Carbohydrate Disposal

1. Carbohydrate Disposal This version is quite “information dense” to save paper.

2. Sources of Dietary Carbs • Starch – polymer of glucose • Amylose • linear, forms helices, difficult to digest, flatulence • Amylopectin • branched, easy to digest

3. Sources of Dietary Carbs • Disaccharides • Lactose • galactose and glucose • consequences of lactase deficiency = lactose intolerance • Sucrose • fructose and glucose • Maltose • glucose and glucose • Monosaccharides • Glucose • Fructose • especially these days with high fructose corn syrup

4. Glucose responses Results of consuming a standard 50 g glucose load 10 Intolerant Blood Glucose (mM) Tolerant 5 0 1 2 Time (h)

5. Consequences of Intolerance • Post-prandial hyperglycemia is a problem • If occurs after each meal and persists for several hours then there will be problems • The person will rarely be euglycemic! • Leads to complications of hyperglycemia • Protein glycosylation • Root cause may be insulin resistance • Impaired ability of tissues to respond to insulin • Underlies Type II Diabetes • Control of glucose intolerance • Consumption of slowly absorbed starches

6. Starch Digestion 10 Different Glycemic Responses Amylopectin Blood Glucose (mM) Amylose 5 0 1 2 Time (h)

7. The Glycemic Index • Describes the post-prandial glucose response • Area under the ‘test’ food glucose curve divided by • Area under a ‘reference’ food glucose curve • Reference food is normally 50 g gluocse • Test food given in an amount that will give 50 g digestible carbohydrate • Expressed as a % • GI of modern, processed, amylopectin foods >80 • GI of legumes < 30

8. The Glycemic Index • Useful knowledge for controlling blood glucose • Especial relevance to diabetes • QUALITY of carbohydrate (GI) as important as total amount of carbohydrate

9. GI critics say.. • Area under slowly absorbed may be the same as quickly absorbed • Look closely at previous figure • The GI should not apply to foods other than starches • Sugary foods are low GI • Because half the carbohydrate is fructose • Similarly, fructose containing foods are low GI • Dairy foods are low GI • Because half the carbohydrate is galactose • And protein elicits insulin secretion  lipogenesis

10. GI critics say.. • Some Low GI values • Due to inaccurate estimation of digestible carbohydrate portion • Claims of “slow burning energy” ?? • What regulates energy expenditure and ‘supply’ of substrates? • Even if supply was important, the classic “persistently but subtly” raised post-prandial glucose response is hardly ever seen

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

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

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

14. Glycogen Synthase • Catalyses the addition of ‘activated’ glucose onto an existing glycogen molecule • UDP-glucose + glycogenn UDP + glycogenn+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, PPI) • 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

15. Phosphofructokinase • Catalyses the second ‘energy investment’ stage of glycolysis • F6P + ATP  fructose 1,6 bisphosphate + ADP • Regulated allosterically • Simulated by low energy charge • Energy charge is balance of ATP, ADP & AMP • 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. • Small change in ATP/ADP causes large change in AMP via adenylate kinase reaction • Many other molecules affect PFK allosterically but all are effectively indicators of ‘energy charge’

16. Coupling (again!) • The stimulation of glycogen synthesis by insulin creates an ‘energy demand’ • Glycogenesis is anabolic • The activation of glucose requires ATP • This drops the cellular [ATP] and increases the [ADP] & [AMP] • Drop in ‘energy charge’ is stimulates PFK • Anabolic pathway requires catabolic pathway • Insulin has ‘indirectly’ stimulated PFK and glucose oxidation • So signals to store fuels also cause fuels to be burnt

17. 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 

18. 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

19. Glycogenesis • In both liver and muscle • 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

20. Hexokinases • 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

21. Lipogenesis Overview glucose Fat ESTERIFICATION GLUT-4 No GS X fatty acids glucose G6P Consumes reductant and ATP GLYCOLYSIS PPP LIPOGENESIS Produces reductant pyruvate acetyl-CoA pyruvate acetyl-CoA PDH Key steps (eg, GLUT-4, PDH, lipogenesis) are stimulated when insulin binds to its receptor on the cell surface KREBS CYCLE NADH release ultimately produces ATP CO2

22. Pyruvate Dehydrogenase Pyruvate + CoA + NAD  acetyl-CoA + NADH + CO2 • 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

23. 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

24. Fate of Acetyl-CoA • Burnt in the Krebs Cycle • Carbon atoms fully oxidised to CO2 • 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

25. 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?

26. 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!

27. 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 + CO2 malonyl-CoA • Reaction requires ATP  ADP + phosphate • Participation of the cofactor, biotin • Biotin is involved in other carboxylation reactions

28. 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

29. 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

30. 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

31. Fatty Acyl Synthase

32. FAS - simplified

33. 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

34. 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’ CO2 • FAs start getting ‘released’ as FA-CoA when chain length is C14 • Desaturation is done AFTER FAS

35. 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)

36. 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

37. 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!!

38. Regulatory Overview Fat glucose ESTERIFICATION GLUT-4 No GS X fatty acids glucose G6P G6PDH glycerol 3-P FAS LIPOGENESIS GLYCOLYSIS ACC pyruvate acetyl-CoA Acetyl-CoA transport stimulated by increased production of citrate pyruvate acetyl-CoA PDH citrate G6PDH stimulated by demand for NADP KREBS CYCLE Insulin stimulates GLUT-4. PDH and ACC. Also switches on the genes for FAS and esterification enzyme. CO2 Krebs cycle will be stimulated by demand for ATP