Gluconeogenesis week 2 Flashcards
Why is gluconeogenesis necessary for survival?
Net synthesis of glucose from various substances is termed gluconeogenesis. Blood glucose levels must be maintained for organs that use glucose as the primary substrate for fuel, such as brain, red blood cells, kidney medulla, lens, cornea, testis. Gluconeogenesis allows this maintenance after dietary glucose is used up.
What organs are capable of gluconeogenesis?
What are substrates for gluconeogenesis?
liver (90%), kidney (10%)
Gluconeogenesis can use various amino acids, lactate, pyruvate, propionate and glycerol to produce glucose. Glucose is also synthesized from galactose and fructose (these sugars can be isomerized/epimerized to form glucose but this is not considered gluconeogenesis)
2 Lactate → 2 Pyruvate ← Amino acids
↓
Amino acids → 2 Oxaloacetate ← 2 Propionate
↓
2 Glycerol → 2 Triose phosphate ← Fructose
↓
Galactose → Glucose
What are the Alanine and Cori cycles?
How much energy is used to run these cycles?
What additional cycles must run when the Alanine cycle runs? How energetically expensive is it?
These cycles can provide continuous glucose supply to tissues that require glucose as their primary energy source.
Peripheral tissues release Ala (from pyruvate in the muscle) or lactate (RBC, muscle, etc) as the end product of glucose metabolism for these cycles to work. These molecules are returned to the liver by the blood stream and there enter gluconeogenesis. Glucose produced in the liver is delivered by blood back to the tissues. To make glucose from lactate in the Cori cycle or glucose from pyruvate in the Ala cycle, 6 ATPs are needed in both cases.
However, the Ala cycle also brings amino nitrogen to the liver, which must be removed by the synthesis of urea. Four ATPs are needed to make one urea in the Ala cycle.
What are the 3 irreversible glycolytic enzymes?
What gluconeogenic enzymes circumvent their rxns?
Gluconeogenesis from lactate requires 6 ATPs.
2 lactate + 6 ATP + 6 H2O –> glucose + 6 ADP+ 6 Pi
Many enzymes of glycolysis are also common to gluconeogenesis, except for the three kinases:
pyruvate kinase (PK),
6-phosphofructo-1-kinase (PFK1)
hexokinase.
These enzymes are circumvented by the following enzymes in gluconeogenesis:
Pyruvate carboxylase and PEP
carboxykinase (1 & 2)
Fructose 1,6-biphosphatase (3)
Glucose 6-phosphates (4)
Explain the reactions of pyruvate carboxylase and PEP carboxykinase.
Indicate where these enzymes are localized, what cofactors are needed, and if energy is required for their rxns.
Step 1. Lactate is converted to pyruvate by Lactate dehydrogenase (LDH). NADH is generated and is also needed for a subsequent step. This enzyme works in reverse direction in glycolysis.
Step 2. Pyruvate cannot be converted to phosphoenolpyruvate (PEP) by PK because the reaction is irreversible under intracellular conditions. Pyruvate is converted into PEP by coupling two reactions that require energy in the form of high-energy phosphate.
The first reaction is catalyzed by pyruvate carboxylase (P–> OOA), which is a biotin-dependent enzyme and found only in mitochondria (ATP is needed for this reaction) and the second reaction is catalyzed by PEP carboxykinase (OOA –> PEP), which is found in mitochondria and cytosol (GTP is needed for this reaction).
Pyruvate + ATP + CO2 –> oxaloacetate + ADP + Pi Oxaloacetate + GTP Æ Phosphoenolpyruvate + GDP + CO2
Note: Conversion of Pyruvate to PEP by PEP carboxykinase costs 2 ATPs, (4 ATPs per glucose) while conversion of PEP to P during glycolysis yields only one ATP.
Explain the mechanism and compartmentalization of the rxns that pyruvate carboxylase and PEP carboxykinase catalyze.
Cytosolic pyruvate enters mitochondria because pyruvate carboxylase is present only in the mitochondria. Pyruvate is converted to oxaloacetate (OOA) by pyruvate carboxylase, which will transfer a CO2 molecule to pyruvate by its cofactor, biotin. Since OAA cannot cross the mitochondrial membrane, OOA is converted into malate, which is transported out into the cytosol (malate OAA shuttle).
In the cytosol, malate is converted back to OOA (by malate dehydrogenase) and then OOA is decarboxylated to PEP by PEP carboxykinase.
Explain the reason for the symptoms the patient presented with.
How can this disease be treated therapeutically? What is the typical outcome of a patient with this disease?
The metabolic function of pyruvate carboxylase is to provide oxaloacetate for both gluconeogenesis and Krebs cycle (anaplerotic reaction). Without oxaloacetate, neither of these pathways will work correctly. Alanine concentration is increased in plasma, since it could be converted to pyruvate but pyruvate cannot be converted further. Lipid accumulation is because pyruvate can’t go to glucose synthesis so it is converted to acetylCoA by pyruvate dehydrogenase and used for lipid synthesis. Lactic acidosis - glycolysis is highly upregulated but pyruvate cannot enter the Krebs cycle so it is converted to lactate by LDH. Glutamine greatly stimulated the growth of fibroblasts from a patient with pyruvate carboxylase deficiency. The function of the Krebs cycle can be stimulated by the addition of other intermediates. Glutamine can be deaminated to glutamic acid, which then transaminates to alphaketoglutarate. This, and asparagine could be used in therapy, since they can cross the blood-brain barrier, however, these are very expensive and not substantial for long-term care. Most of the patients with pyruvate carboxylase deficiency die at a very early age and those who survive are mentally retarded.
Explain the steps of gluconeogenesis from PEP to glucose formation. Indicate what enzymes are used, cofactors, and energy required if applicable.
Step 3. The steps from PEP to fructose 1,6-bisphosphate include steps of the glycolytic pathway in reverse. Thus, PEP is converted to 2 phosphoglycerate, which is converted to 3- phosphoglycerate, then to 1,3-biphosphoglycerate (uses 2 ATPs per glucose), which is converted to glyceraldehyde-3P (uses 2 NADH per glucose), which equilibrates with dihydroxyacetone phosphate, which then is converted to fructose-1,6-biphosphate. The NADH in cytosol needed to reverse GAPDH is generated by LDH converting lactate to pyruvate.
Step 4. Since PFK-1 (6-Phosphofructo-1-kinase) catalyzes an irreversible step in glycolysis it cannot be used. A way around this is fructose 1,6 bisphosphatase, which catalyzes irreversible hydrolysis of fructose 1,6-bisphosphate to F6P.
Step 5. In the next step, phosphoglucose isomerase is freely reversible and functions in both glycolysis and gluconeogenesis. This isomerizes F6P to G6P.
Step 6. In the last step, glucose 6-phosphatase is used instead of glucokinase (and hexokinase) and catalyzes an irreversible hydrolytic reaction under intracellular conditions to convert G6P to glucose. Note that this enzyme is only found in the liver and kidney for the purpose of gluconeogenesis.
What is the cellular localization of glucose-6-phosphatase? Describe the process of how G6P gets to this enzyme.
What transporters are involved in this process?
What genetic defects are known involving this process? What 2 cellular processes do they interfere with? What disease do they lead to?
G6Phosphatase is a membrane bound enzyme within the ER with its active site on the luminal surface. A translocase for G6P is required to move G6P from the cytosol tot eh ER for G6Phosphatase attack. Special transporters are needed to transport inorganic phosphate and glucose (GLUT7) to the cytosol and glucose to the blood stream (GLUT2).
Genetic defects in the translocase and the phosphatase are known. These deficiencies interfere with gluconeogenesis and glycogenolysis, resulting in accumulation of glycogen in the liver (glycogen storage disease). These deficiencies also result in hypoglycemia.
What are the only 2 amino acids that cannot be used for gluconeogenesis? What process can they be used for?
If the catabolism of an amino acid can yield either ____ or ____ then net glucose synthesis can occur.
What are rxns that lead to net synthesis of TCA cycle intermediates called? How/why do they support gluconeogenesis?
What cycle runs closely in conjunction with gluconeogenesis from amino acids?
All amino acids, except for Leu and Lys, can supply carbon for net synthesis of glucose by gluconeogenesis.
Leu and Lys cannot function as carbon sources for net synthesis of glucose. These amino acids are ketogenic (make ketone bodies) and not glucogenic. Acetyl CoA is the end product of Lys metabolism and acetoacetate and acetyl CoA are end products of Leu metabolism. No pathway exists for converting acetoacetate or acetyl CoA into pyruvate or OOA in humans and other animals because the reaction catalyzed by the PDH complex is irreversible.
All other amino acids are glucogenic or gluco ketogenic. Glucogenic amino acids give rise to net synthesis of either pyruvate or OOA, while amino acids that are both glucogenic and ketogenic also yield ketone bodies, acetoacetate or acetyl CoA, which is readily converted into ketone bodies.
If the catabolism of an amino acid can yield either
Pyruvate or OAA, then net glucose synthesis can occur.
Reactions that lead to net synthesis of TCA cycle intermediates are called anaplerotic reactions and support gluconeogenesis because they provide net synthesis of OAA.
Since gluconeogenesis from amino acids imposes N load on the liver, a close relationship exists between urea synthesis and glucose synthesis from amino acids.
Why are children (esp premies and small-for-gestational-age neonates) more susceptible to hypoglycemia?
Premature and small-for-gestational-age neonates have a greater susceptibility to hypoglycemia than full term, appropriate-for-gestational-age infants. Several factors appear to be involved. Children in general are more susceptible than adults to hypoglycemia, simply because they have larger brain/body weight ratios and the brain utilizes disproportionately greater amounts of glucose than the rest of the body. Newborn infants have a limited capacity for ketogenesis, apparently because the transport of longchain fatty acids into liver mitochondria of the neonate is poorly developed. Since ketone body use by the brain is directly proportional to the circulating ketone body concentration, the neonate is unable to spare glucose to any significant extent by using ketone bodies. The consequence is that the neonate’s brain is almost completely dependent on glucose obtained from liver glycogenolysis and gluconeogenesis.
The capacity for hepatic glucose synthesis from lactate and alanine is also limited in newborn infants. This is because the rate-limiting enzyme phosphoenolpyruvate carboxykinase is present in very low amounts during the first few hours after birth. Induction of this enzyme to the level required to prevent hypoglycemia during the stress of fasting requires several hours. Premature and small-for-gestational-age infants are believed to be more susceptible to hypoglycemia than normal infants because of smaller stores of liver glycogen. Fasting depletes their glycogen stores more rapidly, making these neonates more dependent on gluconeogenesis than normal infants.
Why can odd chain (and FA with methyl branches) FA be used for glucose synthesis but even numbered chains cannot?
Explain the process of the generation of glucose from odd chain FA.
How can glycerol (from TGs) be used for gluconeogenesis?
Lack of a pathway that would form OAA from acetyl CoA also means that glucose cannot be synthesized from fatty acids. However, this statement is true only for straight chain even carbon number fatty acids that yield only acetyl CoA.
An exception applies to fatty acids with methyl branches or odd number of carbons that also yield propionyl CoA, which is a good precursor for gluconeogenesis by generating OOA.
Propionate –> Propionyl CoA –> Methylmalonyl CoA –> Succinyl CoA –> ½glucose
Triacylglycerols when liberated from adipose storage also yield one glycerol, which is also a substrate for gluconeogenesis. Phosphorylation of glycerol by glycerol kinase produces glycerol 3-P which can be converted by glycerol 3-P DH into DHAP.
Explain how fructose, galactose, and mannose can be converted to glucose.
Fructose
Humans consume large quantities of fructose in the form of sucrose, which is hydrolyzed in the small bowel to fructose and glucose. In the liver, fructose is phosphorylated by a special ATP linked kinase (fructokinase) yielding fructose 1-P. A special aldolase (F1P aldolase, also called aldolase B) then cleaves F-1-P to make one dihydroxyacetone phosphate (DHAP) and one glyceraldehyde. The latter is reduced to glycerol. Two molecules of DHAP from one fructose can be converted to glucose by enzymes of the gluconeogenesis pathway or alternatively, into pyruvate or lactate by the last stage of glycolysis.
Fructose
↓
F-1-P
↓
DHAP + Glyceraldehyde
↓
Glycerol
↓
DHAP <– G-3-P
↓
Glucose
Galactose: Milk sugar or lactose provides galactose and glucose. Galactose is metabolized to galactose 1-P by galactokinase, then to glucose 1-P, then to glucose 6-P then to glucose. UDP-glucose serves as a recycling intermediate.
Mannose: Mannose is in limited quantities in our diet. It is phosphorylated by hexokinase and then converted into fructose 6-P by mannose phosphate isomerase. This can then be used in either glycolysis or gluconeogenesis.
How many ATPs are required to make glucose from 2 lactate? How many are required to make glucose from alanine?
How many NADHs are needed?
What process usually provides energy for gluconeogenesis?
6 ATPs are needed to make glucose from two lactate, and 10 ATPs from Alanine. Additionally, 2 NADH is also needed. This cost in energy is usually provided by fatty acids in blood that are oxidized by liver mitochondria with the production of large amounts of ATP.
What enzymes of gluconeogenesis are regulated? What factors stimulate/inhibit them?
Both glycolysis and gluconeogenesis take place in the cytosol of liver and kidney. Reciprocal control is needed to avoid futile cycle.
When glycolysis is upregulated –> gluconeogenesis is down regulated
The enzymes involved in the irreversible steps of glycolysis are the ones that are regulated in gluconeogenesis:
- pyruvate carboxylase (stimulated by Acetyl CoA)
- PEP carboxykinase (Glucagon induces the synthesis of enzyme)
- fructose 1,6-bisphosphatase (AMP, F2,6-BP inhibit; glucagon stimulates by upregulating enzyme protein synthesis)
- glucose-6-phosphatase: glucagon induces synthesis
What glycolytic and gluconeogenic enzymes does glucagon effect the transcription of? Explain the cellular mechanism behind this process.
What is the mechanism of insulin regulation of enzyme levels?
Effect of glucagon on gene transcription levels:
Upregulates
- PEP carboxykinase
- fructose 1,6 bisphosphatase
- glucose-6 phosphatase
- various aminotransferases
Downregulates
- glucokinase
- PFK-1
- pyruvate kinase
The gene induction occurs through glucagon activating PKA. PKA phosphorylates the cAMP response element binding protein (CREB), a trans acting factor that when phosphorylated, can bind to a cAMP response element (CRE), a cis acting element within the regulation region of responsive genes. This promotes transcription of genes such as PEP carboxykinase.
Insulin opposes the action of glucagon by activating an insulin response element binding protein (IREB), which binds to an insulin response element (IRE) and inhibits transcription.
What effect can alcohol consumption have on gluconegenesis?
Excess levels of NADH in the cytosol derived from alcohol oxidation, that has to be transferred into the mitochondria by the malate-Asp shuttle, forces the equilibrium of the LDH and MDH reactions in the directions of lactate and malate formation (may cause lactic acidosis). Pyruvate is forced to form lactate (to consume NADH) and OAA is forced to form malate (to consume NADH). This inhibits glucose synthesis by limiting the availability of pyruvate and OAA.
A similar situation can occur in a normal person, who drinks alcohol after exercise, or in fasting children who ingest alcohol.
