Gluconeogenesis refers to a group of metabolic reactions, some of them highly exergonic and irreversible, which are regulated both locally and globally (by insulin, glucagon, and cortisol). The purpose of this system, localized in both the cytosol and mitochondria, is to maintain blood glucose level constant throughout fasting state. The balance between stimulatory and inhibitory hormones regulates the rate of gluconeogenesis. Liver and secondarily the kidney are the organs that supply circulating blood and consequently, various tissues with glucose. Many tissues depend primarily on glucose to maintain adequate energy levels for their proper function during fasting.
Several tissues, including the brain, erythrocytes, renal medulla, the lens and cornea of the eye, testes, skeletal muscles during exercise, require continuous glucose supply. Among these tissues, the brain uses glucose exclusively in both fed state and fasting state except for prolonged fasting, which uses ketones. Notably, the daily amount of glucose used by the brain accounts for 70% of the total glucose produced by the liver in a normal fasting person.
Initially, during the first hours of fasting, hepatic glycogenolysis is the primary source of glucose. Glucose stored as glycogen can cover the energy needs roughly for one day; the amount of glucose supplied by glycogen reserves is 190 g while the daily needs for glucose are 160 g. After several hours of starvation, gluconeogenesis and glycogenolysis contribute equally to blood glucose. The amount of glucose supplied by glycogen decreases rapidly while the increase in the glucose fraction contributed by gluconeogenesis results in keeping constant the total amount of glucose produced. Estimates are that 54% of glucose comes from gluconeogenesis after 14 hours of starvation, and this contribution raises to 64% after 22 hours and up to 84% after 42 hours. However, hours later that glycogen stores deplete, the body uses as glucose sources lactate, glycerol, glucogenic amino acids, and odd chain fatty acids. In prolonged fasting, kidney participation in gluconeogenesis is increased and is responsible for about 40% of total gluconeogenesis.
Alanine, produced in skeletal muscles by protein catabolism and subsequent transamination reactions, is shuttled out in blood and taken up by the liver. Inside hepatocytes, alanine undergoes transamination into pyruvate, used for gluconeogenesis. Glucose produced in the liver is shuttled out in circulation and taken up by muscle cells for use in ATP production (Cahill cycle). Other gluconeogenic amino acids (e.g., methionine, histidine, valine) as well as gluconeogenic and ketogenic (e.g., phenylalanine, isoleucine, threonine, tryptophane) become transaminated into different intermediates of the gluconeogenic pathway.
In red blood cells and other tissues (lens) that lack mitochondria as well as the exercising muscle tissue that favors anaerobic metabolism, glucose is converted to pyruvate and subsequently to lactate. Lactate is secreted into plasma and picked up by the liver for conversion into glucose (Cori cycle) via a redox reaction catalyzed by lactate dehydrogenase.
Fatty acids are stored as triglycerides and mobilized by the hormone-sensitive lipase (HSL); glycerol from the triglyceride structure is released in blood to be taken up by the liver, phosphorylated by glycerol kinase and oxidized into dihydroxyacetone phosphate -an intermediate of gluconeogenesis/ glycolysis pathway- by glycerol phosphate dehydrogenase. Odd-chain fatty acids, in contrast to the ketogenic even- chain fatty acids, are converted with beta-oxidation into propionyl CoA. The latter converts after several steps into methylmalonyl CoA. Methylmalonyl CoA mutase/B12 catalyzes the conversion of the latter into succinyl-CoA. Succinyl-CoA is an intermediate of TCA cycle that is eventually converted into oxaloacetic acid and enters as such the gluconeogenesis pathway. Even-chain fatty acids and purely ketogenic amino acids (leucine, lysine) that convert to acetyl-CoA cannot enter gluconeogenesis as no pathway can reverse the step catalyzed by pyruvate dehydrogenase (PDH).
It is worth mentioning that in certain conditions, such as ischemic strokes and brain tumor development, astrocytes have increased activity of gluconeogenic enzymes, and they use as substrates lactate, alanine, aspartate, glutamate.
I) Glucagon regulates gluconeogenesis through:
- Changes in allosteric regulators (reduces the levels of fructose-2,6 bisphosphate)
- Covalent modification of enzyme activity (phosphorylation of pyruvate kinase results in its inactivation)
- Induction of enzymes gene expression (glucagon via CRE response elements increases the expression of PEPCK
II) Acetyl CoA activates pyruvate carboxylase allosterically
III) Substrate availability might increase or decrease the rate of gluconeogenesis
IV) AMP inhibits fructose-1,6 bisphosphatase allosterically
For gluconeogenesis to occur, the ADP/ATP ratio must be very low, since gluconeogenesis is an energy demanding process requiring high energy molecules to be spent in several steps. In between meals, during early fasting, when cell via TCA cycle has generated sufficient ATP levels, the increased ATP levels inhibit several highly regulated TCA cycle enzymes (citrate synthase, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase). Acetyl-CoA is the indicator of cells metabolic activity and functions as a gluconeogenesis regulator at a local level. Acetyl-CoA levels back up and allosterically activate pyruvate carboxylase. In this way, the cell makes sure that gluconeogenesis and TCA cycle will not happen simultaneously.
Pyruvate generation from phosphoenolpyruvate is the last irreversible step of gluconeogenesis. Once cells are committed into the gluconeogenesis pathway, the reverse reaction occurs in two steps to go around the irreversible step and synthesize phosphoenolpyruvate from pyruvate.
1) The first step involves pyruvate carboxylase (PC), a ligase, adding a carboxyl group on pyruvate to create oxaloacetate. The enzyme consumes one ATP molecule, uses as a cofactor biotin (vitamin B7) and uses a CO2 molecule as a source of carbon. Biotin is bound to a lysine residue of PC. After ATP hydrolysis, an intermediate molecule PC-biotin-CO2 forms, that carboxylates pyruvate forming oxaloacetate. This reaction, apart from forming an intermediate for gluconeogenesis, provides oxaloacetic acid to TCA cycle (anaplerotic reaction). In muscle cells, PC is used mainly for anaplerotic reasons. The enzyme also requires magnesium. Pyruvate carboxylation happens in mitochondria; then via malate shuttle, oxaloacetate is being shuttled into the cytosol to be phosphorylated. Malate can cross the inner mitochondrial membrane while oxaloacetic acid cannot. In cytosol along with the oxidation of oxaloacetic acid into malate, NAD+ gets reduced into NADH. The produced NADH is used in a subsequent step when 1,3 bisphosphoglycerate converts into glyceraldehyde-3 phosphate.
2) The next exergonic reaction catalyzed by PEP carboxykinase (PEPCK), a lyase, uses GTP as a phosphate donor to phosphorylate oxaloacetate and form PEP. Glucocorticoids induce PEPCK gene expression; cortisol after binding its steroid receptor intracellularly moves inside the cell nucleus and binds with its zinc finger domain, the glucocorticoid response element (GRE) on DNA.
3) The rest of the reactions are reversible and common with gluconeogenesis. Enolase, a lyase, cleaves carbon-oxygen bonds and catalyzes the conversion of PEP into 2-phosphoglycerate. Phosphoglycerate mutase, an isomerase, catalyzes the conversion of 2-phosphoglycerate to 3-phosphoglycerate by transferring a phosphate from carbon-2 to carbon-3. Phosphoglycerate kinase using ATP as a phosphate donor and Mg+2 to stabilize with its positive charge the phosphotransfer reaction converts 3-phosphoglycerate to 1,3- bisphosphoglycerate. Glyceraldehyde 3-phosphate dehydrogenase catalyzes the reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate. NADH is oxidized as it donates its electrons for the reaction. As described earlier, glycerol phosphate from triglyceride catabolism is converted eventually into DHAP. Triosephosphate isomerase converts DHAP into glyceraldehyde 3-phosphate. Aldolase A converts glyceraldehyde 3-phosphate into fructose-1,6 bisphosphate.
4) The following irreversible step involves the conversion of fructose 1,6 bisphosphate into fructose-6 phosphate. This step is important as it is the rate-limiting step of gluconeogenesis. Fructose-1,6 bisphosphatase catalyzes the dephosphorylation of fructose-1,6 bisphosphate, requiring bivalent metal cations (Mg+2, Mn+2); this is a highly regulated step both globally and locally. Locally, increased ATP levels, as well as increased levels of citrate (the first intermediate of TCA cycle), activate the enzyme, while increased AMP and increased fructose-2,6 bisphosphate (F2,6BP) inactivate the enzyme. Glucagon by binding to its receptor, a GPCR, activates adenylate cyclase. The resulting increase in cyclic AMP (cAMP) levels leads to the activation of protein kinase A (PKA). PKA phosphorylates fructose 2,6 bisphosphatase (F2,6BPase) and phosphofructokinase-2 (PFK-2). Phosphorylated PFK-2 is inactive while F2,6BPase is active and catalyzes the dephosphorylation of fructose 2,6 bisphosphate. Dephosphorylated F-2,6BP is inactive; hence, it does not have any negative effect on F1,6BPase.
5) The last irreversible reaction involves glucose-6 phosphatase catalyzing the hydrolysis of glucose-6 phosphate into glucose. This enzyme is expressed primarily in liver as well as in kidneys and intestinal epithelium. The reaction happens in the endoplasmic reticulum of the cells. Muscle cells do not express glucose-6 phosphatase as they produce glucose to maintain their own energy needs.
Von Gierke Disease- Glycogen storage disease type 1
Liver cells lack glucose-6 phosphatase, the enzyme required to release glucose from liver cells by dephosphorylating them. Von Gierke disease is a condition affecting both glycogenolysis and gluconeogenesis since the missing enzyme is common in both pathways resulting in accumulation of glucose-6 phosphate in liver cells. Symptoms include:
- Hepatomegaly and kidney enlargement due to glycogen accumulation
- Severe fasting hypoglycemia since liver cells cannot release glucose in blood postprandially
- Lactic acidosis since accumulated glucose-6 phosphate blocks gluconeogenesis and consequently lactate uptake
- Hypertriglyceridemia, since increased levels of glucose-6 phosphate favor glycolysis and acetyl-CoA production, leading to increased malonyl-CoA synthesis and subsequent inhibition of carnitine acyltransferase 1 (the rate-limiting mitochondrial enzyme of fatty acid beta-oxidation);
Hyperuricemia is the result of increased uric acid production (glc-6P that via HMP shunt is converted into ribose-5P and purines) and decreased uric acid excretion (uric acid competes with lactate for excretion via the same organic acid transporter in proximal renal tubules). Other symptoms include protruding abdomen (hepatomegaly), truncal obesity and short height, muscle wasting as well as a rounded doll’s face.
Pyruvate Carboxylase deficiency
Pyruvate carboxylase deficiency is a condition where cells lack pyruvate carboxylase or have an altered enzyme and manifest with lactic acidosis, hyperammonemia, and hypoglycemia. Hyperammonemia is due to pyruvate not being converted into oxaloacetic acid. Oxaloacetic acid gets transaminated into aspartate; reduction in aspartate levels results in the reduced introduction of ammonia into the urea cycle.