Trans Metab Regulation

Dr. Joven Tanchuco, MD

2

OS 201

Correlative Human Cell Biology

Lec 8: Metabolic Regulation – Proprionic acid (terminal 3-carbon molecule from beta oxidation of oddnumbered fatty acids) may be converted into dihydroxyacetone phosphate and diphosphoglyceraldehyde, but there are few odd-numbered fatty acids as sources • Amino acids are the major contributors in glucose synthesis – May result in muscle wasting during starvation as muscles are mainly proteins

Outline I. A.

B.

Regulation of Carbohydrate Regul Metabolism ation of Carbohydrates Carb 1. Characteristics ohydr ate  Available for the body in the form of glucose  Transported in the blood as glucose  Stored as glycogen and triglycerides  Only source of ATP when there is no oxygen  Carbohydrates: up to 80-85% daily caloric intake in less developed countries  Lowest amount by weight (compared to CHON and fats) in the body – about 2 kg in a 70 kg person 2. Functions I.

a. Most important: energy production  Polar  easily soluble in water, easily transportable  Easily diffused through cell membrane (small molecule size overrides polarity, presence of glucose transporters, concentration gradient after a meal)  Abundance of CHO means most cells can use glucose as energy source in the postprandial stage (i.e. after a meal) – Broken down in glycolysis – Stored as glycogen or fatty acids b. Component in cell membranes as glycoproteins  ex. ABO blood groups c. Source of: oxaloacetic acid which replenishes TCA cycle substrates, NADPH for fatty acid synthesis d. Provides precursor of glucoronic acid e. Important component of nucleic acids (5 carbon sugars, such as ribose, deoxyribose, are used for nucleic acid synthesis) f. Glucose in particular is the only major biomolecular energy source in the absence of oxygen  Prevents accumulation of NADH2 and FADH2 in their reduced forms  2 ATPs produced in glycolysis may be lifesaving for the cell (especially neurons which cannot regenrate)  Some cells can only use anaerobic glycolysis as its energy source (RBCs which lack mitochondria) Overview of Carbohydrate Metabolism 1. Interconversion of Carbohydrates • Interconversion and intermittent feeding account for the fact that CHO are abundant in the diet but only constitute a small amount in cells (glucose  glycogen, fatty acids) • Glucose must be maintained normally within a narrow concentration range thus other biomolecules can also be converted to glucose in between feedings (amino acids  glucose) • Major glucose storage is in fatty acids – Glycogen is more hydrated because it remains polar thus its synthesis is limited by space requirements – Fatty acids in the body are saturated and reduced and thus can be stored in a “dehydrated” form • Fatty acid to glucose conversion is not possible

Anna, Everly, Edge, Julie

Monday, Aug.24, 2009

Figure 1. Overview of Carbohydrate Metabolism

Notes: – Lactate formation has a limit because the cell cannot tolerate high concentrations of lactate as it may result in lactate acidosis; lactate is converted back to glucose – Conversion of pyruvate to acetyl CoA is one-way; acetyl CoA can’t be converted back to pyruvate, fat can’t be converted to carbohydrate Remember: The liver is the only organ that will share its glycogen because it is the only organ with glucose-6-phosphotase. Glucose-6-phosphatase dephosphorylates glucose in the liver when other tissues and organs are in need of it. • Glucose Homeostasis • Many hormones in metabolic processes are controlled by glucose levels (ex. insulin, thyroid hormone, epinephrine, glucagon) • Hypoglycemia is a more clinically dangerous situation because RBC, renal medulla cells, and nervous tissue rely almost exclusively on glucose for energy • Insulin released when serum glucose levels go up (post-prandial state) – Anabolic hormone thus stimulates glucose entry and synthesis of glycogen, fatty acids, protein – Inhibits lipolysis, beta oxidation, gluconeogenesis – Responds to serum glucose levels but regulates also fatty acid and amino acid metabolism – Works together with glucagons to regulate CHO metabolism • As serum glucose and insulin levels drop: – Glycogen breakdown and gluconeogenesis begin to maintain serum glucose levels – Inhibition on lipolysis removed to allow energy production from beta oxidation

Page 1 of 1

Dr. Joven Tanchuco, MD

2

OS 201

Correlative Human Cell Biology

Lec 8: Metabolic Regulation

2. • •

•

•

•

– Amino acid breakdown aids in gluconeogenesis and may act as alternative energy source Carbohydrate Absorption and Transport Carbohydrates in the diet are mostly from plants and dairy products for lactose Digestion starts in the mouth with salivary amylase though much of it occurs in the small intestine with pancreatic amylase and other dissacharides Carbohydrates absorbed primarily as monoand disaccharides Absorption in the intestine takes place mainly via facilitated transport – Co-transport with sodium – Non-insulin dependent – Further down the intestine absorption is via active transport Entry into hepatic tissue (first organ encountered after entering portal circulation) is non-insulin dependent

TRANSPORTER GLUT-1

Small intestine C o t r a n s p o r t

F a c il it a t e d

Table 1. Different glucose transporters found inside the body

• GLUT-4 stimulated by insulin  entry of

LOCATION Ubiquitous, expressed to the largest degree in brain and placenta β-cells in the pancreas, liver, kidney

D if f u s i o n

GLUT-3

 Amount of glucose intake of these organs are proportional to the amount of glucose in the blood  Ensures that glucose is only taken up by the liver only when it is abundant Ubiquitous Adipose tissue, heart, skeletal muscle

 Only glucose transporter regulated by INSULIN  Km is as high as the average [serum glucose] which is 5mM GLUT-4

 When the [serum glucose] is high, insulin causes translocation of these transporters from an intracellular store to the cell membrane  Fatty acids decrease the activity of GLUT-4 because it is the preferred source of energy of cells

GLUT-5

Kidneys  Glucose in the urine must be reabsorbed

 High Km (15-20 mM) GLUT-2

Nadepend ent

C. 1.

glucose into skeletal muscle and adipose is insulin-dependent and occurs during high glucose levels • Entry of glucose into pancreas, liver, and renal tubules is non-insulin dependent – Pancreas is responsible for secreting insulin – Renal tubule reabsorption of glucose must always be present especially in times of low serum glucose and insulin – Liver is the first organ encountered and cannot wait for an insulin signal • Phosphorylation of glucose into glucose-6phosphate inside the cell prevents its exit from the cell and decreases the amount of glucose within, allowing for more entry of glucose from the blood – Glucose-6-phosphate may (1) enter pentose-phosphate pathway, (2) be used in glycogen synthesis, (3) undergo glycolysis Regulation of Glycolysis Introduction GLYCOLYSIS • Major energy producing pathway in carbohydrate metabolism • Mainly a catabolic pathway but is also initial stage of glucose to fatty acid conversion • Major pathway for storage of energy available in glucose • Glucose may also come from glycogen, not just the blood • Only glycolytic reactions with the highest free energy change need to be regulated because they are irreversible REGULATION 1. Regulation of glucose uptake through glucose transporters (see table of transporters above)  Glycolysis can be regulated through the availability of the substrate 2. Glycolysis can be regulated through the control of specific reactions. Rate-limiting Step: Characteristics

 In special situations: Anaerobic conditions, presence of respiratory poisons (e.g. Cyanide), uncoupling of oxidative phosphorylation Small intestine

a. b. c. d.

Anna, Everly, Edge, Julie

Monday, Aug.24, 2009

 Need to transport against a gradient from lumen to epithelial cells. Kahit busog na ang cells mo with glucose, kailangan pa rin nya kuhanin ang glucose sa blood. 

Page 2 of 2

Small activity (small Vmax) Small mass: action ratio Products / reactants Large negative free energy change. (NOTE: this is the most important criteria)

Dr. Joven Tanchuco, MD

2

OS 201

Correlative Human Cell Biology

Lec 8: Metabolic Regulation There are 10 steps in the Glycolytic Pathway. Of these ten steps, three involve large amounts of negative free energy. These three steps are presented in the table below. Table 2. Three Reactions of Glycolysis with the Largest Negative Free Energy

Reacti on #

Enzyme

Description

-∆G

Phosphorylation -33.5 1 Hexokinase glucose kj/mol PhosphofruPhosphorylation of -22.2 3 ctokinase fructose-6-phosphate kj/mol Transfer of phosphate Pyruvate -33.4 10 from phosphoenolkinase kj/mol pyruvate to ADP These three reactions are catalyzed by enzymes whose activities are irreversible. This is the primary reason why they are potential sites of regulation in the glycolytic pathway.

CoA for FA synthesis (both are stimulated by insulin) – Liver thus undergoes little glycolysis as it does not absorb much glucose, sparing for other organs that need it • Catalyzed by hexokinase in non-hepatic tissue – Low Km allows extraction of serum glucose even in low concentrations – Low Km also assures phosphorylation of glucose even if only small amounts are taken into the cell – At low insulin levels, glucose undergoes glycolysis; at high insulin levels, it is stored as glycogen and fatty acids The signal for glycogenolysis in skeletal muscle is epinephrine. Always remember that the higher the Km value, the lower the affinity of the enzyme for its substrate.

In general, insulin increases the transcription while glucagon decreases the transcription of these three enzymes. The effects of these hormones take place over a period of hours to days and generally indicate whether a person is well-fed or starving. In general, if forward reaction is stimulated then backward reaction is inhibited 2.

Regulated Reactions a. Glucose Glucose-6-phosphate Hexokinase and Glucokinase

Figure 3. Km Value

Table 3. Comparison of Glucokinase and Hexokinase

Glucokinase High Km (10 mM) Not inhibited by its product High Vmax Highly responsive in changes in glucose serum concentration (more than hexokinase) Hepatic Tissues Phosphorylation occurs only at high glucose levels

Hexokinase Low Km (0.1 mM) Inhibited by its product (Glucose-6-phosphate) Low Vmax

b. Fructose-6-phosphate Fructose-1,6phosphate • Most critical for balance between gluconeogenesis and glycolysis • Signal molecule for regulation is glucagons

• Simultaneous stimulation of one direction and inhibition of the other  allows greater changes in flux and a more rapid response to cell needs – Greater substrates committed  greater waste in overall effect  greater response ability – Ability to respond to changes more important than baseline efficiency • Forward and backward reactions catalyzed by different enzymes: phosphofructokinase (PFK1) for the forward and fructose-1,6biphosphatase (FBP) for the reverse Phosphofructokinase

Extrahepatic Tissues Phosphorylation occurs even at low glucose levels Not regulated by insulin or Insulin increases glucagon (the speed of its synthesis of this enzyme reaction makes it hard to regulate) Significance: Glucokinase in the liver has a higher Km than hexokinase in extrahepatic tissues so that glucose will not be phosphorylated in the liver (it will not be trapped in the liver). The high Vmax of glucokinase also allows the liver to effectively store excess glucose circulating in the blood.

Phosphofructokinase catalyzes the ratelimiting step in glycolysis and is the most important control point.

Elaboration:

a. Allosterically inhibited by ATP

• Catalyzed by glucokinase in the liver – Glucokinase has higher Km and is not inhibited by glucose-6-phosphate thus glucose is quickly metabolized in the liver only when its levels are high – High insulin levels mean that glucose is stored as glycogen c/o glycogen synthetase and fatty acids c/o malonyl CoA synthetase which diverts acetyl

Anna, Everly, Edge, Julie

Monday, Aug.24, 2009

b.

c. Page 3 of 3

meaning glycolysis is slowed when cellular concentrations are high Excess H+ inhibits glycolysis because it favors the low-affinity state of PFK  this helps minimize risk of lactic acidosis Citrate, an intermediate of the TCA cycle, also inhibits PFK  signals

Dr. Joven Tanchuco, MD

2

OS 201

Correlative Human Cell Biology

Lec 8: Metabolic Regulation that biosynthetic precursors are abundant d. Allosterically activated by high levels of AMP  AMP means that there is a low source of ATP. When ATP has already been used up, a high concentration of ADP is generated. From ADP, the cell can generate ATP again through the reaction: ADP + ADP  ATP + AMP, thus AMP is a signal that ATP concentration is down e. Allosterically activated by Fructose 2,6-bisposphate f. Covalent regulation of PFK by glucagon  high glucagon causes phosphorylation of PFK2 which catalyzes the forward reaction of Fructose 6-Phosphate  Fructose 2,6-Phosphate (which we know can activate PFK) Remember: ATP, Citrate and H+ inhibits PFK. AMP and Fructose 2,6-bisphosphate activates PFK. • Bifunctional enzyme II has a kinase domain (active when dephosphorylated) and a phosphatase (active when dephosphorylated) – Phosphorylation regulated by blood glucose level, mediated by glucagon and insulin – Fructose-6-phosphate can be metabolized to fructose-2,6biphosphate c/o phosphofructokinase 2 (PFK2) – Fructose-2,6-biphosphatase (FBP2) catalyzes conversion of fructose2,6-biphosphate to fructose-6phosphate – Major regulator is glucagon – Fructose-2,6-biphosphate stimulates PFK1 (positive feedback) and inhibits FBP (negative feedback)

1.

Kinases and phosphatases are different domains of one bifunctional polypeptide enzyme. 2. The substrate cycle involving Fructose 2,6bisphosphate is a potent regulator of both glycolysis and gluconeogenesis 3. This ensures that gluconeogenesis and glycolysis are not occurring simultaneously in order to avoid a futile cycle 4. Fructose 2,6-bisphosphate is the substrate that has the regulatory effect a. Most potent activator of PFK I b. Inhibitor of Fructose 1,6-phosphatase Phosphofructokinase II (PFK II)

•

In the unphosphoryated state, has kinase function • In the phosphorylated state, has phosphatase function • Part of covalent modification  inhibits fructose biphosphate and stimulates fructokinase Glucagon •

Hormone which is produced when blood glucose is low • Stimulates gluconeogenesis • binds to plasma membrane receptors on liver cells, activating membrane-localized adenylate cyclase, leading to an increase in the conversion of ATP to cAMP Recall: cAMP  protein kinase b  protein kinase a • • • Insulin

Protein kinase phosphorylates F 2,6-phosphatase (FBP II) and inhibits PFK II, leading to a decrease in F 2,6-biphosphate No more inhibition of F 1,6-bis pase (FBP), which dephosphorylates F 1,6 biphosphate to F 6 phosphate Fructose 6 phosphate is converted to glucose (a.k.a. GLUCONEOGENESIS)

• •

opposes all effects of glucagons leads to the dephosphorylation of PFK II (active) which will eventually stimulate FPK through F 2,6-biphosphate • also leads to a decrease of cAMP Allosteric modification of FBP •

high amounts of AMP: stimulates PFK – inhibits the production of glucose through gluconeogenesis and favors the production of ATP (energy) through glycolysis • high amounts of ATP, citrate and H+: inhibits PFK – breakdown of glucose through glycolysis is no longer needed Feedforward Stimulation

•

Figure 4. Substrate Cycle as a Means of Control of Glycolysis and Gluconeogenesis

Pyruvate Kinase a. b. c.

• low fructose-2,6-biphosphate, high fructose6-phosphate  gluconeogenesis Glucagon, acting on adenyl cyclase receptor complex and through a protein kinase, can inhibit PFK2 and stimulate FBP2

Activated by Fructose 1,6-bisphosphate Allosterically inhibited by ATP and alanine Covalently modified by glucagon (glucagons causes phosphorylation which renders the enzyme inactive)

– –

Note:

Anna, Everly, Edge, Julie

Monday, Aug.24, 2009

Page 4 of 4

Low glucose high glucagonlow fructose-2,6-biphosphatehigh fructose-6-phosphate Flux is towards gluconeogenensis because of lack of inhibition of FBP by fructose-2,6-biphosphate

Dr. Joven Tanchuco, MD

2

OS 201

Correlative Human Cell Biology

Lec 8: Metabolic Regulation • high fructose-2,6-biphosphate, high fructose -1,6-biphosphate  glycolysis Low glucagon (high blood sugar)  dephosphorylation of the enzyme II conversion to fructose-2,6-biphosphate from fructose-6-phosphate  stimulatory effect on PFK1 glycolysis

○

Phosphoenol pyruvate pyruvate

• • • • • •

Amino acids as the source of pyruvate  prevents futility of the process Includes the Malate shuttle (malate exits mitochondrion) Will happen simultaneous with the breakdown of carbohydrates Strictly irreversible Last reaction in glycolysis which is regulated. Catalyzed by pyruvate kinase and produces 1 ATP c.

Pyruvate can be converted to oxaloacetate via pyruvate carboxylase

○ ○ ○

○ ○ d.

○ Figure 5. The Pyruvate-Phosphoenol Pseudocycle ○ Phosphoenol pyruvate converted to pyruvate via pyruvate kinase ○

○ ○

○ b.

This enzyme is stimulated by fructose1,6-biphosphate and inhibited by ATP, glucagon, alanine, and phenylalanine (these 2 amino acids are substrates for gluconeogenesis) Inhibition achieved through covalent modification with the phosphorylated pyruvate kinase being the inactive form Alanine makes pyruvate kinase more susceptible to covalent modification PEPpyruvate cycle produces 1 ATP

Pyruvate formed enters mitochondrion to be converted to acetyl CoA c/o pyruvate dehydrogenase (PDH), another strictly irreversible reaction ○ ○

Reason why fatty acids cannot be converted to glucose Acetyl CoA from fatty acid breakdown cannot regenerate pyruvate and instead

Anna, Everly, Edge, Julie

Monday, Aug.24, 2009

Stimulated by low glucose levels  low insulin  PDH becomes inactive This allows conversion of pyruvate to oxaloacetate by pyruvate carboxylase Pyruvate carboxylase is stimulated by acetyl CoA from beta oxidation of fatty acids when serum glucose and insulin levels are low Pyruvate carboxylase is however inhibited by ADP The reaction initiates regeneration of phosphoenol pyruvate from pyruvate

Oxaloacetate is converted to malate via malate dehydrogenase ○

a.

enter the TCA cycle where its 2 carbons are released as CO2 Enzyme also regulated by covalent modification  Also active in the dephosphorylated form c/o PDH phosphatase which is stimulated by insulin  Inactivation via PDH kinase which is stimulated by acetyl CoA, NADH, ATP  Pyruvate inhibits PDH kinase and activates PDH  Dephosphorylation of PDH is done by specific PDH phosphatase  PDH phosphatase is stimulated by insulin  Insulin promotes conversion of pyruvate to acetyl CoA

○ ○

Oxaloacetate is converted to malate inside the mitochondrion by malate dehydrogenase Malate is transported out of the mitochondrion into the cytosol (malate shuttle) Oxaloacetate regenerated from malate via same enzyme (malate dehydrogenase) Oxaloacetate converted to phosphoenol pyruvate via PEP carboxykinase Synthesis of PEP carboxykinase is stimulated by glucagon in the presence of glucocorticoids  Inhibited by ADP, insulin and high level of glucose

e.

Pyruvate can be converted to acetyl coA by pyruvate dehydrogenase. ○ Pyruvate is transferred to mitochondrion ○ Converted to acetyl CoA by pyruvate dehydrogenase (irreversible reaction) ○ Acetyl CoA enters TCA ○ Carbons are released as CO2

•

The whole pyruvatePEP cycle consumes 2 ATPs equivalents

Page 5 of 5

Dr. Joven Tanchuco, MD

2

OS 201

Correlative Human Cell Biology

Lec 8: Metabolic Regulation •

The reverse process of the PEP pyruvate produces only 1 ATP (there is a net requirement of another unit of ATP)

•

Insulin and glucagon also regulate protein synthesis and amino acid breakdown, respectively a. Promote gluconeogenesis and amino acid breakdown b. High glucagon/insulin ratio  promotes synthesis of PEP carboxykinase, glucose-6-phosphatase, other aminotransferases c. inhibits glucokinase synthesis and pyruvate kinase

3.

Blood-borne glucose is converted by exercising muscle to lactate which diffuses into the blood. This lactate is taken up by the liver and converted to glucose which is released back to the circulation or returns to the muscle to replenish their glycogen. ○ 2 lactate  2 pyruvate  glucose (Liver)  glucose  2 pyruvate  2 lactate (muscle) ○ Significance: Cramps is a signal for the body to stop using energy. Too much lactate is being produced. b. Glucose alanine cycle ○

○

Gluconeogenesis  Formation of glucose from nonhexose precursor  Gluconeogenesis is NOT the reverse of glycolysis and vice versa.

Glycolysis Glucose to pyruvate Glucose = substrate Product: 2 molecules of pyruvate, ATP and NADH

Gluconeogenesis Pyruvate to glucose BUT with detour Glucose = product

Makes 2 moles of ATP

Consumes 6 ATP

○ ○ ○ ○

c. Entry of glycerol derived from triglycerides into gluconeogenesis

Substrate: amino acids (ketoacids)

○



    



Glucagon – main controlling hormone Fructose-6-phosphate:fructose-1,6-biphosphate substrate cycle = determines flux between glycolysis and gluconeogenesis Release of glucose from inside the cell would be made possible with the removal of phosphate done by glucose -6-phosphate (found only in liver and renal cells) Reactions converting amino acids into glucose depends on type of amino acid and their corresponding ketoacids About 150 grams of glucose per day can be made in gluconeogenesis. Only liver and kidney are capable of gluconeogenesis. Glucagon - major hormone controlling gluconeogenesis New glucose come from: ○ Breakdown of AA except leucine and lysine (pyruvate) ○ Lactate (Cori cycle) ○ Glycerol backbone of TAG ○ Propionyl CoA (succinyl CoA) Regeneration of glucose ○ Glucose can be regenerated in the liver from lactate and alanine in the muscle. a. Cori Cycle (lactate) ○

○ ○ ○ ○

Monday, Aug.24, 2009

Controlled by the regulation of key enzymes glycogen phosphorylase (involved in glycogenolysis) and glycogen synthetase (involved in glycogenesis) Glycogen synthesis and breakdown occurs in cytosol. Glycogenolysis – stimulated by high levels of AMP and inorganic phosphate (Pi) and inhibited by ATP and glucose-6-phosphate To prevent futile cycles, simultaneous regulation of both pathways has to be present: Glycogen phosphorylase •

○

facilitates conversion of glycogen to glucose • stimulated by high levels of AMP and Pi; • inhibited by ATP and glucose-6phosphate • Active enzyme is independent of these but requires Ca2+ • Epinephrine in muscle and glucagon in liver activates the enzyme • Less sensitive to allosteric modifier Glycogen synthase • • • •

Glucose is regenerated in the liver from lactate

Anna, Everly, Edge, Julie

Glycerol is converted to dihydroxyacetone phosphate (DHAP), which could then be converted to glucose

Regulation of glycogen metabolism



○  

Amino acids in the muscle are converted to alanine (small molecules) which are easily passed on to the liver to create new glucose Nitrogen (carried by alanine) is eliminated through the urea cycle which occurs only in the liver Alpha keto acid is used for energy Glucose can be exported from the liver to the other cells. Gluconeogenesis in the liver allows AA to be broken down in the muscle.

Page 6 of 6

Involve in glycogen synthesis Facilitates conversion of glucose to glycogen Stimulated by insulin allosterically actived by glucose-6phosphate, a precursor of glucose-1-

Dr. Joven Tanchuco, MD

2

OS 201

Correlative Human Cell Biology

Lec 8: Metabolic Regulation • • •

•

phosphate, the building block of glycogen. Has seven sites of phosphorylation catalyzed by seven protein kinase Phosphorylation of these sites lead to progressive inactivated of the enzyme Encourages phosphorylation is a covalent modification of glycogen phosphorylase

•

Hormonal - presence of insulin, glucagon, fight/flight hormones)

[the following are from 2013 trans ]



Allosteric Regulation of glycogen synthesis and degradation

○

Glycogen synthase and glycogen phosphorylase respond to levels of: • metabolites / substrates • energy needs Glycogen synthesis = stimulated when substrate availability and energy levels are high Glycogen degradation = stimulated when energy levels and available glucose are low

○ ○

a. • •

Figure 6. Covalent Modification of the glycogen phosphorylase enzyme. Glycogen Phosphorylase is activated by the linkage of a phosphate prosthetic group. This is catalyzed by phosphorylase kinase which is also activated by phosphorylation of catalyzed by another protein kinase. Activation of protein kinase b is done by presence of high levels of cAMP, formed from ATP by adenyl cyclase located in the cell membrane and associated by cell membrane receptors. In glycogen phosphorylase enzyme system, cell receptor is specific for glucagon (glycogenolysis in liver) or epinephrine (glycogenolysis in skeletal muscle). The roundabout way of activating glycogen phosphorylase with a signal from glucagon or epinephrine is called amplification.

b. Regulation in muscle by calcium b.1. In muscle contraction: rapid and urgent need for ATP – Nerve impulses cause membrane depolarization  Ca2+ release from sarcoplasmic reticulum into sarcoplasm of muscle cells – Ca2+ binds to calmodulin – Binding of 4 molecules of Ca2+ triggers a conformational change = Ca2+ calmodulin complex – Ca2+ - calmodulin complex binds to and activates phosphorylase kinase (without phosphorylation) b.2. in muscle relaxation – Ca2+ returns to the sarcomplamsic reticulum  phosphokinase becomes inactive

The difference in signal molecule illustrates basic difference in the functions of major tissue involved in glycogenolysis. Skeletal muscles involved primarily in movement would need more energy. Epinephrine would allow glycogenolysis to take place to provide lot of glucose needed for rapid production of energy.

✔ ✔ ✔



Regulation of Glycogen Synthesis and Degradation

Regulation of both glycogen synthesis and degradation are important in maintaining blood glucose levels ○ Liver • Glycogen synthesis = occurs during well-fed state • Glycogen degradation = occrs during periods of fasting ○ Muscle • Glycogen synthesis = occurs during active exercise • Glycogen degradation = occurs as soon as muscle is again at rest\ ○ Regulation occurs and is accomplished via glycogen synthase and glycogen phosphorylase on 2 levels: • Allosteric- allosteric enzymes having different conformation induced by the binding of modulators

•

○

Anna, Everly, Edge, Julie

Monday, Aug.24, 2009

Regulation in the well-fed state Glycogen synthase: allosterically activated by glucose 6-phosphate (G6P) Glycogen phosphatase: allosterically inhibited by G6P, ATP, and in the liver, glucose

•



c. Regulation in muscle by AMP Glycogen phosphatase is active in the presence of high AMP concentrations (e.g. during anoxia and ATP depletion) AMP binds to the inactive form of glycogen phosphatase  activated (without phosphorylation)

Activation of glycogen degradation by cAMPdirected pathway ○

Binding of hormones (e.g. glucagon or epinephrine) to membrane receptors signals the need for glycogen breakdown to: • elevate blood glucose levels • provide energy for exercising muscle a.

Page 7 of 7

activation of protein kinase • binding of hormones to receptors cAMP-mediated activation of cAMPdependent protein kinase

Dr. Joven Tanchuco, MD

2

OS 201

Correlative Human Cell Biology

Lec 8: Metabolic Regulation – –

b.

C. • – – • • • • 

• protein kinase: tetramer 2 regulatory subunits 2 catalytic subunits

•

cAMP binds to the regulatory subunit dimer  release of individual catalytic subunits (active)

Activation of phosporylase kinase • Active cAMP-dependent protein kinase phosphorylates inactive phosphorylase kinase  activation Activation of glycogen phosphatase Glycogen phosphatase exists in 2 forms: Inactive “b” form (GP B) Active “a” form (GP A) Active phosphokinase phosphorylates GP B to active GP A GP A begins glycogen breakdown Reconversion of GP A to GP B: hydrolysis of its phosphate by protein phosphatase 1 AMPLIFICATION of hormonal signal

Inhibition of glycogen synthesis by cAMP-directed pathway • Glycogen synthase: regulated enzyme in glycogen synthesis • 2 forms: a) a form: not phosphorylated, most active b) b form: phosphorylated, inactive • glycogen synthase is converted to b form by phosphorylations at a # of sites  catalyzed by a cAMP-dependent protein kinase • protein kinase phosphorylates and thereby inactivates glycogen synthase

4. Pentose-Phosphate Pathway *He skipped this, but it was part of his PowerPoint presentation and was also in the 2013 trans so...



Also called the hexose-monophosphate shunt or 6-phosphogluconate pathway  Occurs in the cytosol  Has two irreversible oxidative reactions: A. Dehydrogenation of glucose 6-phosphate ○ Glucose 6-phosphate dehydrogenase (G6PD) is strongly inhibited by NADPH ○ Insulin enhances G6PD gene expression ○ Flux through the pathway increases in the wellfed state ○ Produces first NADPH B. Formation of ribulose 5-phosphate ○ Produces second NADPH and 1 CO2  Reversible nonoxidative reactions (sugar– phosphate interconversions) ○ Permit ribulose 5-phosphate to be converted to either: ○ Ribose 5-phosphate – When demand for ribose for incorporation into nucleotides and nucleic acids is greater than for NADPH

Anna, Everly, Edge, Julie

Monday, Aug.24, 2009

• •

– When G6PD is inhibited, ribose can be synthesized glyceraldehyde 3-phosphate and fructose 6-phosphate – When more ribose is needed, ribose may be regenerated anew from glucose 6-phosphate from exogenous glucose  Intermediates of glycolysis (fructose 6phosphate and glyceraldehyde 3phosphate) – When demand for NADPH is greater than for ribose 5-phosphate (for reductive biosynthetic reactions) No ATP is directly consumed or produced Products

○

CO2 from carbon 1 of glucose 6-phosphate 2 NADPH per glucose 6-phosphate entering the oxidative part of the pathway – Provides a major portion of the body’s NADPH – NADPH: biochemical reductant; needed in fatty acid synthesis ○ Ribose 5-phosphate – For biosynthesis of nucleotides • Found in tissues active in reductive biosynthesis: Liver, adipose, adrenal cortex, gonads, erythrocytes IV. Regulation of Fatty Acid and Cholesterol Metabolism ○

A.Introduction  Fatty acids and cholesterol belong to the lipid group of biomolecules. They are the best way for storing energy in the cell.  Most of the lipids found in humans are in the form of triglycerides. a. One of the most efficient forms of storing energy b. Contain six times the amount of calories on a weight basis as compared to carbohydrates c. Less oxidized (contains less oxygen per carbon atom) d.Less hydrolysed because they are non-polar  allow more oxidation to take place, stored in compact form without much water. e. Body stores 80% of the energy available to it as fats. B.Overview of Lipid Metabolism

Figure 7. Overview of Fatty Acid Metabolism

 The hydrophobic property of fatty acids allow them to pass easily across the cell and organell membranes.

Page 8 of 8

Dr. Joven Tanchuco, MD

2

OS 201

Correlative Human Cell Biology

Lec 8: Metabolic Regulation  To make the fatty acids impermeable, that is to trap them and make them commit to a metabolic pathway, they are combined with a large molecule, the Coenzyme-A (CoA) by the action of the enzyme Acyl CoA synthetase.  However, beta-oxidation occurs in the inner mitochondrial matrix. The enzyme Carnitine Palmitoyl Transferase (CPT-1) transports the fatty Acyl CoA from the cytosol into the inner mitochondrial matrix.  Once inside the mitochondrial matrix, the FA is already committed to take the beta-oxidation pathway.  Fatty acid from the diet is converted to its storage form, the triglycerides through esterification.  Ketogenesis in the liver occurs when TCA intermediates are inadequate (e.g. low [glucose]) to allow complete oxidation of FAs. This allows the energy of the FAs to be extracted and redistributed in the form of more soluble ketone bodies.  Fatty acids are also derived from glucose and amino acids  even if fat intake is reduced, excess calories from CHOs are stored as fats. C.Lipolysis and Esterification  Esterification – FA molecules are added to the glycerol backbone; esterase enzyme  Lipolysis – hydrolytic removal of the FA moiety from the glycerol backbone of triglycerides; lipase enzyme. a. Lipoprotein lipase – located on the endothelial surface of blood vessels; breaks up TAGs coming from the intestines; allow entry of FAs into the adipose cells. b. Hormone sensitive lipase – located in the cytosol; breaks up TAGs stored inside the adipose cells.

insulin

Figure 8. Lipolysis and Esterification. Encircled portion indicates pathway that proceeds when there is low blood glucose. Solid arrows-activation; broken arrows-inhibition.

c. High [glucose]  high [insulin]  TAGs from the intestines must gain entry into adipose cells for storage  lipoprotein lipase is active. d.Low [glucose]  low [insulin]  there is no inhibition of the hormone-sensitive lipase (it is active)  breakdown of TAGs in the adipose cell  FFA made available to other cells as a source of energy.

Table 1. Hormonal regulation of the Hormone-sensitive and Lipoprotein lipase enzymes. Enzyme Regulatory Agent Effect Hormone Sensitive Lipase

Lipoprotein Lipase

Lipolytic hormones (epinephrine, glucagons, ACTH) Insulin

Stimulation by cAMPmediated phosphorylation of relatively inactive enzyme

Prostaglandin

Inhibition

Lipoprotein

Inhibition Activation

Apoprotein C-II Insulin

Anna, Everly, Edge, Julie

Monday, Aug.24, 2009

Page 9 of 9

Activation

Dr. Joven Tanchuco, MD

2

OS 201

Correlative Human Cell Biology

Lec 8: Metabolic Regulation



    

Figure 9. Covalent modification of hormone-sensitive lipase.

D.Beta-oxidation



the needed TCA intermediates to complete the oxidation of FA. There is now a difficulty occurring in the TCA cycle because there is no enough intermediates to process the acetyl-CoA coming in. A pseudo high-energy state is created because of the accumulation of acetyl CoA (as what happens during starvation) Ketogenesis relieves the TCA cycle of the stress as 2 molecules of Acetyl-CoA form acetoacetate  inhibited accumulation. Ketone bodies are also more soluble and easily transported. Occurs only in the liver because of the high levels of activity of the enzymes HMG-CoA synthase and HMG-CoA lyase. Factors affecting Ketogenesis: a.High lipolysis  serum [FFA]  liver is more saturated  high ketogenesis b.High insulin/glucagons ratio  (+) acetyl CoA carboxylase  high [malonyl CoA]  (-) CPT-1  decreased low ketogenesis. (Activity of CPT-1 determines whether the initial steps of b-oxidn and ketogenesis takes place or not) c. Gluconeogenesis in the liver also utilizes oxaloacetate  lesser ability of the TCA cycle  increased ketogenesis Ketoacidosis – results when there is excessive ketogenesis; seen in people with diabetes mellitus or under starvation

Figure 10. Interrelationships between glycolysis and fatty acid metabolism. Solid circle-activation; hollow circle-inhibition.

 CPT-1 is inhibited by malonyl coA, an intermediate of FA sythesis.  Insulin indirectly inhibits CPT-1 by activating acetyl CoA carboxylase which in turn converts more acetyl CoA to malonyl coA.  Acetyl CoA carboxylase is the committed enzyme in FA synthesis.  Acetyl CoA, which needs to be recycled, is transported out of the mitochondrion through the citrate shuttle.  FFA (palmitic acid in the diagram) serves as a signal that there are adequate amounts of FA thus inhibiting FA synthesis through acetyl CoA carboxylase.  During fasting, glucose is converted to energy and not to FA.  Too much FFA: a. Tissue would prefer FFA, especially skeletal muscle. b. Glucose will be spared. c. Cells will not need glucose d. Glucose uptake will not be significant. e. Insulin levels could remain high (Hyperinsulinemia). E.Ketogenesis  FA break down occurs in conditions of relatively low glucose. However, glucose is needed to generate

Anna, Everly, Edge, Julie

Figure 11. Ketogenesis

F. Lipogenesis  High in people with high carbohydrate intake  Low in people with restricted caloric intake, high fat diet and low insulin levels

Monday, Aug.24, 2009 Page 10 of 10

Dr. Joven Tanchuco, MD

2

OS 201

Correlative Human Cell Biology

Lec 8: Metabolic Regulation  Hyperinsulinemia  Hypercholesterolemia

Julie: Hi Dana and Laureen! Hi Psych my loves! Hi Bryan, Ricky and Joreb! Thanks for keeping me company; no thanks for making me watch that stupid slasher movie. Hi Dior, Chax, Nino, JB Boy, Jana, Ruby, Shasha, kung sinuman sa inyo ang maging buddy ko! Anna: Kamusta naman? Siguro ay nakangiti ka habang binabasa mo ito, nagbabakasakali na ikaw ay mababati.. hehe. Kaya para naman ikaw ay masiyahan, kahit sino ka man, buong galak kitang binabati sa trans na ito. AJA! (>’-’)> sa mga behsci pips -robertojegargracestephrenz -tandaan nyo ang theme song natin.. ang walang kamatayan (If we hold on together!) ahaha.. pipahatid ko din ang pagbati sa masiyahing tao sa kabilang pampang – jeshjaybeadavidalphiusms.drogerlance – kaway naman jan! sa mga masisipag na rso, carry on! at sa aking magigiting na seatmates, tulugan na! 

Figure 12. Lipogenesis and Glycolysis.

 Main regulatory points: a. Acetyl CoA carboxylase ○ Stimulation by citrate indicates wide availability of glucose and raw materials for FA synthesis ○ Inhibited by acyl CoA means that FAs are coming in from the diet b. Pyruvate dehydrogenase

Everly: Sorry sa mahabang trans. Kasalanan ni ed! Hello to the IB people especially to Bea, Marvin and Al. Godbless sa metab exam natin.

○ Inhibited by high [acetyl-CoA], [NADH] and [ATP] through covalent modification; indicates a high-energy state. G.Cholesterol Metabolism

Figure 13. Regulation of Cholesterol Metabolism.  Negative feedback mechanism: cholesterol inhibits HMG CoA reductase by inhibition of the enzyme synthesis or activation of the enzymes that degrade HMG CoA reductase.  Activation by insulin is through covalent modification.  HMG CoA reductase is active at the dephosphorylated state.  Level of cholesterol in the cell: a.Increased uptake by the number of LDL receptors  inhibition of cholesterol synthesis b. Cholesterol inside the cell downregulates number of LDL receptors. c. Decreased by loss of cholesterol from cell membranes as HDL, esterification and synthesis into cholesterol derivatives.

Anna, Everly, Edge, Julie

Monday, Aug.24, 2009 Page 11 of 11