Lecture 17 and 18
Describe the rate of gluconeogenesis
-the rate of gluconeogenesis is often limited by the availability of substrate, including the rate of proteolysis in muscle or, in some cases, muscle mass
What is the overall stoichiometry for gluconeogenesis from pyruvate
2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 6 H2O g--> glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+ + 2 H+
Describe fructose metabolism
Approximately 10 % of the calories of a western diet come from fructose. The major source of fructose is sucrose which, when cleaved in the intestine, releases equimolar amounts of fructose and glucose. Entry of fructose into cells is insulin-independent and in contrast to glucose does not promote the secretion of insulin. For fructose to enter the pathways of intermediary metabolism it must first be phosphorylated which can be accomplished by hexokinase or fructokinase (ketohexokinase). Hexokinase has a high km for fructose and it is usually saturated with glucose. Therefore, it can only phosphorylate fructose when the levels of fructose are very high. As such fructokinase provides the primary mechanism for fructose phosphorlyation. It is found in the liver, kidney, and small intestine and converts fructose into fructose-1-phosphate (F-1-P)using ATP. F-1-P is not converted to F-1,6-P but is cleaved by aldolase B to yield dihydroxyacetone (DHAP) and glyceraldehyde. DHAP can directly enter glycolysis, gluconeogenesis.
What organs use glucose as the primary substrate for fuel
Brain RBCs Kidney Medulla Lens Cornea Testis
How do we receive glucose homeostasis?
By utilizing our diet, short term storage, (glycogenolysis) or new synthesis (gluconeogenesis).
T/F: all 20 of the amino acids, except leucine and lysine, can be degraded to pyruvate or TCA cycle intermediates.
FALSE: all 20 of the amino acids, except leucine and lysine, can be degraded to pyruvate or TCA cycle intermediates. When glycogen stores are depleted in muscle during exertion and liver during fasting, catabolism of muscle proteins to amino acids contributes the major source of carbon for maintenance of blood glucose levels.
T/F: since gluconeogenesis is conceptually the opposite of glycolysis, we can essentially just turn glycolysis backwards and have the process.
FALSE:Gluconeogenesis is conceptually the opposite of glycolysis, but proceeds by a slightly different pathway, involving both mitochondrial and cytosolic enzymes. Thus, we need to find a way to bypass the three kinase reactions (since all three of these consist of such a - free energy)
Briefly list the 3 allosteric effectors of gluconeogenesis
Fructose 2,6-bisphosphate and AMP Citrate Acetyl CoA
Describe the reactions that are bypassed in gluconeogenesis and how
Fructose-1, 6-bisphosphate is dephosphorylated by fructose-1,6-bisphosphatase, releasing free phosphate, and bypassing phosphofructokinase. Fructose-6-phosphate is converted to glucose-6-phosphate by a phosphoglucose isomerase (readily reversible). Glucose-6-phosphate is then converted to glucose by glucose-6-phosphatase, bypassing glucokinase (liver) (hexokinase-- kidney). Glucose-6-phosphatase is a membrane bound enzyme within the ER and the active site is on the luminal surface of the membrane. A translocase for G6P is required to move G6P from cytosol to the ER for phosphatase attack. Special transporters are then needed to transport inorganic phosphate and glucose (GLUT- 7) to the cytosol from the ER and glucose to the blood stream (GLUT-2). Genetic defects in the translocase or the phosphatase are known and interfere with gluconeogenesis. The overall stoichiometry for gluconeogenesis from pyruvate is: 2 pyruvate + 4 ATP + 2 GTP + 2 NADH + 6 H2O g--> glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+ + 2 H+
Describe glucagon's affects on pyruvate kinase
Glucagon inhibits pyruvate kinase, and here's how: Glucagon activates adenylate cyclase to produce cAMP, which activates PKA (protein kinase A), that phosphorylates and inactivates pyruvate kinase (Liver isoform only). Inactivation of PK stimulates gluconeogenesis by blocking futile conversion of PEP to pyruvate. In addition, glucagon activates lipases in adipose tissue, promoting release of fatty acids into the bloodstream. These fatty acids are broken down in the mitochondria of liver, resulting in high concentrations of acetyl CoA. 1) Remember, acetyl CoA acts as an allosteric activator of pyruvate carboxylase and an inhibitor of PDH. 2) Also, some the acetyl CoA will be utilized in the Krebs cycle to generate the ATP required for gluconeogensis
Describe the basic concept behind gluconeogensis
Gluco---sugar, New----new, Genesis------ make/synthesize Gluconeogenesis is a metabolic pathway that results in the generation of glucose from non-carbohydrate carbon substrates. This process occurs during periods of fasting, starvation, low-carbohydrate diets, or intense exercise. Approximately 90 % takes place in the liver with the rest occurring in the cortex of the kidneys under normal conditions. During prolonged starvation the kidney can account for up to 40%. Gluconeogenesis is conceptually the opposite of glycolysis, but proceeds by a slightly different pathway, involving both mitochondrial and cytosolic enzymes.
Describe the second gluconeogenesis bypass 1: convesion of pyruvate into PEP
In the second reaction phosphoenolpyruvate carboxykinase (PEPCK) simultaneously decarboxylates and phosphorylates oxaloacetate to generate phosphoenolpyruvate (PEP). GTP is used as the phosphoryl donor. Therefore bypass 1 can be written as follows: Pyruvate + ATP + CO2 g oxaloacetate + ADP + Pi Oxaloacetate + GTP g Phosphoenolpyruvate + GDP + CO2 Each reaction requires an ATP equivalent and for each glucose molecule to be synthesized (6 carbon) we need 2 pyruvates (3 carbon), therefore the initial bypass costs a total of 4 ATPs. 1) CO2 is activated and transferred to pyruvate by pyruvate carboxylase producing oxaloacetate. 2) Oxaloacetate cannot cross the mitochondrial membrane so it is reduced to malate that can. 3) In the cytosol, malate is deoxidized to oxaloacetate, which is converted to phosphoenolpyruvate by PEP carboxykinase
Describe fructose 2,6 bisphosphate regulation of gluconeogenesis
In the well fed state, insulin is present and the PFK-2/FBP2 enzyme is not phosphorylated (open circles). This causes the PFK2 portion of the enzyme to be active, fructose 2,6 bisphosphate is synthesized promoting glycolysis and inhibiting gluconeogenesis. Conversely, in the starved state (need), glucagon will cause the phosphorylation (yellow filled circles) of the enzyme which will then cause the dephosphorlyation of fructose 2,6 bisphosphate via the FBP2 portion of the enzyme. This stops the promotion of glycolysis and relieves the inhibition of gluconeogenesis.
Describe the cori cycle
Lactate, which comes primarily from RBCs and muscle, is a predominate source of carbon atoms for glucose synthesis by gluconeogenesis. In the Cori cycle, the lactate produced by the lactate dehydrogenase reaction (LDH) during anaerobic glycolysis 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.
What are the sources (substrates) for gluconeogenesis?
Lactate: cori cycle Pyruvate: glucose-alanine cycle Amino acids (especially in starved state) Glycerol Proprionate (3 C molecule) OTHER sugars -among these, muscle protein (amino acids) is the major precursor of blood glucose -the rate of gluconeogenesis is often limited by the availability of substrate, including the rate of proteolysis in muscle or, in some cases, muscle mass During prolonged fasting, malnutrition or starvation, we lose both adipose and muscle mass
Describe the enzymes decreased by insulin
PEP carboxykinase
Describe the first gluconeogenesis bypass 1: conversion of pyruvate into PEP
Pyruvate is converted into PEP by coupling two reactions that require energy in the form of high-energy phosphate. The first is catalyzed by pyruvate carboxylase (pyruvategoxaloacetate), which is biotin-dependent and found only in mitochondria (ATP is needed). Note: It is the biotin that binds the CO2. To be used for gluconeogenesis, the oxaloacetate must be transferred back into the cytoplasm; however, mitochondria lack an efficient transporter for oxaloacetate. Therefore, oxaloacetate is reduced to malate by malate dehydrogenase which converts one molecule of NADH to NAD+. Malate is then transported out and reoxidized to oxaloacetate, regenerating NADH from NAD+ in the cytoplasm.
Describe the glucose/alanine cycle
Pyruvate, generated in muscle and other peripheral tissues, can be transaminated (TA) to alanine which is returned to the liver for gluconeogenesis (GNG). The glucose-alanine cycle is, therefore, an indirect mechanism for muscle to eliminate nitrogen while replenishing its energy supply (alanine released from muscle --> blood --> liver). Within the liver the alanine is converted back to pyruvate and used as a gluconeogenic substrate (if that is the hepatic requirement) or oxidized in the TCA cycle. The amino nitrogen is converted to urea in the urea cycle and excreted by the kidneys. [alanine + alpha-ketoglutarate <> pyruvate + glutamate]
Describe how glycerol, from triacylglycerol, can be used for gluconeogenesis
The catabolism of triacylglycerol yields glycerol (highlighted in yellow) and acetylCoA. Oxidation of 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. The glycerol backbone of lipids can be used for gluconeogenesis. The liver (and only liver) has the enzyme glycerol kinase which phosphorylates glycerol to yield glycerol-3-phosphate. This is then followed by the dehydrogenation to dihydroxyacetone phosphate (DHAP) by glyceraldehyde-3-phosphate dehydrogenase (G3PDH,same enzyme as in the shuttles) which can then feed into gluconeogenesis.
Describe how propionate can be used as a substrate for gluconeogenesis
The last round of beta oxidation from fatty acids with an odd number of carbon atoms and the oxidation of some amino acids generates as the terminal oxidation product, propionyl-CoA. Propionyl-CoA is converted to the TCA intermediate, succinyl-CoA, which can feed into the TCA cycle and therefore into gluconeogenesis.
Describe how ethanol ingestion can inhibit gluconeogenesis
The oxidation of alcohol can cause excessive levels of NADH in the cytoplasm. Ethanol + NAD+ -----> acetaldehyde + NADH + H+ Thus, the increase in nadh will limit gluconeogenesis The equilibrium of the LDH reaction is shifted toward lactate, limiting gluconeogenesis from pyruvate from either lactate or alanine. The cell will transfer the excess NADH into the mitochondria in an attempt to resolve the metabolic imbalance. It does this through use of the shuttles which results in a shift of cytosolic oxaloacetate toward malate, reducing gluconeogenesis from citric acid cycle intermediates. Increased NADH will also cause a shift of dihydroxyacetone phosphate toward glycerol-3-phosphate, reducing gluconeogenesis from glycerol.
Describe the allosteric regulation of gluconeogenesis
The regulation of gluconeogenesis will be in direct contrast to the regulation of glycolysis. 3 allosteric effectors have opposite effects on fructose 1,6-bisphosphatase and PFK-1. Fructose 2,6-bisphosphate and AMP stimulate PFK-1, which results in increased glycolysis. Conversely, they inhibit fructose 1,6-bisphosphatase, which results in decreased gluconeogenesis. In contrast, citrate inhibits PFK-1, which leads to decreased glycolysis but stimulates fructose 1,6-bisphosphatase leading to increased gluconeogenesis. Acetyl CoA is a positive allosteric effector of pyruvate carboxylase, which diverts pyruvate into the gluconeogenic pathway rather than the citric acid cycle. The liver also contains glucokinase inhibitor protein, which is activated by fructose-6 phosphate(F6P). When bound to F6P, glucokinase inhibitor protein sequesters and inactivates glucokinase, shutting down the first step in glycolysis.
From where to where are the steps of the glycolytic pathway in reverse
The steps from PEP to fructose 1,6-bisphosphate are the steps of the glycolytic pathway in reverse. Thus, PEP is converted to 2-phosphoglycerate, which goes to 3- phosphoglycerate. In the the next step the formation 1,3-bisphosphoglycerate uses an ATP equivalent (2 per glucose). This is subsequently converted to glyceraldehyde-3P (uses 2 NADH per glucose), which equilibrates with dihydroxyacetone phosphate, and then is combined to form fructose-1,6-biphophate.
In gluconeogenesis, how are glucokinase and phosphofructokinase bypassed
These are bypassed by phosphatase reactions. Fructose-1, 6-bisphosphate is dephosphorylated by fructose-1,6-bisphosphatase, releasing free phosphate, and bypassing phosphofructokinase. Fructose-6-phosphate is converted to glucose-6-phosphate by a phosphoglucose isomerase (readily reversible). Glucose-6-phosphate is then converted to glucose by glucose-6-phosphatase, bypassing glucokinase (liver) (hexokinase-- kidney). Glucose-6-phosphatase is a membrane bound enzyme within the ER and the active site is on the luminal surface of the membrane. A translocase for G6P is required to move G6P from cytosol to the ER for phosphatase attack. Special transporters are then needed to transport inorganic phosphate and glucose (GLUT- 7) to the cytosol from the ER and glucose to the blood stream (GLUT-2). Genetic defects in the translocase or the phosphatase are known and interfere with gluconeogenesis.
What are the major changes between glycolysis and gluconeogenesis
These steps have to be bypassed in gluconeogenesis. Pyruvate kinase is bypassed in two steps and the PFK-1 and glucokinase/hexokinase reactions are by passed by using phosphatase reactions. The majority of the glycolytic and gluconeogenic reactions occur in the cytoplasm and therefore need to be reciprocally regulated. This prevents the formation of a futile cycle. Something borrowed, something new: seven steps of glycolysis are retained.... steps 2 and 4-9 (borrowed). Three steps are replaced (new); the regulated steps. --The new reactions provide for a spontaneous pathway (changeG negative in the direction of sugar synthesis), and they provide new mechanisms of regulation.
Gluconeogenesis
Use carbon sources to generate glucose
Describe difference between hypoglycemic coma and diabetes mellitus
When blood glucose decreases to less than 40 mg/dL, hypoglycemic coma occurs; when it remains consistently greater than 125 mg/dL we have diabetes mellitus
What percentage of the daily glucose does the brain consume?
about 70% of the 200 g of glucose consumed in the body per day.
Describe the enzymes decreased by glucagon/epinephrine
glucokinase, phospho-fructokinase, pyruvate kinase
Describe the enzymes in glycolysis/gluconeogenesis that are increased by insulin
glucokinase, phospho-fructokinase, pyruvate kinase
Describe the enzymes increased by glucagon/epinephrine
glucose-6-phosphatase, fructose- 1, 6- bisphosphatase, PEP carboxykinase
Describe the enzymes when insulin is released. Describe the enzymes when glucagon is released
well fed----> activated enzymes nonphosphorylated (for the most part, insulin will cause the dephospho rylation of responsive enzymes through the activation of protein phosphates. Therefore, enzymes that are positively regulated by insulin will be active in their non-phosphorylated form. need------> activated enzymes phosphorylated. In contrast, to insulin, glucagon, and epinephrine stimulate the formation of cAMP which in turn activates protein kinases and hence responsive enzymes activated by these hormones are active in their phosphorylated states.
