gluconeogenesis is the process that occurs mainly in the liver in which glucose is produced from non-carbohydrate sources during times of fasting, exercise, or stress. it is essentially the opposite of glycolysis, in that instead of producing pyruvate from glucose, it synthesizes glucose from pyruvate. pyruvate itself is supplied by several different sources: lactate can be oxidized into pyruvate and is available from anaerobic glycolysis or by adipocytes in red blood cells. alanine can be transaminated into pyruvate and is produced from other amino acids released from the muscle. glycerol also serves as the precursor to an intermediate of gluconeogenesis, DHAP. the reactions of gluconeogenesis can be divided into three major sections:
conversion of pyruvate to phosphoenolpyruvate (PEP)
1. pyruvate is formed from alanine or lactate in the cytosol.
2. pyruvate diffuses into the mitochondria and is carboxylated to oxaloacetate via pyruvate carboxylase (recall that this is an anaplerotic reaction of the TCA cycle)
3. oxaloacetate is either transaminated to aspartate, or reduced to malate (using NADH as an electron source) and transported back out of the mitochondria.
4. oxaloacetate is reformed in the cytosol either by transamination of aspartate or oxidation of malate.
5. oxaloacetate is converted to phosphoenolpyruvate via phosphoenolpyruvate carboxykinase, using one GTP.
conversion of PEP to fructose 1,6 bisphosphate (reverse of glycolysis)
6. PEP is converted to 2-phosphoglycerate
7. 2-phosphoglycerate is converted to 3-phosphoglycerate
8. 3-phosphoglycerate is converted to 1,3-bisphosphoglycerate
9. 1,3-bisphosphoglycerate is converted to G3P.
10. for every two molecules of G3P that are formed, one isomerizes to DHAP
11. G3P condenses with DHAP to form fructose 1,6 bisphosphate.
conversion of fructose 1,6 bisphosphate to glucose (reverse of glycolysis)
12. fructose 1,6 bisphosphate is converted to fructose 6-phosphate via fructose 1,6 bisphosphatase.
13. fructose 6-phosphate is isomerized to glucose 6-phosphate via phosphoglucoisomerase.
14. glucose 6-phosphate is converted to glucose via glucose 6-phosphatase.
the reactions in bold are irreversible, endergonic reactions that use enzymes that are not used in the reverse glycolytic pathway. this is significant because the relative activity of these competing enzymes determines whether the reaction will proceed in the glycolytic or gluconeogenic pathway. the regulation of the first of these reactions, the conversion of oxaloacetate to PEP, is the most complex and is regulated by several enzymes in upstream reactions beginning with pyruvate production. the first, pyruvate dehydrogenase, is normally responsible for oxidizing pyruvate to acetyl CoA but is deactivated during gluconeogenesis, allowing pyruvate to instead be carboxylated into oxaloacetate. pyruvate carboxylase, the enzyme that catalyzes this reaction, is in turn activated by acetyl CoA, which is produced during the fatty acid oxidation which occurs during fasting or stress. these two reactions work in tandem during fasting conditions to ensure production of oxaloacetate from pyruvate rather than acetyl CoA.
the third enzyme which takes place in the regulation of the production of PEP is PEP carboxykinase, which converts oxaloacetate to PEP. in fasting conditions, glucagon and epinephrine stimulate cAMP to increase transcription of PEPCK enzymes, increasing the quantity of enzyme in the cell (called inducing). finally, the last enzyme involved is pyruvate kinase, which normally converts PEP back into pyruvate (recall the last step of glycolysis). high glucagon levels causes phosphorylation of the enzyme (using a mechanism involving cAMP and protein kinase A) and inactivates it-- thus allowing PEP to be used for gluconeogenesis instead of being uselessly cycled back to pyruvate. these four enzymes basically act as "switches" which first turn on the gluconeogenic pathway by allowing pyruvate to be converted to PEP.
the next places for enzymatic regulation of the gluconeogenic pathway are: the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate, and the conversion of glucose 6-phosphate into glucose. both reactions are similar in that (as mentioned earlier) they use enzymes that are not the same as the reverse glycolytic reaction. in fasting conditions, the enzymes that catalyze the glycolytic reaction are deactivated, allowing the reaction to proceed in the gluconeogenic direction.
the book then talks about what happens in the liver and body tissues during, after, and long after a meal. during a high carbohydrate meal, blood glucose levels can rise from the normal 80-100 mg/dL to a high of 140 mg/dL. during this time insulin is secreted from the beta cells in the pancreas, and glucagon levels decrease. the net result is a storage of glucose in the liver as glycogen. within a few hours after eating, blood glucose and insulin levels fall back down, and glucagon levels start to rise- this initiates the process of glycogenolysis, which is the conversion of the stored glycogen in the liver back into glucose to maintain blood glucose levels. glucagon stimulates glycogenolysis and inhibits glycogen storage concurrently via production of cAMP, which stimulates protein kinase A to inactivate the enzyme related to glycogen synthesis as well as activate the glycogenolytic pathway. within 4 hours after a meal, as the liver's glycogen supply is decreasing (it takes about 30 hours to deplete the liver's supply of glycogen), gluconeogenesis is also stimulated by glucagon and falling blood sugar levels.
questions
1. what happens in the liver during fasting?
2. what is gluconeogenesis?
3. what are the three carbon sources for gluconeogenesis in humans?
4. describe the role of lactate as a gluconeogenic precursor.
5. describe the role of alanine as a gluconeogenic precursor.
6. describe the role of glycerol in gluconeogenesis.
7. describe the conversion of pyruvate to PEP.
8. what determines the path in which oxaloacetate will be converted and transported across the mitochondrial membrane?
9. describe the conversion of PEP to fructose 1,6 bisphosphate.
10. describe the conversion of fructose 1,6 bisphosphate to glucose.
11. describe the conversion of glycerol to DHAP.
12. what are other factors that can stimulate gluconeogenesis?
13. what are the three main reactions that are regulated in gluconeogenesis?
14. how does the fasting state deactivate pyruvate dehydrogenase?
15. how does the fasting state activate pyruvate carboxylase?
16. how is PEP carboxykinase regulated?
17. what is pyruvate kinase and how is it regulated?
18. describe the regulation of the reaction from fructose 1,6 bisphosphate to fructose 6-phosphate.
19. describe the regulation of the reaction from glucose 6-phosphate to glucose.
20. what is the energy consumption during gluconeogenesis and where does it happen?
21. what are normal blood glucose levels for fasting, right after a meal, 2 hours after a meal, and starvation?
22. describe the pancreas's actions after ingestion of a high glucose meal.
23. glycerol, glucagon, glycogen.
24. describe the stimulation of glycogenolysis in the liver.
25. describe what happens roughly 4 hours after a meal.
26. describe what happens during prolonged starvation.
27. how long does it take to deplete liver glycogen stores? (and therefore halt glycogenolysis)
answers
1. liver releases glucose into the blood via glycogenolysis and gluconeogenesis.
2. the process by which glucose is created in the liver from non carbohydrate sources.
3. lactate, glycerol, and amino acids- particularly alanine.
4. lactate is produced by anaerobic glycolysis through reduction of pyruvate or by adipocytes in the fed state or by red blood cells. lactate is oxidized into pyruvate, which is a precursor for gluconeogenesis.
5. alanine is produced in the muscle from other amino acids (whenever insulin is low or stress hormones are high) and from glucose. it is converted to pyruvate via alanine aminotransferase.
6. glycerol is released from adipose tissue whenever insulin levels are low or stress hormones are high. it is converted to DHAP, which is a gluconeogenetic intermediate (as well as a glycolytic one)
7. pyruvate is created from alanine or lactate in the cytosol, and then travels into the mitochondria, where it is carboxylated to oxaloacetate via pyruvate carboxylate (an anaplerotic reaction of the TCA cycle). oxaloacetate is then transaminated to aspartate or reduced to malate and transported back out into the cytosol, and reformed back into oxaloacetate (via oxidation or transamination). in the cytosol, oxaloacetate is decarboxylated by phosphoenolpyruvate carboxylkinase to form PEP.
8. the reduction of oxaloacetate into malate requires reducing equivalents; if the mitochondria has need for reducing equivalents for other reactions, it will use the other venue, the conversion to aspartate.
9. PEP is converted to fructose 1,6 bisphophate through a reversal of the glycolytic reactions. PEP is converted into 2-phosphoglycerate, to 3-phosphoglycerate, to 1,3 bisphosphoglycerate, and reduced to G3P. for every two molecules of G3P produced, one isomerizes to DHAP. G3P and DHAP condense to form fructose 1,6 bisphosphate.
10. fructose 1,6 bisphosphate has a phosphate removed by fructose 1,6bisphosphatase to form fructose 6 phosphate. fructose 6 phosphate is isomerized to glucose 6 phosphate by phosphoglucose isomerase. glucose 6 phosphate has a phosphate removed by glucose 6-phosphatase, producing glucose.
11. glycerol is converted to glycerol 3-phosphate via glycerol kinase, and then oxidized to DHAP.
12. prolonged exercise, stress, and a high protein diet.
13. OAA to PEP, fructose 1,6 bisphosphate to fructose 6 phosphate, glucose 6 phosphate to glucose. all three reactions use regulatory enzymes which are not involved in the reverse glycolytic pathway.
14. during the fasting state, fatty acids are released from adipose tissue and undergo beta oxidation, producing NADH, acetyl CoA, and ATP. the higher ATP / ADP ratio phosphorylates pyruvate dehydrogenase into the inactive form.
15. fatty acid oxidation produces acetyl CoA, which activates pyruvate carboxylase.
16. glucagon is released during fasting and EP is released during exercise/stress, both of which stimulate production of cAMP, which increases transcription of PEPCK genes.
17. pyruvate kinase is the enzyme that catalyzes the conversion of PEP back into pyruvate. when glucagon levels are high, pyruvate kinase is phosphorylated and inactive through a mechanism involving cAMP and protein kinase A.
18. this reaction occurs via the fructose 1,6 bisphosphotase enzyme, and normally would compete with the reverse reaction from glycolysis, fructose 6-phosphate to fructose 1,6 biphosphate via PFK-1. however, under conditions favoring gluconeogenesis, the enzymes that stimulate PFK-1 are inactive, allowing the reaction to head towards the production of glucose.
19. low insulin and glucose levels deactivate the enzyme for the glycolytic forward reaction and allow the glucose synthesis to occur.
20. for every mole of glucose that is produced, 6 moles of ATP and 2 moles of NADH are used. 2 moles of ATP at the conversion of pyruvate to oxaloacetate, 2 moles of ATP at the conversion of oxaloacetate to PEP, 2 moles of ATP at the conversion from 3-phosphoglycerate to 1,3 bisphosphoglycerate, and 2 moles of NADH at the reduction of 1,3 bisphospholycerate to G3P. (2 moles at each reaction because 2 molecules of pyruvate combine into one molecule of glucose)
21. fasting: 80-100mg/dL. right after a meal: up to 140mg/dL. 2 hours after a meal: back to 80-100mg/dL. starvation: not lower than 65mg/dL.
22. during a meal, the high glucose concentration in the blood stimulates the beta cells of the pancreas to increase insulin production. glucagon levels decrease in response to a high carbohydrate meal but increase in response to a high protein meal.
23. glycerol is released from adipose whenever levels of insulin are low and levels of glucagon is high-- and is converted into DHAP. glucagon is a hormone released by the alpha cells of the pancreas in response to decreasing blood glucose levels-- stimulating gluconeogenesis. glucagon also activates production of cAMP in liver cells, which activates protein kiase A, which inactivates glycogen synthase-- thus high glucagon levels inhibit glycogen production. glycogen is synthesized from glucose and stored in the liver.
24. high glucagon levels stimulate adenylate cyclate, which synthesizes cAMP. cAMP activates protein kinase A, which inactivates glycogen synthase, and activates phosphorylase kinase. phosphorylase activates phosphorylase b, which converts glycogen to glucose 1-P, which is then converted to glucose 6-P and then free glucose in the liver, which can then enter the blood.
25. in addition to supplementing blood glucose levels with glycogenolysis, gluconeogenesis is stimulated by the release of precursor material such as glycerol, alanine, and lactate from peripheral body tissues.
26. the body switches to fatty acid and ketone body oxidation and requires much less glucose.
27. ~30 hours
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