Regulation of glycogen synthesis and breakdown Ferchmin 2018 Regulation of glycogen synthesis and breakdown Integration of lipid and carbohydrate metabolism Slides content 1-3 Generalities 4-6 Regulation of glycogen synthase, phosphorylase and phosphorylase b kinase 7-9 Stepwise integration of glycogen metabolism in successive slides 10-12 Role of insulin 14 Slides 14 and beyond link glycogen metabolism with glycolysis, PPP, phosphofructokinase-2, lipid synthesis, and gene regulation.

I was informed that you are knowledgeable in the field of cell-signaling, second-messengers, receptors, and, protein kinases and phosphatases. Based on my faith in you, I will go straight into the subject matter. Cell-signaling applied to glycogen synthesis is not too complicated. Besides, I will be available to clarify doubts through my email or at my office to clarify doubts. If you have not done it yet, review glycogen structure. All the metabolism of glycogen takes place in the granule of glycogen. The granule of glycogen contains all the enzymes necessary for the synthesis and breakdown, and regulation of these processes. However, the granule is not a multi-enzymatic complex, like the pyruvate dehydrogenase complex (PDC), because the enzymes are not in fixed proportion nor are interconnected specifically like in the PDC.

The sketch below illustrates a general situation where a protein kinase and a protein phosphatase regulate the concentrations of the non-phosphorylated and phosphorylated forms of an enzyme. This sketch will be used three times in this lecture. Moreover, it will be utilized in more complex settings several times. Therefore, it would be time-effective if you understood it now. Admittedly, there are other ways to present the same idea. However, I prefer this one. Glycogen metabolism is part of the normal homeostatic regulation of glycemia and also of the fight-or-flight response. Hypoglycemia or fast require glycogen breakdown. This is achieved by phosphorylation of all the enzymes involved in glycogen synthesis. On the contrary, satiety will initiate glycogen synthesis which is accomplished by dephosphorylation of the same set of enzymes. The principal regulatory enzymes involved are glycogen synthase, glycogen phosphorylase, and the phosphorylase b kinase. The phosphorylase b kinase is inaccurately named sort of like pyruvate kinase

This cartoon shows the phosphorylation and dephosphorylation of To start we will consider glycogen synthase. This cartoon shows the phosphorylation and dephosphorylation of glycogen synthase The inactive glycogen synthase becomes active in the presence of glucose-6P Glycogen synthase can be D, or dependent on glucose-6-P, or I, independent of the presence of glucose -6-P. Please, remember that the immediate precursor of glycogen is UDP-glucose not glucose-6-P. The latter is only an allosteric regulator of glycogen synthase.

b stands for inactive Phosphorylase a, was the first enzyme ever discovered to be activated by phosphorylation. So, it was called a for active. The regulation of liver glycogen phosphorylase is different from that of muscle because the role of the liver in glucose homeostasis is to release free glucose to blood and for other tissues when the blood-glucose level is low.

This enzyme is regulatory. Does not affect “real” metabolites. On top of it its is “misnamed”. See below. Glycogen phosphorylase b kinase also phosphorylates the synthase and should actually be called synthase phosphorylase b kinase. The phosphorylation of the alpha subunit regulates the dephosphorylation of the beta subunit. The delta subunit is calmodulin. The direct interaction of Ca2+ with calmodulin activates this enzyme. This effect is specially relevant in muscle.

Integration of the regulation of glycogen synthesis and breakdown Glucagon Epinephrine Receptors Adenylyl Cyclase cAMP ATP AMP Phosphodiesterase R2C2 R2(cAMP)4 2C ACTIVE PKA Glycogen synthase Phosphorylase b Kinase revise the slide # Glycogen Phosphorylase activation inhibition

Integration of the regulation of glycogen synthesis and breakdown Glucagon Epinephrine Receptors Adenylyl Cyclase cAMP ATP AMP Phosphodiesterase R2C2 R2(cAMP)4 2C ACTIVE PKA Glycogen synthase Phosphorylase b Kinase Glycogen Phosphorylase Protein Phosphatase activation Protein Phosphatase Inhibitor inhibition

Integration of the regulation of glycogen synthesis and breakdown Glucagon Epinephrine Receptors Adenylyl Cyclase Obviously, insulin has a role in this metabolism. See next slides. cAMP ATP AMP Phosphodiesterase R2C2 R2(cAMP)4 Ca2+ 2C ACTIVE PKA Glycogen synthase Phosphorylase b Kinase Glycogen Phosphorylase Protein Phosphatase activation Protein Phosphatase Inhibitor inhibition

Pancreatic β-cells and hepatocytes have the same glucokinase and GluT2 transporter. What for? To accurately sense Glycemia! GluT2 maintains the concentration of glucose in the blood in equilibrium with the glucose in the beta cells. 2) Glucokinase keeps the concentration of glucose-6-P at concentrations proportional to the levels of blood glucose. 3) Metabolism of glucose and other substrates synthesizes ATP in amounts commensurate to glucose concentration. 4) ATP blocks the hyperpolarizing potassium channels. 5) The depolarization caused by the blocking of potassium channels activates the voltage sensitive calcium channels. 6) The influx of calcium starts the vesicular exocytosis of insulin. 7) Insulin is released and glycemia is adjusted to proper levels. Mutations in the glucokinase (GK) gene cause defects in blood glucose homeostasis. Maturity-onset diabetes of the young or (MODY) refers to hereditary forms of diabetes caused by mutations in an autosomal dominant gene disrupting insulin production. MODY is a monogenic diabetes to distinguish it from the classic types 1 and 2 of diabetes which involve complex combinations of causes involving multiple genes and environmental factors

Insulin activates glycogen synthesis You must remember the mechanism of insulin receptor. It is by Tyr phosphorylation on the receptor and on the substrates of this receptor. You have to know that the insulin receptor is a membrane receptor that binds insulin and at the same time is a tyrosine kinase capable to auto phosphorylate and to phosphorylate other tyrosine containing . The insulin receptor is a member of a family of receptors of growth factors and similar peptides. Insulin inhibits gluconeogenesis in liver through a complex signaling by decreasing cAMP, however, in diabetic patients the signal of insulin is low and allows for gluconeogenesis.

Insulin stimulates glucose transport in muscle and adipose cells by stimulating translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The cell signaling of insulin is partially equal to the one shown in the previous slide The regulation of GluT4 is unexpected because it is regulated not by allosteric signals but by removal from the membrane and sequestration in the cytoplasm. In the above slide identify IRS, PI3K, PDK, AKT, AS160 and the Rab G protein. You do not have to remember this from my classes but have a notion that insulin acts by cell-signaling. You do not have to cram those intermediates which you have already or will study them in the near future.

or inhibition of inhibition is as good as activation Integration of the regulation of glycogen synthesis and breakdown Glucagon Epinephrine Receptors Heart, muscle and other Insulin-R PI3-K PKB/Akt GSK-3 Adenylyl Cyclase Remember that (-1) x (-1)=+1 or inhibition of inhibition is as good as activation cAMP ATP AMP Phosphodiesterase R2C2 R2(cAMP)4 Ca2+ 2C ACTIVE PKA Glycogen synthase Phosphorylase b Kinase This slide shows the integrated regulation of glycogen metabolism. Also, insulin enhances glycogenesis by increasing the uptake of glucose in muscle and by decreasing the antagonistic effect of cAMP. The mechanism by which insulin decreases the effect of cAMP was not fully clarified. Glycogen Phosphorylase Protein Phosphatase activation Protein Phosphatase Inhibitor inhibition Akt inhibits glycogen synthase kinase 3 (GSK-3) which then stops inhibiting the glycogen synthase

In liver L-pyruvate kinase is one of the leading enzymes involved in lipid synthesis. In muscle the function of pyruvate kinase is to make ATP.

2) Nonoxidative steps of pentose phosphate shunt Misplaced slide? No, it is integration of glucose metabolism with lipid synthesis 2) Nonoxidative steps of pentose phosphate shunt Xylulose-5P the pentose that makes you FAT Xylulose-5P the pentose that makes you FAT

Phosphofructokinase-2 Fructose-2,6-bis-phosphatase Regulation of fructose-2,6-bis phosphate synthesis and break down HEXOSES Fructose-6-P Fructose-2,6-bis-phosphate ATP ADP Lipogenesis by activation of hepatic glycolysis PKA ATP Phosphofructokinase-2 PP2A Pi cAMP Bifunctional enzyme. Phosphorylated is phosphatase Dephosphorylated is kinase PKA is just that, protein kinase A Fructose-2,6-bis-phosphatase Fructose-2,6-P Fructose-6-P H2O Pi xylulose-5-P comes from PPP when there is plenty of glucose PP2A is protein phosphatase 2A

Role of 2,6-Fructose-bisphosphate and phosphofructokinases-1 and -2 Phosphofructokinase-1 is the well-known glycolytic enzyme; phosphofructokinase-2 is exclusively a regulatory enzyme. PFK-2, when it is not phosphorylated, has kinase activity and catalyzes the phosphorylation of fructose-6-P in carbon 2 thus making fructose-2,6-P. When phosphorylated, phosphofructokinase-2 has phosphatase activity and dephosphorylates the carbon 2 of FDP-2,6 making fructose-6-P. The role of 2,6-FDP is to "convey" to the liver that there is plenty of hexoses and that glycolysis has to be activated to support fatty acid synthesis. Simultaneously, insulin inhibits gluconeogenesis by lowering 3,5-cyclic-AMP. In liver, glycolysis is inhibited indirectly by cAMP which, inhibits liver pyruvate kinase and phosphofructokinase-1 by lowering 2,6-FDP and by activation of 1,6-FDP phosphatase. In the muscle, cAMP activates glycolysis by activation of glycogenolysis. This difference in regulation reflects the different roles of glycolysis in both organs. During stress muscle requires energy, but the liver has to regenerate glucose. In cardiac muscle phosphofructokinase-2 is a different isozyme than in liver and the phosphorylated muscle phosphofructokinase-2 is active allowing cyclic-AMP to activate glycolysis. Insulin inhibits gluconeogenesis in liver through a complex signaling by decreasing cAMP, however, in diabetic patients the signal of insulin is low and allows for gluconeogenesis.

Abbreviated glycolysis, gluconeogenesis, and pentose shunt pathways and roles of Xu5P and Fru-2,6-P2 in lipogenesis Kabashima T et al. PNAS 2003;100:5107-5112 The roles of Xu5P in the activation of PP2A and PFK-2. The scheme illustrates the formation of Xu5P from fructose 6-phosphate (F6P) and glyceraldehyde 3-phosphate (GAP) by transketolase, which activates PP2A. PP2A activates 6-phosphofructokinase-2 which increases Fru-2,6-P resulting in activation of PFK-1. The same PP2A also activates ChREBP in the nucleus increasing the expression of enzymes involved in lipid synthesis, including liver pyruvate kinase, citrate lyase (ACL), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS).

Can fat make you slim? The old dogma that all calories are equally responsible of gaining weight is not entirely true. Polyunsaturated fatty acids (PUFA) negatively regulate lipid synthesis, and could decrease the epidemics of obesity and metabolic syndrome,. No all factors that mediate the obesity are is understood.