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

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

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

Glucose responses Blood Glucose (mM) 5 10 Time (h) 012 Intolerant Tolerant Results of consuming a standard 50 g glucose load

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

Starch Digestion Blood Glucose (mM) 5 10 Time (h) 012 Amylose Amylopectin Different Glycemic Responses

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

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

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

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

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

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”

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

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, 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

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’

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

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

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

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

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

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

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

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

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

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?

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!

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

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

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

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

Fatty Acyl Synthase

FAS - simplified

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

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

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)

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

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!!

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