Metabolism Pathways II: Regulation

Question 1

Leptin

Answer 1

: Hormone released by adipose tissue, regulates control of feeding by hypothalamus

Question 2

Insulin

Answer 2

: Hormone released by the pancreas, regulates metabolism of nutrients.

Question 3

Gut Hormones

Answer 3

: Including secretin, cholecystokinin (CCK) and substance P. Regulates GIT and hypothalamus.

Question 4

Vagus nerve:

Answer 4

Regulates via brainstem, responds to physical signals (Eg Stomach distension).

Question 5

Nutrient Signals:

Answer 5

Including glucose and fatty acids, diffuse regulation throughout the brain.

Question 6

Regulation of Feeding: Well Fed State

Answer 6

• Well-fed state lasts for approximately 4 hours post- consumption of a meal.
• Rise of glucose levels in the bloodstream results in increased insulin release from the pancreas.
• Increased glucose absorption at the liver.
• Increased conversion of glucose to glycogen in liver and muscle tissue.

Question 7

Regulation of Feeding: Well Fed (Metabolism)

Answer 7

Under well fed conditions, liver and muscle glycogen stores are replenished. In liver (and to a lesser extent in adipose tissue), excess glucose is converted to fat (via acetyl CoA). Under normal well-fed conditions FFA’s are converted to TAG and packaged into VLDL particles for transport to the adipose tissue and, depending on energy requirements, the muscle. The liver is in a ‘glycolytic’ or ‘storage’ mode (stimulated by high insulin:glucagon- this also serves to regulate storage of fat by adipose tissue).

Rise of glucose and protein levels in the bloodstream.
• Increased glucose absorption at the liver.
• Increased conversion of glucose to glycogen in liver and muscle tissue.
• Increased production of fat in the liver and transport to the adipose tissue.

Question 8

Regulation of Feeding: Fasting State

Answer 8

Fasting state lasts for the period between meals where all food has been digested, absorbed and stored.
• Drop of glucose levels in the bloodstream results in increased glycogen release from the pancreas.
• Inhibition of glycogen synthesis in the liver.
• Increased conversion of glycogen to glucose in liver and muscle tissue.

Question 9

Regulation of Feeding:hungry (Metabolism)

Answer 9

Decrease in glucose and protein levels in the bloodstream.
• Reduced glucose absorption at the liver.
• Increased protein metabolism to form glucose.
• Release of fatty acids from adipose tissue, producing energy within the liver.

The Cori cycle continues to operate but provides no net increase in glucose levels (mere cycling, maintaining glucose supply to tissues). Complete oxidation of glucose occurs in brain and so needs to be replaced. Muscle does not remove glucose from the blood (low insulin) and instead relies on fatty acid oxidation. Substrates for gluconeogenesis come largely from breakdown of muscle protein and also from glycerol released from the adipose by activation of HSL. Muscle oxidises protein and releases alanine and glutamine. Alanine is transported to liver and converted to pyruvate (substrate for gluconeogenesis and synthesis of ketone bodies via acetly-CoA). Enterocytes of the gut utilise glutamine, transamination of glutamine to the corresponding keto-acid (alpha-keto glutarate) provides a substrate for entry to the TCA cycle. The amino group is transferred to pyruvate, producing alanine, which is then used by the liver. Fatty acid breakdown in adipose provides substrates (FFA’s) for the synthesis of ketone bodies by the liver and can be used by the muscle under aerobic conditions by beta-oxidation. Free glycerol provides a substrate for gluconeogenesis in the liver.

Question 10

Making new molecules and destroying old ones

Answer 10

– this allows changes both in metabolic capacity (how much activity) and in capability (do different things). This form of control is relatively slow (hours to days) and is governed by changes in gene expression

Question 11

Changing the activities of existing molecules

Answer 11

– enzyme activity can be changed quickly (milliseconds to minutes) by covalent modification or by allosteric interactions, or both.

Usually occurring at those steps that catalyse irreversible steps, often these reactions involve ATP and/or a transport step.

Question 12

BIOCHEMICAL MODIFICATION

Answer 12

Changing the activity of pre-existing enzymes
• Fast onset
• Short duration

Question 13

GENETIC MODIFICATION

Answer 13

Changing the amount of an enzyme
• Slow onset
• Long duration

Question 14

Regulation of Metabolism: Short-Term Regulation

Answer 14

Changing the activity of pre-existing enzymes:
• Substrate availability
• Product inhibition
Allosteric regulation: Reciprocal activation and inhibition of alternate pathways

pH and enzyme conformation
• PH and active site protonation

• Covalent modification: Post-translational modification changing function

Question 15

Allosteric regulation:

Answer 15

Reciprocal activation and inhibition of alternate pathways

Question 16

Covalent modification:

Answer 16

Post-translational modification changing function

Question 17

Regulation of Metabolism: Allosteric (Well-fed)

Answer 17

F-2,6-P2 is key allosteric regulator of glycolysis and gluconeogenesis.
• Enhances production of F1-6-P2 and inhibits degradation. Resulting in higher levels of Pyruvate.
• Citrate is the key allosteric regulator of fatty acid (Fat) synthesis.
• Enhances Methyl CoA production, resulting in higher fatty acid synthesis and inhibits degradation. Resulting in higher levels of fat.
The conversion of F-6-P to F-1,6-BP is the first committed step specific to glycolysis (and irreversible). The other key irreversible steps are also sites of allosteric regulation. Many of the other steps are dependent only on the prevailing substrate concentration. F-2,6-BP is produced under conditions where F-6-P levels are high, binding of this to PFK significantly increases its catalysis of the formation of F-1,6-BP (lowering the Km and increasing the observed rate). Conversely, binding to FBPase has the opposite effects, increasing its Km for F-1,6-BP and decreasing the observed rate. This step is also regulated by allosteric binding of citrate (produced at higher levels under a glycolytic and lipogenic state).

High glucose concentrations also stimulate glycogen synthesis (although the primary regulation of this step is the hormonal regulation by covalent modification (phosphorylation) of glycogen synthase). High [citrate] promote fatty acid synthesis, the first of these steps produces malonyl co-A which inhibits CPT-1 and prevents fatty acid oxidation.

Question 18

Regulation of Metabolism: Allosteric (Fasting)

Answer 18

Increased lactate production from active tissues results in increased levels pf Pyruvate.
• Acetyl CoA is a key allosteric regulator, increasing Aspartate production, inhibiting Pyruvate degradation. Resulting in higher glucose production.
• Acetyl CoA inhibits production of fatty acids resulting in increased concentration for Pyruvate production.

Under fasting conditions, gluconegenesis becomes active; primarily hormonal control is exerted by an increase in glucagon:insulin. Substrate levels also increase rate of gluconeogensis vs glycolysis.

Increased muscle utilisation of glycogen stores (and glucose supplied by liver) increases lactate output of tissues and muscle, providing a substrate for gluconeogenesis in the liver (the Cori cycle). Lactate is converted to pyruvate by LDH; Pyruvate enters mitochondria and is converted to oxaloacetate by pyruvate carboxy kinase (located only in the mitochondria). Oxaloacetate is transported to the cytosol via conversion to malate and aspartate and back to oxaloacetate in the cytosol. Oxaolacetate is converted to phosphoenolpyruvate by PEPCK which enters gluconeogensis.

Fatty acid oxidation leading to ketone body formation increases acetyl coA which allosterically increases the conversion of pyruvate to oxaloacetate, promoting glucose output whilst maintaining fatty acid oxidation and preventing the conversion of pyruvate to acetyl coA. This step is irreversible, hence why FFA’s cannot act as substrates for gluconeogensis; inhibition of PDH activity ensures gluconeogenic substrates from the Cori and glucose:alanine cycles do enter ketogenesis or TCA cycle- the production of acetyl coA from FFA represents a committed step in the production of ketone bodies (or entry to TCA) under these conditions.

Question 19

Allosteric Regulation Summary

Answer 19

Binding of allosteric regulators affect substrate binding and observed reaction rates
• Allosteric activators: lower Km and increase observed rate
• Allosteric inhibitors: increase Km and decrease observed rate

Question 20

Fructose-2,6-BP

Answer 20

• Key regulator of glycolysis
• Allosteric ‘activator’ of phosphofructokinase
• Allosteric inhibitor of fructose- bisphosphatase
• ATP:ADP also important regulators here

Question 21

Citrate

Answer 21

• Allosterically increase production of malonyl-CoA

* Negative regulator of PFK

Question 22

Malonyl coA

Answer 22

• Key regulator of fatty acid synthesis/oxidation

* Allosteric inhibitor of CPT1 (or CAT1)

Question 23

Acetyl coA

Answer 23

• Regulator of pyruvate dehydrogenase (indirect) and pyruvate carboxylase
• Prevents conversion of pyruvate derived from cori and glc:ala cycle into acetyl coA
• Key substrates for gluconeogenesis

Question 24

Regulation of Metabolism: Covalent

Answer 24

Enzymes subject to covalent modification undergo phosphorylation by a protein kinase
• Dephosphorylation of the modified enzyme is carried out by a phosphoprotein phosphatase
• Phosphorylation can activate or deactivate enzymes
• Affects the Vmax, i.e slow or fast turnover: “on” or “off” at six key sites
• Important note: effects of insulin:glucagon are indirect and are mediated by downstream activation of protein kinases and protein phosphatases

Question 25

Regulation of F- 2,6-bp by phosphorylation of PFK2

=

Answer 25

reduced PFK2 activity / increased FBPase2 and reduced levels of F-2,6-bp (decreasing rate of glycolysis)

Question 26

Regulation of Metabolism: Covalent (Fatty Acids)

Answer 26

Acetyl coenzyme A carboxylase (ACC) is the principle site of insulin regulation of the pathway
• This is the committed step in fatty acid synthesis.
• The reaction product is malonyl-coA which inhibits fatty acid oxidation and is the precursor for fatty acid synthes

Question 27

Regulation of Metabolism: Covalent (Well-fed)

Answer 27

1. Glycogen phosphorylase
2. Glycogen synthase
3. 6-Phosphofructo-2-kinase/ Fructose- 2,6-bisphosphatase
4. Pyruvate kinase
5. Pyruvate dehydrogenase 6. Acetyl CoA carboxylase

Covalent modification of enzymes in these pathways is tightly regulated by the insulin:glucagon ratio. A high ratio in the well fed states promotes the dephosphorylation of glycogen synthase (active) and glycogen phosphorylase (inactive) to promote glucose storage as glycogen. PFK2 is also active (dephosphorylated) and increases the levels of F2,6BP which a key allosteric regulator of glycolysis. Utilisation of glucose is promoted by dephosphorylation of pyruvate kinase and pyruvate dehydrogenase and diversion into fatty acid synthesis is promoted by the dephophorylation of acetyl Co-A carboxylase which increases the production of malonyl Co-A and FFA synthesis. Recall that malonly Co-A serves to allosterically inhibit CPT1 preventing fatty acid oxidation

Question 28

Regulation of Metabolism: Covalent (Fasting)

Answer 28

1. Glycogen phosphorylase
2. Glycogen synthase
3. 6-Phosphofructo-2-kinase/ Fructose-2,6- bisphosphatase
4. Pyruvate kinase
5. Pyruvate dehydrogenase 6. Acetyl CoA carboxylase

Insulin: LOW Glucagon: HIGH

Question 29

Long-term Regulation: Well-fed

Answer 29

High [glucose] increases the activity of the carbohydrate- response-element-binding protein (ChREBP)
• Mediated by a protein phosphatase in response to glucose
• Increase expression of lipogeneic genes (CarRE: carbohydrate response element)
• Opposed by glucagon

Insulin signalling increases activity of sterol- response-element-binding protein (SREBP-1c)
• Inactivation of forkhead transcription factor (mediated by PKB/Akt) results in its degradation
• This results in decreased expression of gluconeogenic genes and those for fatty acid oxidation

Question 30

Long-term Regulation: Fasting

Answer 30

Increased PKA leads to activation of cAMP- response-element-binding protein (CREB)
• Leads to increased expression of genes involved in gluconeogenesis and decreased expression of genes involved in lipogenesis