Carbohydrate metabolism: regulation of glycolysis week 2 Flashcards
T or F: Glucose uptake by cells is regulated.
True.
When do GLUT1 and GLUT3 tranport glucose? Do they transport it into or out of cells? (or both?) Under what conditions do they transport glucose?
GLUT1 and GLUT3 are general glucose transporters. They are only regulated by blood glucose concentration and transport glucose into cells when blood [glucose] is high.
What tissues is GLUT2 located in? What is special about this glucose transporter?
GLUT2 is found in the plasma membranes of the liver, pancreas, intestine, and kidney. GLUT2 is a bidirectional transporter of glucose.
In these organs, glucose is put back into circulation. When glycogenolysis (liver) or gluconeogenesis (90% liver, 10% kidney) kick in, must transport glucose out. Pancreas: produces insulin. Stimulation of insulin production is the amount of incoming glucose. Some of the glucose is excess (because not all was used for energy generation but as a sensor of blood [glucose]). The excess is transported back into the blood by GLUT2.
What tissues is GLUT4 located in? What is special about GLUT4?
GLUT4 is found in skeletal muscle, heart, adipose tissue, and blastocysts.
Activity in muscle and adipose tissue is stimulated by insulin, and in muscle by hypoxia and diet.
When blood glucose is high, these tissues will take up lots of glucose. when glucose is low these organs will take in very little glucose and use lipids for energy.
Explain the effects of insulin on GLUT4 at the cellular level.
When insulin signals to tissues with this receptor (muscle, heart, adipose, blastocysts), vesicles of GLUT4 transporters fuse with the cell membrane to bring more glucose into the cells. This is in addition to the glucose being brought in by GLUT1 which is already incorporated into the cell membrane (remember GLUT1 is a general glucose transporter).
Where is the GLUT5 transporter located? What is special about the GLUT5 transporter?
GLUT5 is located in the muscle, spermatozoa, intestine, and liver. This transporter only transports fructose. Sperm cells only use fructose for energy generation. The intestine must have this transporter to absorb fructose from foods we eat and the liver must have this transporter to process fructose before it (and glucose it is converted to) is sent into the blood.
What 3 glycolytic enzymes are regulated? Are their rxns reversible?
What 2 classes of effectors regulate these enzymes?
- Glucose uptake is regulated.
- Glycolytic enzymes are regulated. The regulatory enzymes of glycolysis are:
o hexokinase
o 6-phosphofructo-1-kinase (PFK1; committed step)
o pyruvate kinase
Regulated by: small molecular weight effectors (allosteric) and hormones
What 3 tisses regulate glycolysis in repsonse to insulin and glucagon?
In what 2 ways do hormones regulate enzymatic activity?
Glycolysis in liver, muscle, and adipose are regulated by insulin and glucagon. Most other cells are not because they need glucose for energy generation.
Hormones can regulate the activity (by phosphorylation/dephosphorylation) and the gene expression level of an enzyme.
How do the Kms of hexokinase and glucokinase compare to one another? What is the significance of this?
Compare the specificities of these two enzymes.
Where is glucokinase found? (in addition to the liver)
The hexokinase isoenzymes found in most tissues and have a low Km for glucose (<0.1 mM) relative to the glucose concentration in blood (about 5 mM). It means that it works at saturation at physiological blood glucose concentration. It is good for tissues, such as brain, that needs glucose even when blood glucose concentration is low.
Liver parenchymal cells contain glucokinase, an isoenzyme of hexokinase with a higher Km for glucose (~7 mM). It “turns on” only when blood glucose concentration is high.
Hexokinase can phosphorylate other sugars. Glucokinase is specific to glucose.
Glucokinase can also be found in the islet cells of the pancreas. This is the way that these cells “sense” increased load of glucose and in response, secrete insulin.
How is hexokinase regulated? What is the significance of the way in which it is regulated?
Hexokinase is strongly inhibited by the product, glucose 6-P. This prevents the cell tying up all the inorganic phosphate in G6P.
Explain the regulation of glucokinase.
a. Glucokinase is not inhibited by G6P. However, it is indirectly inhibited by fructose 6-P (one step further down in glycolysis). It sends the signal that glycolysis is saturated, thus G6P can be used for other pathway, e.g. glycogen synthesis. Inhibition of glucokinase is accomplished through a regulatory protein that is responsible for sequestering glucokinase into the nucleus. F6P promotes binding of glucokinase to the regulatory protein. This inhibitory effect of F6P can be completely overcome by a large increase of glucose concentration. Glucose triggers dissociation of the enzyme from the regulatory protein.
b. Fructose (a sugar component of vegetables and fruits) promotes hepatic glucose utilization. Fructose is phosphorylated by fructokinase to fructose 1-phosphate, and glucokinase is activated by F1P through the same regulatory protein. It promotes the dissociation of glucokinase from the regulatory protein and translocation of the enzyme to the cytosol. This effect of F1P might be responsible for the adverse effect, e.g. hypertriacylglycerolemia, which is associated with excessive dietary fructose consumption. Note that this pathway is energy expensive. Production of F1P requires usage of ATP. Glucokinase also requires ATP usage.
c. Glucokinase is inducible by insulin within a few hours. Insulin increases transcription of the glucokinase gene. Since an increase in blood glucose stimulates release of insulin, glucose indirectly induces glucokinase gene expression.
What are the allosteric regulators of PFK1? Explain how/why these regulators work/why they are used.
What are the hormonal regulators of PFK1?
6-Phosphofructo-1-kinase Is the Major Regulatory Site
6-phosphofructo-1-kinase (PFK1) is the rate-limiting step and the most important regulatory site of glycolysis.
- Citrate, ATP and H+ (not lactate) are the most important negative allosteric effectors
- AMP and fructose 2,6-bisphosphate are the most important positive allosteric effectors.
These effectors represent signals by
a. cellular energy level (ATP-high energy, and AMP-low energy level),
b. internal environment of the cell (H+). glycolysis produces lactate under anaerobic conditions. Low pH signals possibility of lactic acidosis–>inhibition of glycolysis
c. availability of alternate fuels such as fatty acids and ketone bodies (citrate). Some tissues prefer to use fatty acids and ketone bodies as fuel in place of glucose. This helps preserve glucose for tissues such as the red blood cells or brain that are absolutely dependent on glucose as a fuel. When both fatty acids and ketone bodies are oxidized in the mitochondria, cytosolic citrate (coming from acetyl CoA and OAA in mitochondria and crossing to cytosol through a transporter) is elevated and this inhibits 6-phosphofructo-1-kinase to decrease glucose utilization.
d. insulin/glucagon ratio in the blood (through fructose 2,6-bisphosphate levels)
e. insulin/glucagon ratio in the blood also influences PFK-1 gene expression
Hormonal Control of PFK1 is manifested through the action of Fructose 2,6- bisphosphate an allosteric regulator of the enzyme:
Fructose 2,6-bisphosphate (F2,6-BP), like AMP,
- is a positive allosteric effector of PFK1
- and is a negative allosteric regulator of fructose 1,6-bisphosphatase (the opposing enzyme of gluconeogenesis)
This compound is very important for control since without it PFK-1 would not be active enough and fructose 1,6-bisphosphatase (converts F-1,6-BP back to F6P in gluconeogenesis) would be too active. Thus, this compound shifts the process toward glycolysis.
Explain the activity of PFK2 and what the effects of insulin and glucagon are on its activity.
How does epinephrine effect PFK2 activity in the heart?
Note that fructose 2,6-bisphosphate is not an intermediate of glycolysis. It is made from F6P (a glycolysis intermediate) by 6-phosphofructo-2-kinase. It can be converted back to F6P by F 2,6 bisphosphatase in a futile cycle. The phosphatase and kinase are part of a bifunctional enzyme (PFK2) and oppose each other’s effect.
In the liver, hormonal regulation of PFK-2 is based upon phosphorylation and dephosphorylation of a specific serine residues in the PFK-2 kinase domain of the bifunctional enzyme. Phosphorylation of this serine residue by protein kinase A triggers comformational change in the enzyme and results in the inactivation of the kinase and activation of the phosphatase domain. Vice versa, when the phosphate group is removed by a phosphatase, the kinase domain is active and the phosphatase domain is inactive. You need PFK-2 to be dephosphorylated in order to synthesize F-2,6 biphosphate, which in turn, will activate PFK-1, thus glycolysis will run.
Glucagon –> Phosphorylation –> inactivation of the kinase and activation of the phosphatase (glycolysis inhibition, stimulation of gluconeogenesis)
Insulin –> Dephosphorylation –> activation of the kinase inactivation of the phosphatase (stimulation of glycolysis, inhibition of gluconeogenesis)
Interestingly, in heart muscle, the phopshorylation site is in the phopshatase subunit. Thus, by the action of epinephrine, the phosphatase subunit will be inativated, thus the kinase subunit will be active. Consequently, F2,6-BP will be synthesized, PFK 1 will be active and glycolysis will run.
see reverse
Explain the intracellular mechanism of glucagon signaling as it pertains to PFK2 inhibition.
Glucagon is released from α-cells of pancreas and circulates in blood until it reacts with glucagon receptors on the outer surface of liver plasma membranes. This binding activates adenylate cyclase on the inner region of the plasma membrane, stimulating conversion of ATP to cAMP. cAMP activates protein kinase A, which phosphorylates a Ser amino acid residue in the kinase domain of PFK-2, which leads to inactivation of the kinase function (thus diminishing the synthesis of F2,6-BP) and activation of the phosphatase function of the PFK-2, which reduces the levels of F 2,6- BP and reduces PFK-1 activity and thus, halts glycolysis.
Explain the intracellular mechanism of insulin signaling as it pertains to PFK2 stimulation.
Insulin will activate phosphodiesterase to reduce intracellular level of cAMP, thus inactivating protein kinase A. It will also activate phosphoprotein phosphatase to remove phosphate group from the bifunctional enzyme, thus activating the kinase activity of PFK2 to synthesize F-2,6-BP. F2,6-BP can now accumulate and activate PFK-1 to increase the rate of glycolysis.
What allosteric effectors regulate pyruvate kinase (PK) activity?
a. PK is drastically inhibited by physiologic concentrations of ATP.
b. PK is inhibited by alanine, an amino acid, which is the starting material for gluconeogenesis.
c. The isoenzyme found in liver is greatly activated by fructose 1,6-bisphosphate (a glycolysis intermediate), thus linking regulation of PK to what happens to PFK-1. Thus, if conditions favor flux through 6-phosphofructo-1-kinase, F1,6-BP increases and activates PK. This is a feed forward effect, which pushes intermediates through glycolysis once glycolysis is committed at the level of PFK1.
Explain hormonal regulation of pyruvate kinase.
a. Protein kinase A acts on PK. PK is active in the dephosphorylated state but inactive in the phosphorylated state. Thus, glucagon will inhibit PK through cAMP–> activation of protein kinase A –> phosphorylation of PK. Insulin acts in a reverse manner.
b. PK, like glucokinase, is induced to higher steady state concentration in liver by a combination of high carbohydrate intake and high insulin level. Glucagon acts in a reverse manner.
What is the effect of pyruvate kinase deficiency on RBCs?
Why do reticulocytes not have issues with PK deficiency?
Mature erythrocytes are absolutely dependent on glycolytic activity for ATP production. ATP is needed for the ion pumps, especially the Na+, K+ –ATPase, which maintains the biconcave disk shape of erythrocytes, a characteristic that helps erythrocytes slip through the capillaries as they deliver oxygen to the tissues. Without ATP the cells swell and lyse. Anemia due to excessive erythrocyte destruction is referred to as hemolytic anemia. Pyruvate kinase deficiency is rare but is by far the most common genetic defect of the glycolytic pathway known to cause hemolytic anemia.
Most pyruvate kinase-deficient patients have 5–25% of normal red blood cell pyruvate kinase levels and flux through the glycolytic pathway is restricted severely, resulting in markedly lower ATP concentrations. The expected crossover of the glycolytic intermediates is observed; that is, those intermediates proximal to the pyruvate kinase catalyzed step accumulate, whereas pyruvate and lactate concentrations decrease.
Normal ATP levels are observed in reticulocytes of patients with this disease. Although deficient in pyruvate kinase, these “immature” red blood cells have mitochondria and can generate ATP by oxidative phosphorylation. Maturation of reticulocytes into red blood cells results in the loss of mitochondria and complete dependence on glycolysis for ATP production. Since glycolysis is defective, the mature cells are lost rapidly from the circulation. Anemia results because the cells cannot be replaced rapidly enough by erythropoiesis.
