing 1 g of glucose consumes 2 g of muscle protein, slowing down the Rate of protein breakdown significantly enhances the survival of the starving organism.

Chapter 15 provides a detailed Structure/133.html">Discussion of the glucose-alanine cycle (see Fig. 15.9), illustrating how alanine synthesized in muscles and delivered to the liver is converted there into glucose. However, in this pathway, the pyruvate required for alanine synthesis is generated through glycolysis. This involves the following sequence of events: glucose in liver —> glucose in muscle —> alanine in muscle —> alanine in liver —> glucose in liver.

Clearly, such a cycle does not result in a net increase in glucose or solve The problem of tissue supply; rather, it serves merely as a transport mechanism for amino nitrogen from the muscles to the liver.

Another source of pyruvate for gluconeogenesis is significant not so much during starvation as under normal physiological conditions. We are referring to lactate, which is produced during the anaerobic glycolytic breakdown of glucose or glycogen (see p. 101). Certain cells, such as those in the renal medulla and retina, are effectively anaerobic, while mature erythrocytes lack mitochondria altogether and are therefore incapable of Oxidative Phosphorylation. Under normal nutritional conditions, the primary source of lactate is glycolysis in vigorously working muscles. Under these circumstances, mitochondria cannot keep pace with the reoxidation of accumulating NADH. As a result, reducing equivalents are transferred to pyruvate, converting it into lactate. Lactate is then transported via the blood to the liver, where it is reconverted into pyruvate and subsequently into glucose and glycogen. This physiological cycle is named the Cori cycle (Fig. 11.4) after its discoverer.

Fig. 11.4. The Cori cycle is a physiological cycle operating between muscles and the liver. Because muscles contain very low levels of the three enzymes required for gluconeogenesis, lactate accumulated in Muscle tissue must be transported to the liver for conversion into glucose. Excess lactate is generated in muscles during active contraction via anaerobic glycolysis

The Cori cycle serves two vital Functions: conserving lactate for subsequent use and preventing so-called lactic acidosis. If large amounts of lactic acid enter the bloodstream, the blood's buffering capacity can be overwhelmed, leading to a dangerous drop in pH. This is prevented by the conversion of lactate into glucose, a process that consumes two protons (1 proton (H⁺) and 1 molecule of NADH are utilized for the reduction of 1,3-bisphosphoglycerate).

Synthesis of Glucose from Glycerol

Another carbon source for gluconeogenesis is glycerol, produced by the hydrolysis of triglycerides primarily within adipose tissue. Glycerol is taken up by The Liver and converted into glucose According to the pathway shown in Fig. 11.5. The initial step—the phosphorylation of glycerol—is catalyzed by glycerol kinase, which is present in much lower amounts in adipose tissue than in the liver. All subsequent reactions are also localized in the liver. This makes complete sense, as the liver is tasked with solving the problem when glucose production becomes vital. Consequently, fat cells do not utilize glycerol themselves.

Fig. 11.5. Conversion into glucose of glycerol released during the hydrolysis of neutral fats. The bulk of glycerol is produced in fat cells, but lacking glycerol kinase, these cells cannot perform gluconeogenesis from glycerol, which therefore takes place in the liver. This process ensures the formation of glucose from glycerol during starvation

During prolonged starvation, after a minor initial decline, blood glucose levels are maintained remarkably stable for several weeks thanks to the continuous supply of fatty acids from adipose cells. Surprisingly, however, unlike plants and Bacteria, animals were not endowed by nature with a simpler mechanism that avoids "consuming" their own muscles. Presumably, evolution had its own inscrutable reasons for this.

Synthesis of Glucose via The Glyoxylate cycle

E. coli thrives using acetate as its sole carbon source. Unlike animals, bacteria are capable of converting acetyl-CoA into C₄ acids involved in the citric acid cycle, utilizing them to synthesize glucose and other essential cellular components. Germinating plant seeds similarly utilize stored triglycerides for glucose synthesis. How do bacteria and plants manage this?

These organisms possess a standard citric acid cycle, but they can divert some of its intermediates into alternative pathways not found in animals. For instance, in the citric acid cycle, two carbon atoms from acetyl-CoA are first condensed with an oxaloacetate molecule (C₄) to form citrate (C₆), and then two carbon atoms are released as two molecules of CO₂ (transition from C₆ to C₄ acids) to yield succinate. This irreversible loss is circumvented by the so-called glyoxylate pathway, which allows two carbon atoms to be diverted from the citric acid cycle not as CO₂, but as glyoxylate, formed directly through the Cleavage of isocitrate into succinate and glyoxylate:

Glyoxylate (C₂) then reacts with acetyl-CoA to form malate, a standard intermediate of the citric acid cycle.

A general scheme illustrating the interrelationship of all the processes discussed is presented in Fig. 11.6.

Fig. 11.6. The glyoxylate cycle, which plants and bacteria (absent in animals) use to synthesize CARBOHYDRATES from acetyl-CoA

Reactions specific to this cycle are highlighted in color. Dashed lines indicate the citric acid cycle reactions that are not involved in the glyoxylate cycle. This makes it possible to preserve the 2 molecules of CO₂ required to convert citrate into 2 molecules of oxaloacetate, one of which is reused for citrate synthesis, while the other is converted into phosphoenolpyruvate

Their net effect is that acetyl-CoA plus oxaloacetate are converted into malate plus succinate. Both malate and succinate can be converted into oxaloacetate, one molecule of which can be directed toward glucose synthesis without disrupting the cycle. In plants, these reactions take place within the membranes of Organelles called glyoxysomes.

In Conclusion, it is worth recalling that the vast majority of carbohydrates on Earth are produced via Photosynthesis, in which solar energy is used to fix CO₂ in the form of diphosphoglycerate—a compound familiar to us from glycolysis. The mechanisms of this process and the subsequent conversion of diphosphoglycerate into glucose will be discussed in Chapter 14 as components of photosynthesis.

Thus, we have examined the utilization of fats and glucose as Energy Sources, as well as the mechanisms for synthesizing these substances. It should be noted that these metabolic processes do not exist in isolation, but form an integrated metabolic system whose components are all interdependent and require regulation.

Questions for Chapter 11

1. After 24 hours of fasting, liver glycogen stores are depleted, yet the body retains substantial fat reserves. Why does gluconeogenesis occur during fasting when the body has virtually limitless supplies of acetyl-CoA (derived from fatty acids), which are more than sufficient for energy production?

2. Why can phosphoenolpyruvate, which is required for gluconeogenesis, not be produced by the phosphorylation of pyruvate via pyruvate kinase?

3. Starting from phosphoenolpyruvate, hepatic gluconeogenesis proceeds via the reversal of glycolytic reactions. Which two glycolytic enzymes catalyze irreversible reactions? How does the existence of these reactions affect The Mechanism of gluconeogenesis?

4. Is glucose-6-phosphatase present in muscle tissue? Explain your answer.

5. What is the Cori cycle and what is its physiological role?

6. Fat cells contain virtually no glycerol kinase—the enzyme that catalyzes the conversion of glycerol to glycerol-3-phosphate—even though glycerol is produced there (via triglyceride hydrolysis). However, this enzyme is present in the liver. Does this make physiological sense? If so, why?

7. How do bacteria and plants manage to convert acetyl-CoA into carbohydrates, unlike animals?