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Gluconeogenesis- Subjective questions (Solved) – Part-1
Give a brief account of the thermodynamic barriers of gluconeogenesis.
Answer- Gluconeogenesis is the process of converting noncarbohydrate precursors to glucose or glycogen. The major substrates are the glucogenic amino acids, lactate, glycerol, and propionate. These noncarbohydrate precursors of glucose are first converted into pyruvate or enter the pathway at later intermediates such as oxaloacetate and Dihydroxyacetone phosphate. Liver and kidney are the major gluconeogenic tissues.
Gluconeogenesis meets the needs of the body for glucose when sufficient carbohydrate is not available from the diet or glycogen reserves. A supply of glucose is necessary especially for the nervous system and erythrocytes. Failure of gluconeogenesis is usually fatal.
Reactions of Gluconeogenesis
In glycolysis, glucose is converted into pyruvate; in gluconeogenesis, pyruvate is converted into glucose. However, gluconeogenesis is not a reversal of glycolysis. Three nonequilibrium reactions in glycolysis catalyzed by hexokinase, phosphofructokinase and pyruvate kinase are considered thermodynamic barriers which prevent simple reversal of glycolysis for glucose synthesis.
Several reactions must differ because the equilibrium of glycolysis lies far on the side of pyruvate formation. The actual ∆G for the formation of pyruvate from glucose is about -20 kcal mol-1 (-84 kJ mol-1) under typical cellular conditions. Most of the decrease in free energy in glycolysis takes place in the three essentially irreversible steps catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase.
In gluconeogenesis, the following new steps bypass these virtually irreversible reactions of glycolysis:
1. First bypass (Formation of Phosphoenolpyruvate from pyruvate)
Reversal of the reaction catalyzed by pyruvate kinase in glycolysis involves two endothermic reactions. Phosphoenolpyruvate is formed from pyruvate by way of oxaloacetate through the action of pyruvate carboxylase and phosphoenolpyruvate carboxykinase.
Mitochondrial pyruvate carboxylase catalyzes the carboxylation of pyruvate to oxaloacetate, an ATP-requiring reaction in which the vitamin biotin is the coenzyme. Biotin binds CO2 from bicarbonate as carboxybiotin prior to the addition of the CO2 to pyruvate.
Pyruvate carboxylase is a mitochondrial enzyme, whereas the other enzymes of gluconeogenesis are cytoplasmic.
Oxaloacetate, the product of the pyruvate carboxylase reaction, is reduced to malate inside the mitochondrion for transport to the cytosol. The reduction is accomplished by an NADH-linked malate dehydrogenase. When malate has been transported across the mitochondrial membrane, it is reoxidized to oxaloacetate by an NAD+-linked malate dehydrogenase in the cytosol.
Figure-1- showing the transportation of oxaloacetate outside the mitochondrion in the form of Malate
A second enzyme, phosphoenolpyruvate carboxykinase, catalyzes the decarboxylation and phosphorylation of oxaloacetate to phosphoenolpyruvate using GTP as the phosphate donor. In liver and kidney, the reaction of succinate thiokinase in the citric acid cycle produces GTP (rather than ATP as in other tissues), and this GTP is used for the reaction of phosphoenolpyruvate carboxykinase, thus providing a link between citric acid cycle activity and gluconeogenesis, to prevent excessive removal of oxaloacetate for gluconeogenesis, which would impair citric acid cycle activity.
2. Second bypass (Formation of Fructose 6-phosphate is formed from fructose 1,6-bisphosphate)
On formation, phosphoenolpyruvate is metabolized by the enzymes of glycolysis but in the reverse direction. These reactions are near equilibrium under intracellular conditions; so, when conditions favor gluconeogenesis, the reverse reactions will take place until the next irreversible step is reached. This step is the hydrolysis of fructose 1,6- bisphosphate to fructose 6-phosphate and Pi.
Fructose 1,6-bisphosphatase catalyzes this exergonic hydrolysis.
Its presence determines whether a tissue is capable of synthesizing glucose (or glycogen) not only from pyruvate, but also from triose phosphates. It is present in liver, kidney, and skeletal muscle, but is probably absent from heart and smooth muscle. Like its glycolytic counterpart, it is an allosteric enzyme that participates in the regulation of gluconeogenesis.
3. Third bypass (Formation of Glucose by hydrolysis of glucose 6-phosphate)
The fructose 6-phosphate generated by fructose 1,6-bisphosphatase is readily converted into glucose 6-phosphate. In most tissues, gluconeogenesis ends here. Free glucose is not generated; rather, the glucose 6-phosphate is processed in some other fashion, notably to form glycogen. One advantage to ending gluconeogenesis at glucose 6-phosphate is that, unlike free glucose, the molecule cannot diffuse out of the cell. To keep glucose inside the cell, the generation of free glucose is controlled in two ways. First, the enzyme responsible for the conversion of glucose 6-phosphate into glucose, glucose 6-phosphatase, is regulated.
Second, the enzyme is present only in tissues whose metabolic duty is to maintain blood-glucose homeostasis- tissues that release glucose into the blood. These tissues are the liver and to a lesser extent the kidney the enzyme is absent in muscle and adipose tissue, which therefore, cannot export glucose into the bloodstream.
Figure-2- The glucose 6-phosphatase enzyme is stabilized by the Ca2+-binding protein SP .After the cleavage, a set of transporters, T2 and T3, return the products orthophosphate and glucose back into the cytosol.
This final step in the generation of glucose does not take place in the cytosol. Rather, glucose 6-phosphate is transported into the lumen of the endoplasmic reticulum, where it is hydrolyzed to glucose by glucose 6-phosphatase, which is bound to the membrane .An associated Ca2+-binding stabilizing protein is essential for phosphatase activity. Glucose and Pi are then shuttled back to the cytosol by a pair of transporters. The glucose transporter in the endoplasmic reticulum membrane is like those found in the plasma membrane. It is striking that five proteins are needed to transform cytosolic glucose 6-phosphate into glucose.
In the kidney, muscle and especially the liver, G6P be shunted toward glycogen if blood glucose levels are adequate. The reactions necessary for glycogen synthesis are an alternate bypass series of reactions.
The G6P produced from gluconeogenesis can be converted to glucose-1-phosphate (G1P) by phosphoglucose mutase (PGM). G1P is then converted to UDP-glucose (the substrate for glycogen synthase) by UDP-glucose pyro phosphorylase, a reaction requiring hydrolysis of UTP.
Figure-3- showing reactions of gluconeogenesis
Energetics of gluconeogenesis
The overall reaction of gluconeogenesis is-
The overall reaction of glycolysis is-
Six nucleotide triphosphate molecules are hydrolyzed to synthesize glucose from pyruvate in gluconeogenesis, whereas only two molecules of ATP are generated in glycolysis in the conversion of glucose into pyruvate. Thus it is not a simple reversal of glycolysis but it is energetically an expensive affair.
The four additional high phosphoryl-transfer potential molecules are needed to turn an energetically unfavorable process (the reversal of glycolysis, D G°´ = + 20 kcal mol-1 [+84 kJ mol-1]) into a favorable one (gluconeogenesis, D G°´ = -9 kcal mol-1 [-38 kJ mol-1]). This is a clear example of the coupling of reactions: ATP hydrolysis is used to power an energetically unfavorable reaction.
Q.2- How do the substrates of gluconeogenesis gain entry in to the main pathway?
Explain giving reactions involving each substrate.
Answer- The major substrates are the glucogenic amino acids, lactate, glycerol, and propionate.
A) Glucogenic amino acids- Amino acids are derived from the dietary proteins, tissue proteins or from the breakdown of skeletal muscle proteins during starvation. After transamination or deamination, glucogenic amino acids yield either pyruvate or intermediates of the citric acid cycle. Amino acids that are degraded to acetyl CoA or Acetoacetyl CoA are termed ketogenic amino acids because they can give rise to ketone bodies or fatty acids. Amino acids that are degraded to pyruvate, α-ketoglutarate, succinyl CoA, fumarate, or oxaloacetate are termed glucogenic amino acids. The net synthesis of glucose from these amino acids is feasible because these citric acid cycle intermediates and pyruvate can be converted into phosphoenolpyruvate. The entry point of these glucogenic amino acids in to the pathway of gluconeogenesis is as follows-
1) Pyruvate is the point of entry for alanine, serine, cysteine, glycine, Threonine, and tryptophan (Figure-4). The transamination of alanine directly yields pyruvate.
2) Oxaloacetate- Aspartate and asparagine are converted into oxaloacetate, a citric acid cycle intermediate. Aspartate, a four-carbon amino acid, is directly transaminated to oxaloacetate.
3) α-Ketoglutarate is the point of entry of several five-carbon amino acids that are first converted into glutamate.(Figure)
4) Succinyl CoA is a point of entry for some of the carbon atoms of methionine, isoleucine, and valine. Propionyl CoA and then Methylmalonyl CoA are intermediates in the breakdown of these three nonpolar amino acids.
5) Fumarate is the point of entry for Aspartate, Phenyl alanine and Tyrosine.
Figure- 4- Amino acids forming Acetyl co A or Acetoacetyl co A are not considered glucogenic, they are called ketogenic amino acids since acetyl co A is a precursor for ketone bodies. All other amino acids which form pyruvate or intermediates of TCA cycle are considered glucogenic.
B) Lactate- Lactate is formed by active skeletal muscle when the rate of glycolysis exceeds the rate of oxidative metabolism. Lactate is readily converted into pyruvate by the action of lactate dehydrogenase.
Lactate is a major source of carbon atoms for glucose synthesis by gluconeogenesis. During anaerobic glycolysis in skeletal muscle, pyruvate is reduced to lactate by lactate dehydrogenase (LDH). This reaction serves two critical functions during anaerobic glycolysis. First, in the direction of lactate formation the LDH reaction requires NADH and yields NAD+ which is then available for use by the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis. These two reactions are, therefore, intimately coupled during anaerobic glycolysis. Secondly, the lactate produced by the LDH reaction is released to the blood stream and transported to the liver where it is converted to glucose. The glucose is then returned to the blood for use by muscle as an energy source and to replenish glycogen stores. This cycle is termed the Cori cycle.(See details)
C) Propionate- Propionate is a major precursor of glucose in ruminants; it enters gluconeogenesis via the citric acid cycle. After esterificaton with CoA, Propionyl-CoA is carboxylated to D-Methylmalonyl-CoA, catalyzed by Propionyl-CoA carboxylase, a biotin-dependent enzyme (Figure-5). Methylmalonyl-CoA racemase catalyzes the conversion of D-Methylmalonyl-CoA to L-Methylmalonyl-CoA, which then undergoes isomerization to succinyl-CoA catalyzed by Methylmalonyl-CoA mutase. In non-ruminants, including humans, propionate arises from the Beta -oxidation of odd-chain fatty acids that occur in ruminant lipids, as well as the oxidation of isoleucine and the side-chain of cholesterol, and is a (relatively minor) substrate for gluconeogenesis. Methylmalonyl CoA Isomerase/ mutase is a vitamin B12 dependent enzyme, and in deficiency methylmalonic acid is excreted in the urine (methylmalonic aciduria). (Figure-5)
Figure-5- showing the fate of Propionyl co A
D) Glycerol-The hydrolysis of triacylglycerols in fat cells yields glycerol and fatty acids. Glycerol may enter either the gluconeogenic or the glycolytic pathway at Dihydroxyacetone phosphate; however, the carbons of the fatty acids cannot be utilized for net synthesis of glucose. In the fasting state glycerol released from lipolysis of adipose tissue triacylglycerol is used solely as a substrate for gluconeogenesis in the liver and kidneys. This requires phosphorylation to glycerol-3-phosphate by glycerol kinase and dehydrogenation to Dihydroxyacetone phosphate (DHAP) by glyceraldehyde-3-phosphate dehydrogenase (G3PDH). The G3PDH reaction is the same as that used in the transport of cytosolic reducing equivalents into the mitochondrion for use in oxidative phosphorylation. This transport pathway is called the glycerol-phosphate shuttle.
Figure-6- showing the conversion of glycerol to dihydroxy acetone phosphate
Glycerol kinase is absent in adipose tissue, so glycerol released by hydrolysis of triglycerides can not be utilized for re esterificaton, it is a waste product, It is carried through circulation to the liver and is used for gluconeogenesis or glycolysis as the need may be. In fact adipocytes require a basal level of glycolysis in order to provide them with DHAP as an intermediate in the synthesis of triacylglycerols.
Q.3-. “It is incorrect to say that fats can not be converted to glucose”, Comment on the statement.
Answer- It is a justified statement.
Triglycerides (fats) on hydrolysis yield fatty acids and glycerol.Even chain fatty acids are not the glucogenic precursors, Oxidation of these fatty acids yields enormous amounts of energy on a molar basis, however, the carbons of the fatty acids cannot be utilized for net synthesis of glucose. The two carbon unit of acetyl-CoA derived from β-oxidation of fatty acids can be incorporated into the TCA cycle, however, during the TCA cycle two carbons are lost as CO2. Moreover the formation of acetyl CoA from pyruvate is an irreversible step, thus acetyl CoA can not be converted back into glucose. The oxidative decarboxylation of pyruvate to acetyl CoA commits the carbon atoms of glucose to two principal fates: oxidation to CO2 by the citric acid cycle, with the concomitant generation of energy, or incorporation into lipid (Figure-7), Thus, explaining why even chain fatty acids do not undergo net conversion to carbohydrate.
Odd chain fatty acids on oxidation produce Propionyl co A which is a substrate for gluconeogenesis through formation of succinyl co A.
Glycerol component of fats can also be utilized for the formation of glucose through formation of dihydroxy acetone phosphate. Hence therefore except for even chain fatty acids, the other fat components are glucogenic, so the above given statement that “It is incorrect to say that fats can not be converted to glucose”, is a justified statement.
Figure-7- showing the irreversible reaction of Pyruvate to Acetyl co A
Q.4- What is Cori cycle or Lactic acid cycle? How does it differ from Glucose Alanine cycle? Explain how muscle may be used to supply energy in a wasting condition like anorexia nervosa.
Answer- Lactate produced by active skeletal muscle and erythrocytes is a source of energy for other organs. Erythrocytes lack mitochondria and can never oxidize glucose completely. In contracting skeletal muscle during vigorous exercise, the rate at which glycolysis produces pyruvate exceeds the rate at which the citric acid cycle oxidizes it. Under these conditions, moreover, the rate of formation of NADH by glycolysis is greater than the rate of its oxidation by aerobic metabolism.
Continued glycolysis depends on the availability of NAD+ for the oxidation of glyceraldehyde 3-phosphate. The accumulation of both NADH and pyruvate is reversed by lactate dehydrogenase, which oxidizes NADH to NAD+ as it reduces pyruvate to lactate. However, lactate is a dead-end in metabolism. It must be converted back into pyruvate before it can be metabolized. The only purpose of the reduction of pyruvate to lactate is to regenerate NAD+ so that glycolysis can proceed in active skeletal muscle and erythrocytes. The formation of lactate buys time and shiftspart of the metabolic burden from muscle to other organs.
Figure-8- showing Cori cycle
The plasma membrane of most cells contains carriers that render them highly permeable to lactate and pyruvate. Both substances diffuse out of active skeletal muscle into the blood and are carried to the liver. Much more lactate than pyruvate is transported out because the high NADH/NAD+ ratio in contracting skeletal muscle favors the conversion of pyruvate into lactate. The lactate that enters the liver is oxidized to pyruvate, a reaction favored by the low NADH/NAD+ ratio in the cytosol of liver cells. Pyruvate in the liver is converted into glucose by the gluconeogenic pathway. Glucose then enters the blood and is taken up by skeletal muscle. Thus, the liver furnishes glucose to contracting skeletal muscle, which derives ATP from the glycolytic conversion of glucose into lactate. Contracting skeletal muscle supplies lactate to the liver, which uses it to synthesize glucose. These reactions constitute the Cori cycle (Figure-7).
Glucose Alanine cycle
The glucose-alanine cycle is used primarily as a mechanism for skeletal muscle to eliminate nitrogen while replenishing its energy supply. Glucose oxidation produces pyruvate which can undergo transamination to alanine. This reaction is catalyzed by alanine transaminase, ALT (ALT used to be referred to a serum glutamate-pyruvate transaminase, SGPT). Additionally, during periods of fasting, skeletal muscle protein is degraded for the energy value of the amino acid carbons and alanine is a major amino acid in protein. The alanine then enters the blood stream and is transported to the liver. Within the liver alanine is converted back to pyruvate which is then a source of carbon atoms for gluconeogenesis. The newly formed glucose can then enter the blood for delivery back to the muscle. The amino group transported from the muscle to the liver in the form of alanine is converted to urea in the urea cycle and excreted.(Figure-9)
Role of Muscle in wasting conditions, starvation or in Anorexia nervosa
Plasma glucose concentrations are normally maintained within a relatively narrow range, roughly 70–110 mg/dL (3.9–6.1 mmol/L) in the fasting state with transient higher excursions after a meal, despite wide variations in exogenous glucose delivery from meals and in endogenous glucose utilization by, for example, exercising muscle. Between meals and during fasting, plasma glucose levels are maintained by endogenous glucose production, hepatic glycogenolysis, and hepatic (and renal) gluconeogenesis. Although hepatic glycogen stores are usually sufficient to maintain plasma glucose levels for approximately 8 h, this time period can be shorter if glucose demand is increased by exercise or if glycogen stores are depleted by illness or starvation.
Gluconeogenesis requires a coordinated supply of precursors from muscle and adipose tissue to the liver (and kidneys). Muscle provides lactate, pyruvate, Alanine, glutamine, and other amino acids. Triglycerides in adipose tissue are broken down into fatty acids and glycerol, which is a gluconeogenic precursor. Fatty acids provide an alternative oxidative fuel to tissues other than the brain (which requires glucose).
Figure- 9-showing glucose- alanine cycle
This glucose-alanine cycle thus provides an indirect way of utilizing muscle glycogen to maintain blood glucose in the fasting state. The ATP required for the hepatic synthesis of glucose from pyruvate is derived from the oxidation of fatty acids. Thus by interplay of glycolysis and Gluconeogenesis, the energy requirements of different cell types are fulfilled.
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