1. glucose G6P GLUTsGLYCOGENESIS GLYCOLYSIS glucose insulin Translocation Vesicles in Golgi PFK – phosphofructokinase GS – glycogen synthase Muscle & WAT Glucose Uptake

2. Hexose Metabolism P glucose Using ATP hexokinase glucose 6-phosphate P glucose 1-phosphate P PP fructose 6-phosphate fructose 1,6-bisphosphate PP U PFK UDP glucose Using UTP Releases PP PP hydrolysis pulls reaction to completion Pyrophosphate hydrolyses to two phosphates Pulls UDP-glucose conversion over “Activated Glucose”

3. Glycogen Synthesis PP U UDP glucose PP U UDP Glycogen Glycogen with one more glucose Note synthesis is C1  C4 C1 end of glycogen attached to glycogenin UDP needs to be made back into UTP Use ATP for this UDP + ATP  UTP + ADP

4. Glycogen Synthase Catalyses the addition of ‘activated’ glucose onto an existing glycogen molecule –UDP-glucose + glycogen n  UDP + glycogen n+1 Regulated by reversible phosphorylation (covalent modification) –Active when dephosphorylated, inactive when phosphorylated Phosphorylation happens on a serine residue –Dephosphorylation catalysed by phosphatases (specifically protein phosphatase I) –Phosphorylation catalysed by kinases (specifically glycogen synthase kinase) Insulin stimulates PPI –And so causes GS to be dephosphorylated and active –So insulin effectively stimulates GS

5. Phosphofructokinase Catalyses the second ‘energy investment’ stage of glycolysis –F6P + ATP  fructose 1,6 bisphosphate + ADP Regulated allosterically –Simulated by concentration changes that reflect a low energy charge An increase in ADP/AMP and a decrease in ATP These molecules bind at a site away from the active site – the allosteric binding sites. –Many other molecules affect PFK allosterically but all are effectively indicators of ‘energy charge’ Energy charge is balance of ATP, ADP & AMP Small change in ATP/ADP causes large change in AMP via adenylate kinase reaction

6. Coupling (again!) The stimulation of glycogen synthesis by insulin creates an ‘energy demand’ –Glycogenesis is anabolic –The activation of glucose prior to incorporation into glycogen requires ATP –This drops the cellular [ATP] and increases the [ADP] & [AMP] This drop in ‘energy charge’ is reflected by a stimulation of PFK –A good example of how an anabolic pathway requires energy from a catabolic pathway –Insulin has ‘indirectly’ stimulated PFK and glucose oxidation even though it does not have any direct lines of communication to this enzyme –Signals to store fuels also cause fuels to be burnt

7. Liver Glucose Uptake GLUT-2 used to take up glucose from bloodstream –Very high activity and very abundant –[Glucose] blood = [Glucose] liver Glucokinase –Rapidly converts G  G6P –Not inhibited by build up of G6P –High Km (10 mM) for glucose – not saturated by high levels of liver glucose –So [G6P] rapidly increases as blood [glucose] rises G6P can stimulate inactive GS –Even phosphorylated GS –Glucose itself also stimulates the dephosphorylation of GS Via a slightly complex process that involves other kinases and phosphatases which we needn’t go into right now

8. Glycogenesis In liver –The “push” mechanism Glycogenesis responds to blood glucose without the need of insulin Although insulin WILL stimulate glycogenesis further In muscle –[G6P] never gets high enough to stimulate GS “Push” method doesn’t happen in muscle More of a “pull’ as insulin stimulates GS In both cases –2 ATPs required for the incorporation of a glucose into glycogen chain G  G6P and UDP  UTP –Branching enzyme needed to introduce a1  6 branch points –Transfers a segment from one chain to another –Limit to the size of glycogen molecule Branches become too crowded, even if they become progressively shorter Glycogen synthase may need to interact with glycogenin to be fully active

9. A Tale of Two Kinases Glucokinase (GK) –Only works on glucose –High Km for glucose (~10mM) –Not inhibited by G6P –Only presents in liver, beta-cells –Responsive to changes in [glucose] blood Hexokinase (HK) –Works on any 6C sugar –Km for glucose ~0.1mM –Strongly inhibited by its product G6P –Present in all other tissues –If G6P is not used immediately, its build up and inhibits hexokinase –Easily saturated with glucose

10. Fructose Metabolism fructose Using ATP hexokinase P fructose 6-phosphate fructose 1-phosphate PFK P ‘normal glycolysis’ fructokinase CH 2 OH CHOH CHO CH 2 OP C=O CH 2 OH Glyceraldehyde Dihydroxyactone phosphate CH 2 OP CHOH CHO Aldolase B Triose Kinase ‘normal glycolysis’ Glyceraldehyde 3- phosphate

11. Fructose Metabolism Fructose entry into cells does not require insulin In muscle, fructose just enters glycolysis –Or could be made into glycogen if insulin stimulus available! F6P  G6P  G1P  UDP-glucose  Glycogen In liver, fructokinase traps fructose –FK produces F1P –FK is quite fast in comparison to the aldolase B that uses the F1P –F1P can build up –But more seriously producing ‘dead’ F1P traps phoshpate FK reaction consumes ATP Lack of phosphate akes new ATP synthesis difficult ATP levels in liver fall –Even more serious in people with a deficiency in Aldolase B

12. Lipogenesis Overview glucose G6P pyruvate acetyl-CoA pyruvate LIPOGENESIS Fat PDH GLYCOLYSIS GLUT-4 No GS KREBS CYCLE CO 2 fatty acids ESTERIFICATION X Produces reductant PPP Consumes reductant and ATP NADH release ultimately produces ATP Key steps (eg, GLUT-4, PDH, lipogenesis) are stimulated when insulin binds to its receptor on the cell surface

13. Pyruvate Dehydrogenase Pyruvate + CoA + NAD  acetyl-CoA + NADH + CO 2 Irreversible in vivo No pathways in humans to make acetate into ‘gluconeogenic’ precursors –Can’t make glucose from acetyl-CoA –No way of going back once the PDH reaction has happened –Key watershed between carbohydrate and fat metabolism

14. PDH Control Regulated by reversible phosphorylation –Active when dephosphorylated Inactivated by PDH kinase Activated by PDH phosphatase –Insulin stimulates PDH phosphatase Insulin thus stimulates dephosphorylation and activation of PDH

15. Fate of Acetyl-CoA Burnt in the Krebs Cycle –Carbon atoms fully oxidised to CO 2 –Lots of NADH produced to generate ATP Lipogenesis –Moved out into the cytoplasm –Activated for fat synthesis In both cases the first step is citrate formation –Condensation of acetyl-CoA with oxaloacetate Regenerates Coenzyme A –Transport or Oxidation The ‘fate’ will depend on the need for energy (ATP/energy charge) and the stimulus driving lipogenesis

16. ATP-Citrate Lyase Once in the cytoplasm, the citrate is cleaved –By ATP-Citrate Lyase (ACL) –Using CoA to generate acetyl-CoA and oxaloacetate Reaction requires ATP  ADP + phosphate ACL is inhibited by hydroxy-citrate (OHCit) –OHCit is found in the Brindleberry Sold as a fat synthesis inhibitor –Would we expect it to prevent the formation of fatty acids And, if so, would that actually help us lose weight?

17. The Carrier Oxaloacetate produced by ACL needs to return to the matrix –Otherwise the mitochondrial oxaloacetate pool becomes depleted –Remember, oxaloacetate is really just a ‘carrier’ of acetates Both in the Krebs's cycle and in the transport of acetyl-CoAs into the cytoplasm –Oxaloacetate cannot cross the inner mitochondrial membrane Some interesting inter-conversions occur to get it back in!

18. Acetyl-CoA Carboxylase Activates acetyl-CoA and ‘primes’ it for lipogenesis Unusual in that it ‘fixes’ carbon dioxide –In the form of bicarbonate –A carboxylation reaction Acetyl-CoA + CO 2  malonyl-CoA –Reaction requires ATP  ADP + phosphate –Participation of the cofactor, biotin Biotin is involved in other carboxylation reactions

19. ACC Control ACC is stimulated by insulin –Malonyl-CoA is committed to lipogenesis Reversible Phosphorlyation Stimulated allosterically by citrate (polymerisation) Inhibited allosterically by long-chain fatty acyl-CoAs

20. Malonyl-CoA Activated acetyl-CoA –Tagged and primed for lipogenesis –But also a key regulator of fatty acid oxidation –ACC is not only present in lipogenic tissues –Also present in tissues that need to produce malonyl- CoA in ‘regulatory’ amounts Malonyl-CoA inhibits carnitine acyl transferase I –An essential step in fatty acid oxidation –Only way of getting long chain fatty acyl-CoAs into the mitochondria

21. Malonyl-CoA So when ACC is active in, say, muscle –Malonyl-CoA concentration rises –CPT-1 is inhibited –Fatty acid oxidation stops –Cell must use carbohydrate instead –Therefore insulin, by stimulating acetyl-CoA carboxylase, encourages carbohydrate oxidation and inhibits fatty acid oxidation

22. Fatty Acyl Synthase

23. FAS - simplified

24. FAS Fatty acyl synthase (FAS) is multi-functional –Lots of different enzyme activities in the complex –Can you count them all? Bringing in acetyl and malonyl groups, catalysing the reaction between the decarboxylated malonyl and the growing fatty acid chain, the reduction/dehydration/reduction steps, moving the fatty acid to the right site and finally releasing it as FA-CoA Two free -SH groups on an ‘acyl-carring protein’ –Keeps the intermediates in exactly the right position for interaction with the right active sites –Each new 2C unit is added onto the carboxy-end

25. Addition Sequence Each round of 2C addition requires –2 molecules of NADPH … but No ATP (!!) –The release of the carbon dioxide that went on during the production of malonyl-CoA Thus the carboxylation of acetyl-CoA does not result in ‘fixing’ CO 2 FAs start getting ‘released’ as FA-CoA when chain length is C14 –Desaturation is done AFTER FAS

26. Pentose Phosphate Pathway Provides NADPH for lipogenesis –NADPH - A form of NADH involved in anabolic reactions –Rate of NADPH production by PPP is proportional to demand for NADPH Key regulatory enzyme is G6PDH –Glucose 6-phosphate dehydrogenase G6P + NADP  6-phosphogluconolactone + NADPH –The gluconolactone is further oxidised to give more NADPH Decarboxylation to give a 5-carbon sugar phosphate (ribulose 5-phosphate)

27. Pentose Phosphate Pathway Need to put the 5-C sugar back into glycolysis –Accomplished by rearranging and exchanging carbon atoms between 5C molecules –Catalysed by enzymes called transaldolases and transketolases So, 5C + 5C  C7 + C3 by a transketolase (2C unit transferred) Then C7 + C3  C6 + C4 by a transaldolase (3C unit transferred) Then C4 + C5  C6 + C3 by a transketolase (2C unit transferred) –The C6 and C3 sugars can go back into glycolysis Alternatively, PPP used to make ribose 5-phosphate –Important in nucleotide pathways Or generate NADPH as an anti-oxidant –Red blood cells - deficiency in G6PDH can cause anemia

28. Esterification Formation of Fat Glycerol needs to be glycerol 3-phosphate –From reduction of glycolytic glyceraldehyde 3-phosphate –Glycolysis important both for production of acetyl-CoA and glycerol! Esterification enzyme uses FA-CoA –Not just FAs –FAs added one at a time Both esterification enzyme and FAS are unregulated by insulin –Gene expression and protein synthesis FAS is downregulated when lots of fat around –As in a Western diet!!

29. Regulatory Overview glucose G6P pyruvate acetyl-CoA pyruvate LIPOGENESIS Fat PDH GLYCOLYSIS GLUT-4 No GS KREBS CYCLE CO 2 fatty acids ESTERIFICATION X G6PDH G6PDH stimulated by demand for NADP Insulin stimulates GLUT-4. PDH and ACC. Also switches on the genes for FAS and esterification enzyme. Krebs cycle will be stimulated by demand for ATP ACC FAS glycerol 3-P Acetyl-CoA transport stimulated by increased production of citrate citrate