1. 1 Ferchmin 2015 Regulation of glycogen synthesis and breakdown Regulation of lipid synthesis from carbohydrates The content of the slides is described below. 1.Slides 2-5 are a brief introduction to cell-signaling 2.Slides 7-12 are about regulation of glycogen synthase and glycogen phosphorylase 3.A brief introduction to the role of insulin is given is slides 13, 14, and 15. 4.Slide 16 integrates slide 12 with the effect of insulin on glycogen synthesis. 5.The integration of carbohydrate metabolism with lipids starts in slide 17 in spite of the fact that lipids are not even mentioned. 6.The inhibition of hepatic pyruvate kinase by cyclic-3’5’-AMP is in slide 18. 7.The last six slides address the regulation of lipid synthesis from carbohydrates by xylulose-5P and 2,6-fructose-bis-phosphate. The main “strategy” used here is to present you a biochemical process. Next, additional complexity is added. So, you review the previous step in the context of the addition of the new biochemical step. My ambitious purpose is that you learn a lot by these reiterative steps and that only some practice will be needed before the exam. Your comments, suggestions and critiques and warmly welcomed.

2. 2 There are many regulatory protein kinases some of them phosphorylate other protein kinases. For example: 1) Cyclic AMP-dependent or PKA* 2) Calcium/calmodulin-dependent (CaM II) or PK II* 3) Phospholipid-dependent or PKC* 4) Tyrosine kinases and receptor tyrosine kinases or RTK * These kinases phosphorylate serines or threonines. There are Tyr and Ser-Thr protein kinases. The most remarkable example are the cascades of the Mitogen Activated Kinases (MAP kinases or MAPK). You will see them in this and other courses. This cascades are formed by MAPKKK MAPKK MAPK. In the case of ERK (Extracellular Regulated Kinase) cascade. The first kinase (MAPKKK) is Raf. Raf is a serine/threonine kinase that is recruited to the membrane by binding to active (thus, GTP-bound) G-protein Ras. The second is MEK (MAPKK) is a dual specificity kinase since it can phosphorylate both threonine and tyrosine residues, which it does on ERK (MAPK). ERK has cytosolic substrates but the most important role is to be translocated to the nucleus where it phosphorylates specific substrates (proteins) leading to gene expression. Ferchmin 2015 You might find some of the following slides apparently unrelated to each other or to the subject matter. Also there will be no continuity between slides. Just focus on the concept presented in a given side and hopefully “by the end of the day” you will see the application to glycogen regulation Brief introduction to signal transduction needed to understand the regulation of glycogen synthesis and breakdown. The regulation of the synthesis and breakdown of glycogen into glucose is regulated in a complex manner with the intervention of hormones, receptors, G- proteins and protein kinases. The human genome contains about 500 protein kinase genes and they constitute about 2% of all human genes.

3. 3 PKA is the most important protein kinase in glycogen metabolism but a Ca 2+ -calmodulin kinase also participates. In addition several kinases, regulated by insulin, like PKB/Akt and glycogen synthase kinase-3 or GSK-3 are involved. Where there are protein kinases there must be protein phosphatases. 1) Protein phosphatase I 2) Protein phosphatase II There is also a protein phosphatase inhibitor that is activated by phosphorylation by PKA on a threonine and prolongs the effect of PKA. The interaction between kinases and phosphatases is regulated by targeting subunits. Docking proteins are modulatory proteins that attach enzymes to specific targets, or specific locations. Often attaching to the membrane does the trick. These targeting proteins ensure the fidelity of protein phosphorylation and facilitate that the proper protein kinase or phosphatase quickly finds its substrate.

4. 4 CYCLIC AMP REGULATES THE SYNTHESIS AND BREAKDOWN OF GLYCOGEN β-Adrenergic receptor binds epinephrine in muscle and liver and a functionally similar receptor binds glucagon in liver. These receptors are coupled to G-proteins. There are other G proteins that are not receptor coupled. This binding activates the regulatory enzyme adenylyl cyclase. Adenylyl cyclase synthesizes cyclic-AMP which is one of the so called second messengers. The message of cAMP is terminated by phosphodiesterase. Calmodulin is a Ca 2+ binding protein. It binds sequentially 4 Ca 2+ per molecule and acts as second messenger of Ca 2+. Often Ca 2+ and cAMP regulate the same process. There is a guanyl cyclase which synthesizes cGMP. The physiological role of cGMP is less prominent than that of cAMP for glycogen metabolism. The synthesis of cAMP is unfavorable, ΔGE◦'=1.6 Kcal/mole, however it is coupled to the hydrolysis of PP i which releases about -4.6 Kcal/mole. Therefore, the net synthesis of cAMP releases a total of -3 Kcal/mole, (+1.6 -4.6=3). Interestingly, the hydrolysis of cAMP releases -12 Kcal/mole because it relieves the strain imposed by the cyclic ester. The role of PPi hydrolysis as a coupled reaction, is equal to its role in the synthesis of sugar nucleotides.

5. 5 G proteins are a family of GTP binding modulators

6. 6 The previous slides were introductory to glycogen synthesis and regulation. In the next side starts the regulation of glycogen metabolism 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). This is because the enzymes are not in fixed proportion nor are linked like in the PDC.

7. 7 After this hasty introduction to cell signaling we will study the regulation of (1) glycogen synthase, (2) glycogen phosphorylase and of (3) phosphorylase b Kinase. The latter is a regulatory enzyme that indirectly affects glycogen metabolism. For the three enzymes we will use the diagram shown below. Please, understand the diagram. Later we will integrate all in a single graph. 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. This cartoon shows the phosphorylation and dephosphorylation of glycogen synthase The inactive glycogen synthase becomes active in the presence of glucose-6P Let me introduce here the idea of the “monster” that activates adenylate cyclase (adenylyl cyclase) and antagonizes fructose-2,6-di-phosphate. How many enzymes are shown in this sketch?

8. 8 Phosphorylase a, was the first enzyme ever discovered to be activated by phosphorylation. So, it was dubbed a for (active) b stands for inactive

9. 9 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 Ca 2+ with calmodulin activates this enzyme. This effect is specially relevant in muscle. This enzyme is regulatory. Does not affect “real” metabolites. On top of it its is “misnamed”. See below.

10. 10 Integration of the regulation of glycogen synthesis and breakdown R2C2R2C2 R 2 (cAMP) 4 2C ACTIVE PKA Phosphorylase b Kinase Glycogen Phosphorylase Glycogen synthase AMP Phosphodiesterase Glucagon Epinephrine Receptors Adenylyl Cyclase cAMP ATP activation inhibition Slides 7 to 9 are conceptually merged in slide 10. Slides 10, 11 and 12 introduce in a stepwise manner the regulation of glycogen metabolism by epinephrine (in muscles and liver) and by glucagon in liver. Slides 13 and 15 introduce the role of insulin. All of that is integrated in slide 16. If you study sequentially all the slides (7 to 16) you will be able (hopefully) to understand the regulation of glycogen metabolism.

11. 11 Integration of the regulation of glycogen synthesis and breakdown R2C2R2C2 R 2 (cAMP) 4 2C ACTIVE PKA Phosphorylase b Kinase Glycogen Phosphorylase Protein Phosphatase Protein Phosphatase Inhibitor Glycogen synthase AMP Phosphodiesterase Glucagon Epinephrine Receptors Adenylyl Cyclase cAMP ATP activation inhibition

12. 12 Integration of the regulation of glycogen synthesis and breakdown R2C2R2C2 R 2 (cAMP) 4 2C ACTIVE PKA Phosphorylase b Kinase Glycogen Phosphorylase Protein Phosphatase Protein Phosphatase Inhibitor Glycogen synthase AMP Phosphodiesterase Ca 2 + Glucagon Epinephrine Receptors Adenylyl Cyclase cAMP ATP activation inhibition

13. 13 Pancreatic β-cells and hepatocytes have the same glucokinase and GluT2 transporter. Why?

14. 14 Insulin activates glycogen synthesis You must know the mechanism of insulin receptor. It is by Tyr phophorylation on the receptor and on its substrates

15. 15 Insulin stimulates glucose transport in muscle and adipose cells by stimulating translocation of glucose transporter 4 (GLUT4) to the plasma membrane.

16. 16 Integration of the regulation of glycogen synthesis and breakdown R2C2R2C2 R 2 (cAMP) 4 2C ACTIVE PKA Phosphorylase b Kinase Glycogen Phosphorylase Protein Phosphatase Protein Phosphatase Inhibitor Glycogen synthase AMP Phosphodiesterase Ca 2 + Insulin-R PI3-K PKB/Akt GSK-3 Glucagon Epinephrine Receptors Adenylyl Cyclase cAMP ATP activation inhibition Heart, muscle and other Akt inhibits glycogen synthase kinase 3 (GSK-3) which then stops inhibiting the glycogen synthase Remember that (-1) x (-1)=+1 or inhibition of inhibition is as good as activation

17. 17 We will leave glycogen metabolism and return to glycolysis, fructose-2,6-bisphosphate, xylulose-5-P, and expand the regulatory big picture

18. 18 Take home message. When you are chased by a monster your liver won’t synthesize glycogen.

19. 19 2) Nonoxidative steps of pentose phosphate shunt Misplaced slide? No, it is integration of glucose metabolism with lipid synthesis Xylulose-5P the pentose that makes you FAT

20. 20 Phosphofructokinase-2 Fructose-6-P Fructose-2,6-bis-phosphate ATP ADP Fructose-2,6-bis-phosphatase Fructose-2,6-P Fructose-6-P H2OH2O PiPi PKA ATP cAMP HEXOSES PP2A PiPi Lipogenesis by activation of hepatic glycolysis xylulose-5-P comes from PPP when there is plenty of glucose PP2A is protein phosphatase 2A PKA is just that, protein kinase A Regulation of fructose-2,6-bis phosphate synthesis and break down Bifunctional enzyme. Phosphorylated is phosphatase Dephosphorylated is kinase

21. 21 This “repeated” slide was placed for you to practice the metabolism of fructose-2,6-P

22. 22 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 has kinase activity when nonphosphorylated and catalyzes the phosphorylation of fructose-6-P in carbon 2. When phosphorylated, phosphofructokinase-2 has phosphatase activity and catalyzes the dephosphorylation of carbon 2 of 2,6-FDP. The role of 2,6-FDP is to "convey" to the liver cells that there is plenty of hexoses and that glycolysis has to be activated to support fatty acid synthesis. At the same time, gluconeogenesis has to be inhibited. In liver glycolysis is inhibited by cAMP, which indirectly inhibits phosphofructokinase-1 (by lowering 2,6-FDP and activating 1,6-FDP phosphatase) and liver pyruvate kinase (as seen above). In muscle, cAMP activates glycolysis by activation of glycogenolysis. This difference in regulation reflects the different roles of glycolysis in both organs. You should recall from the regulation of glycolysis that 2,6-FDP activates the hepatic PFK- 1 by removing the inhibitory effect of ATP. Also 2,6-FDP enhances the action of PFK-1 by inhibition the 1,6- FDP phosphatase. 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. It is unlikely that the you will find this in the NBE.

23. Abbreviated glycolysis, gluconeogenesis, and pentose shunt pathways and roles of Xu5P and Fru-2,6-P2 in activation of PP2A and PFK, respectively. Kabashima T et al. PNAS 2003;100:5107-5112 ©2003 by The National Academy of Sciences

24. 24 Veech RL (2003) A humble hexose monophosphate pathway metabolite regulates short- and long-term control of lipogenesis. Proc Natl Acad Sci U S A 100:5578-5580.