Wk 2 - Molecular Cell Biology: Liver Function, Protein, Amino Acid and Metabolism

Question 1

What are the liver’s main functions?

Answer 1

1. Metabolism
2. Inactivation and detoxification
3. Biosynthesis and secretion

Question 2

If the liver is removed, what would a person die of?

Answer 2

Hypoglycaemia (liver v imp in continually providing glucose in bloodstream)

Question 3

The liver is an _____ and _____-sensitive tissue and is instructed to regulate ______ concentrations.

Answer 3

The liver is an insulin- and glucagon-sensitive tissue and is instructed to regulate blood glucose concentrations.

Question 4

How does the liver regulate glucose in the ‘fed state’?

Answer 4

In the fed state:

• the liver takes up glucose via GLUT2 transporters
• GLUT2 is a high Km transporter and is not insulin sensitive
• movement of glucose into the hepatocyte is a passive process
• 1. it metabolises glucose via glycolysis
     * Pyruvate converted to acetyl Co-A, a substrate for fat synthesis
• 2. it also metabolises glucose via the pentose phosphate pathway (also called hexose monophosphate shunt)
     * NADPH production for fat synthesis
• 3. it also replenishes glycogen stores by glycogenesis (glycogen synthesis)
     * Liver stores ~ 100g glycogen

Question 5

What does the liver do in response to glucose?

Answer 5

1. Metabolises glucose via glycolysis
   
   • Pyruvate converted to acetyl Co-A, a substrate for fat synthesis
2. Metabolises glucose via the pentose phosphate pathway (also called hexose monophosphate shunt)
   
   • NADPH production for fat synthesis
3. Replenishes glycogen stores by glycogenesis (glycogen synthesis)
   
   • Liver stores ~ 100g glycogen

Question 6

The formation of glycogen is _______.

Answer 6

The formation of glycogen is glucose-dependent.

Question 7

How does the liver store glucose?

Answer 7

As glycogen

Question 8

What is the rate-limiting enzyme in glycolysis?

Answer 8

Glucokinase

Question 9

How does the liver regulate glucose in the ‘fasting state’?

Answer 9

In the resting state:

• Glucose entry into hepatocytes is reduced.
• Glucose is produced from glycogen stores by glycogenolysis.
• The catabolic process pathway for glycogenolysis is not the reverse of glycogenesis and uses a different set of enzymes.
• The liver also takes up lactate produced by other tissues, especially muscles and blood, and converts it back into glucose or glycolytic intermediates via gluconeogenesis.
• This cycling between lactate producing tissues and the liver is called the Cori cycle.
• Gluconeogenesis is not glycolysis in reverse – other enzymes are required and the liver is specialised to perform this function [note that the production of glucose from glycogen - glycogenolysis is NOT termed gluconeogenesis].

Question 10

What process produces glucose from stored glycogen?

Answer 10

Glycogenolysis

Question 11

What is the Cori cycle?

Answer 11

The liver takes up lactate produced by other tissues, especially muscles and blood, and converts it back into glucose or glycolytic intermediates via gluconeogenesis. This cycling between lactate producing tissues and the liver is called the Cori cycle.

Question 12

What role does the liver have in lipid synthesis?

Answer 12

Synthesis of endogenous lipids from smaller molecules

Lipids originate from two main sources – exogenous lipids in the diet, and endogenous lipids, which are synthesized in the liver, usually from smaller molecules.

Question 13

How does the liver synthesise lipids in the fed state?

Answer 13

1. Dietary cholesterol and triglycerides are digested and the fatty acids and glycerol taken up by enterocytes. These cells package triglycerides in chylomicrons, with cholesterol, phospholipids and other molecules.
2. The liver also synthesises triglycerides and cholesterol and packages them in another lipoprotein particle – very low density lipoproteins (VLDLs). These are denser than chylomicrons, having a higher ratio of protein to lipid.
3. All of these are transported to peripheral tissues and their triglyceride is catabolised by lipoprotein lipase.

Question 14

How does the liver synthesise lipids in the fasting state?

Answer 14

• Adipocytes release fats and fatty acids and glycerol.
• The liver then produces new glucose from glycerol (gluconeogenesis) and converts the fatty acids into ketone bodies.
• Ketone bodies are acetoacetate, acetone and β-hydroxybutyrate (here ‘body’ means the compound itself).
• Ketone bodies are an important energy source during prolonged fasting.

Question 15

What is excess glucose converted to in the liver?

Answer 15

Converted into fats by fatty acid synthesis (occasionally lipogenesis) –> packaged in VLDLs and transported to adipose tissue.

Question 16

Muscle is a glycogen store and can use it for energy. But what is the main reason why muscle can’t contribute to circulating glucose?

Answer 16

Muscles lack glucose-6-phosphatase (so cannot produce glucose from gluconeogenesis).

Question 17

How does the liver create ‘sugar’?

Answer 17

1. Creates glucose via gluconeogenesis
2. Uses lactate (Cori cycle)

Question 18

Where do lipids come from?

Answer 18

• exogenous lipids, ingested and processed in the intestine.
• endogenous lipids, synthesized in the liver

Question 19

How does the liver deal with dietary lipids?

Answer 19

1. Dietary cholesterol and triglycerides are packaged into chylomicrons in the intestine, before passing into the bloodstream via lymphatics.
2. Chylomicrons are broken down by lipoprotein lipase (LPL) in the capillaries of muscle and adipose tissue to fatty acids, which then enter the cells.
3. The chylomicron remnants, which have lost much of their triglyceride content, are taken up by the liver for disposal.

Question 20

How does the liver synthesise endogenous lipids?

Answer 20

• The liver synthesizes triglycerides and cholesterol, and packages them as VLDLs before releasing them into the blood.
• When VLDLs (which consist mainly of triglyceride) reach muscle and adipose blood vessels, their triglycerides are hydrolyzed by LPL to fatty acids.
• The fatty acids that are released are taken up by the surrounding muscle and adipose cells. During this process, the VLDLs become progressively more dense and turn into LDLs.
• While most of the resulting LDLs are taken up by the liver for disposal, some circulate and distribute cholesterol to the rest of the body tissues.

Question 21

What happens to triglycerides during the fasting state? Discuss the liver’s role in it.

Answer 21

• During fasting triglycerides released from adipose are converted to fatty acids and glycerol.
• The liver:
  
  • Converts the glycerol into glucose – gluconeogenesis;
  • Converts the fatty acids into ketone bodies

Question 22

Why are ketone bodies important?

Answer 22

Ketone bodies are an important energy source during prolonged fasting, particularly in the brain, but also build up during pathological hyperglycemia in for example diabetes (resulting in ketoacidosis).

Question 23

By what metabolic pathways does the liver reduce blood glucose during the fed state and what hormone controls this?

Answer 23

• Glycogenesis/glycogen synthesis, fatty acid synthesis (and VLDL production)
• Controlled by insulin

Question 24

By what metabolic pathways does the liver increase blood glucose during the fasting state and what hormone controls this?

Answer 24

• Gluconeogenesis, glycogenolysis
• Controlled by glucagon

Question 25

What is the name of the enzyme that oxidises triglycerides in the peripheral tissue?

Answer 25

Lipoprotein lipase

Question 26

What are dietary amino acids used for in the liver?

Answer 26

• Used for synthesis of hepatic and serum proteins
• Biosynthesis of nitrogen-containing compounds which use amino acids as precursors e.g. heme, hormones, neurotransmitters and purine, pyrimidine bases

Question 27

What are excess amino acids converted to?

Answer 27

Excess amino acids can be converted to glycogen or triacylglycerols

Question 28

What are amino acids used for in other peripheral tissue in the body?

Answer 28

Travel to peripheral circulation to be used by other tissues and be used:

• For protein synthesis
• Biosynthetic pathways
• Oxidised for energy

Question 29

During fasting, ______ from ______ and other tissues are transported to the liver and used as a ______ for ______.

Answer 29

During fasting, amino acids from muscle and other tissues are transported to the liver and used as a carbon source for gluconeogenesis.

Question 30

How does the liver access carbon skeletons of the amino acids?

Answer 30

• To access the carbon skeletons the amino group must be removed from each amino acid – TRANSAMINATION
• Oxaloacetate or a-ketoglutarate accept these amino groups and transfer them to the Urea cycle for safe removal in the urine

Question 31

What is alanine?

Answer 31

• Alanine is used as a means of transporting amino acids from the muscle to the liver
• Allows the muscles to get energy from amino acids and gives the liver the job of excreting the excess nitrogen
• Remember – muscle can’t directly contribute glucose to circulation but can do so via this cycle

Question 32

Serum ALT and AST (alanine and aspartate aminotransferases) are biochemical markers of liver damage. Why?

Answer 32

Liver cells have high levels of these enzymes and cell death results in their release into the blood stream

Question 33

Apart from the inter-organ glucose-alanine cycle, (which forms _____ from ______), amino acids can enter energy-yielding metabolic reactions at other points e.g. ______ and ______.

Answer 33

Apart from the inter-organ glucose-alanine cycle, (which forms pyruvate from alanine), amino acids can enter energy-yielding metabolic reactions at other points e.g. gluconeogenic and ketogenic amino acids.

Question 34

Some amino acids are _____, meaning they can be catabolised to form a ______.

Answer 34

Some amino acids are glucogenic, meaning they can be catabolised to form a citric acid cycle intermediate.

Question 35

Some amino acids are ______, meaning they are catabolised to form ______ or a precursor (i.e. _______ or ______).

Answer 35

Some amino acids are ketogenic, meaning they are catabolised to form acetoacetate or a precursor (i.e. acetyl CoA or acetoacetyl CoA)

Question 36

What are amino acids which are catabolised from an intermediate in the citric cycle called?

Answer 36

‘Glucogeneic’

Question 37

What are amino acids which are catabolised to form acetoacetate or one of its precursors called?

Answer 37

‘Ketogenic’

Question 38

What are anaplerotic reactions?

Answer 38

In the liver (black text dashed boxes) [and brain blue text dashed boxes]), TCA cycle intermediates are continuously withdrawn into synthetic pathways.

Question 39

What is the aim of drug metabolism in the liver?

Answer 39

The goal of metabolism is to de-toxify drugs, and make them either more water-soluble (for excretion in the urine) or more fat-soluble (for excretion in the bile, and then into the feces).

Question 40

What enables the liver to metabolise drugs?

Answer 40

• Low substrate specificity of hepatic enzymes produces a wide-ranging capability for drug metabolism. But drug metabolism also occurs in a wide range of other tissues
• Potential ingested toxins – like drugs – are carried to the liver first via the hepatic portal vein.
• Hepatocytes have evolved to have a high capacity to metabolise potentially harmful compounds.

Question 41

What are the main outcomes of drug metabolism?

Answer 41

1. The inactivation of drug so that it no longer has any biological activity (by conjugation, see below)
2. To produce metabolites with less biological activity
3. To produce active metabolites that may be more potent and persist longer than the parent compound
4. To convert an inactive pro-drug to the active form
5. To produce toxic metabolites
6. To produce inactive metabolites that are water soluble for excretion (by conjugation, see below)

Question 42

Name the two phases of drug metabolism.

Answer 42

1. Phase I metabolic reactions: ‘Pre-conjugation reactions’ or functionalisation
2. Phase II metabolic reactions: ‘Conjugation reactions’

Question 43

What happens in phase I reactions?

Answer 43

Oxidation is the most important reaction in the liver (also reduction, hydrolysis). This occurs primarily in microsomes, but can also occur in cytosol or mitochondria.

Question 44

What happens in phase II reactions?

Answer 44

Glucuronidation is the most important reaction (addition of glucuronic acid to the drug or its metabolite). But also acetylation, methylation, sulphate or glutathione conjugation.

Question 45

List the families of Cytochrome P450 that are involved in Phase I metabolism.

Answer 45

1. CYP1
2. CYP2
3. CYP3

Question 46

What is Cytochrome P450’s role in phase I?

Answer 46

Chemically oxidize or reduce drugs (for example through hydroxylation). The polarity of the drug is increased.

Question 47

Give an example of a phase I drug reaction that happens in the liver.

Answer 47

• Diazepam is a tranquiliser. It is hydroxylated and demethylated (to Oxazepam) by cytochrome P450 enzymes, making it more polar.
• Oxazepam is also a tranquiliser, but is eliminated more quickly than diazepam – less potent + excreted more quickly.

Question 48

What happens in phase II conjugation reactions?

Answer 48

Cytoplasmic conjuation enzymes conjugate the functional groups introduced in the first phase reactions, most often by glucuronidation or sulphation (also methylation, acetylation).

Question 49

What does HIV combination therapy (Ritonavir) do to liver function?

Answer 49

One drug used in HIV combination therapy (Ritonavir) reduces the CYP3A-mediated metabolism of some anti-HIV drugs, thereby increasing plasma levels of HIV.

Question 50

Give an example in which glucuronidation enhances drug action.

Answer 50

Some drugs are made more active: morphine 6-glucuronide has greater analgesic potency than the parent compound morphine.

Question 51

What are the routes of metabolite excretion?

Answer 51

• Polar species are excreted in the urine
• Non-polar species are excreted in the stools

Question 52

Give an example in which there is drug hepatotoxicity (ie liver cannot handle it – in excess!).

Answer 52

Acetaminophen (paracetamol) is hepatotoxic in excess

Question 53

What happens in the liver when paracetamol is taken in excess?

Answer 53

• In overdose, the conjugation pathways are overwhelmed
• It is normally oxidised by cytochrome P450 to N-acetyl-p-benzoquinoneimine (NAPQI)
• NAPQI is usually detoxified by conjugation with glutathione – but in an overdose situation, glutathione resources are exhausted
• NAPQI causes free radical mediated peroxidation of membrane lipids, kills hepatocytes

Question 54

What is NAPQI?

Answer 54

Metabolite of paracetamol (oxidised by P450)

Question 55

How does NAPQI induce hepatotoxicity?

Answer 55

NAPQI causes free radical-mediated peroxidation of membrane lipids, which kills hepatocytes

Question 56

How is NAPQI dealt with? And what happens in an overdose of paracetamol?

Answer 56

NAPQI is usually detoxified by conjugation with glutathione – but in an overdose situation, glutathione resources are exhausted.

Question 57

What are the sources of amino acids?

Answer 57

• Amino acids can be digested from ingested proteins and absorbed by the intestines
• Alternatively some amino acids can be synthesised by metabolic processes in all cells – these are ‘non-essential amino acids’
• Those which cannot be synthesised and must be ingested are called ‘essential amino acids’

Question 58

Amino acids that cannot be synthesised and must be ingested are called ‘_____ amino acids’.

Answer 58

Amino acids that cannot be synthesised and must be ingested are called ‘essential amino acids’.

Question 59

What determines the function of a protein?

Answer 59

The 3D structure characteristic of a given sequence of amino acid is what determines a protein’s function.

Question 60

Give an example of amino acid synthesis.

Answer 60

As an example of amino acid synthesis, some amino acids alanine, aspartate and glutamate can be synthesised directly from pyruvate, oxaloacetate and α-ketoglutarate (citric acid cycle intermediates).

Question 61

What are some essential amino acids?

Answer 61

PVT TIM HALL

Phenylalanine, Valine, Threonine, Tryptophan, Isoleucine, Methionine, Histidine, Arginine, Leucine and Lysine.

Question 62

What condition is caused by a deficiency in tyrosinase production?

Answer 62

Albinism

Question 63

Why does tyrosinase deficiency lead to albinism?

Answer 63

• Tyrosinase is required for the production of quinone from tyrosine (via DOPA).
• Quinone is a precursor of melanin.

Question 64

Amino acids are the building blocks of small molecules that contain _____.

Answer 64

Amino acids are the building blocks of small molecules that contain nitrogen.

Question 65

What do skeletal muscles rely on for short bursts of energy as you start exercising?

Answer 65

For short bursts of energy, skeletal muscle relies on its ATP stores and an additional reserve of the high-energy storage compound, phosphocreatine (PCreatine), to regenerate ATP rapidly during the first minutes of exercise as glycogenolysis is activated

Question 66

Where is creatine synthesised?

Answer 66

Creatine is synthesised in the liver from arginine, glycine and methionine

Question 67

How does creatine become phosphocreatine?

Answer 67

Creatine is phosphorylated in skeletal muscle to form phosphocreatine.

Question 68

Why does phosphocreatine decline rapidly after exercise initiation?

Answer 68

Phosphocreatine is unstable and undergoes slow spontaneous (no enzymes) degradation to Pi and creatinine, which is excreted from the muscle into plasma and then urine. Creatine has a half life of < 3 hrs in blood.

Question 69

Creatinine is a biochemical readout of _________, while creatine kinase is a readout of ________.

Answer 69

Creatinine is a biochemical readout of kidney function, while creatine kinase is a readout of muscle damage.

Question 70

What is a possible cause of creatine deficiency?

Answer 70

• Congenital diseases caused by defective creatine deficiency are rare
• 3 genetic diseases have been described, involving mutations in enzymes (eg guanidinoacetate methyltransferase) and transporters

Question 71

What is the most obvious sign of creatine deficiency?

Answer 71

The most obvious sign is developmental delay or regression and mental retardation. Creatine is an important energy source in the brain.

Question 72

What is heme (haem)?

Answer 72

Heme is a constituent of hemoglobin, myoglobin and cytochromes.

Question 73

Where is heme (haem) synthesised?

Answer 73

Can be synthesised by most cells but the liver (~20%) is the major non-marrow source.

Question 74

What is porphyrias?

Answer 74

• Porphyrias are (usually) genetic diseases resulting in decreased activity of one of the enzymes involved in heme synthesis e.g. PBG Synthase, Prophobilinogen Deaminase.
• Without negative feedback from haem (haem feedbacks negatively on its own synthesis), intermediates build up and may be toxic.
• Porphyrins are light-absorbing and fluorescent, and therefore photosensitizing (this is what causes skin damage).

Question 75

How is porphyrias managed?

Answer 75

Most heme synthesis enzymes —even dysfunctional enzymes—have enough residual activity to assist in heme biosynthesis.

• Most porphyrias can be managed by avoiding risk factors
• Acute attacks managed with opiate pain relief and intravenous heme

Question 76

What is the most common subtype of porphyria?

Answer 76

Porphyria cutanea tarda (PCT) is the most common subtype (20% cases caused by mutations in uroporphyrinogen decarboxylase [UROD]).

Question 77

What is serum albumin? What is its function?

Answer 77

• Serum albumin is the primary plasma protein responsible for the transport of:
  
  • Hydrophobic fatty acids –> binds and makes soluble for transport in blood –> each albumin molecule can carry 7 fatty acids
  • Bilirubin –> Binds unconjugated bilirubin (more tomorrow) and renders it non-toxic
  • Drugs
    
    • Salicylates, penicillin, warfarin
  • Steroids and thyroid hormones
    
    • T3 and T4 (less than 0.3% unbound)
    • Testosterone, cortisol
• Albumin also is important in maintaining oncotic pressure (prevents oedema in tissues)

Question 78

What organ produces serum albumin?

Answer 78

The liver produces 12g/day of serum albumin

Question 79

A fall in albumin is a marker of ______.

Answer 79

A fall in albumin is a marker of liver disease.

Question 80

What other transport proteins does the liver synthesise?

Answer 80

• Various globulins bind and transport circulating steroid hormones
  
  • Transcortin (cortisol, sex hormones)
  • eg sex-hormone binding globulin – andogens and estrogens
  • Mutations in these proteins cause intersex phenotypes
• Apolipoproteins – VLDL, HDL
  
  • VLDL are secreted directly into the bloodstream by hepatocytes
  • HDL transport cholesterol to some tissues (glands; adrenal, testes). But also transport cholesterol back to the liver
• Ceruloplasmin major copper carrying plasma protein
  
  • Important in redox reactions and iron use
• Transferrin major iron carrying plasma protein
  
  • Particularly important in carrying iron to erythroid precursors in the marrow
  • Deficiency causes anaemia

Question 81

What is Wilson’s disease?

Answer 81

• Disease of copper transport
• Caused by mutations in an ATPase, resulting in a reduced incorporation of copper into ceruloplasmin – Cu builds up in tissue

Question 82

What are some signs and symptoms of Wilson’s disease?

Answer 82

Patients may have neurological symptoms, liver cirrhosis and have Kaiser-Fleischer rings in the cornea.

Question 83

What are acute phase proteins?

Answer 83

Proteins synthesised in the liver in response to trauma (e.g. infection, inflammation) help protect against infection and tissue damage.

Question 84

What stimulates the production of acute phase proteins in the liver?

Answer 84

Production is stimulated by cytokines, IL-1 and IL-6

Question 85

Give some examples of acute-phase proteins that are synthesised in the liver.

Answer 85

• C-reactive Protein – binds to microbes and damaged cells and assists complement binding
• Complement factors involved in killing bacteria and removing damaged cells
• Protease inhibitors (a 1-antitrypsin) - prevent unrestricted and potentially harmful protease activity
• Coagulation proteins (prothrombin, fibrinogen)

Question 86

What are some common causes of liver damage?

Answer 86

• Liver cirrhosis caused by alcohol abuse
• Viral infection – Hep B and Hep C
• Paracetamol overdose

Question 87

In brief, what happens during liver damage?

Answer 87

Liver damage caused by death of hepatocytes, formation of fibrous scar, and reduction or disruption of blood flow in organ.

Question 88

List some of the results of liver damage.

Answer 88

1. Reduced clotting ability
2. Increased blood ammonia
3. Increased blood amino acids (perhaps causing mental disturbances)
4. Reduced albumin synthesis
5. Reduced gluconeogenesis

Question 89

What are some of the BIOCHEMICAL results of liver damage?

Answer 89

1. Reduced clotting ability - for example, decrease production of plasminogen, vitamin K deficiency
2. Increased blood ammonia (liver is the primary site of urea synthesis
3. Increased blood amino acids (perhaps causing mental disturbances)
4. Reduced albumin synthesis
5. Increased blood bilirubin (more tomorrow)
6. Reduced gluconeogenesis, hypoglycaemia

Question 90

The liver has a function in both protein ____ and ___.

Answer 90

The liver has a function in both protein synthesis and degradation.

Question 91

Why is protein degradation important?

Answer 91

• Supplies amino acids for new proteins
• Removes excess enzymes
• Removes transcription factors that are no longer needed

Question 92

Where does protein degradation take place?

Answer 92

Takes place in lysosomes in the cells or proteasomes - NOT PROTEIN METABOLISM IN THE LIVER.

Question 93

How do lysosomes degrade proteins?

Answer 93

• Lysosomes degrade extracellular proteins such as plasma proteins or those taken up by the cells by endocytosis (e.g. membrane receptors)
• Requires proteases
• Lysosomes also degrade damaged organelles
• Fuses with protein and degrades it

Question 94

How do intracellular proteins get degraded?

Answer 94

Via proteasomes - more targeted way of getting rid of proteins, requires ATP

Question 95

What is ubiquitin?

Answer 95

Ubiquitin is a small protein that can be covalently linked to lysine residues of proteins targeted for intracellular degradation by proteasomes.

Question 96

Describe the ubiquitin-proteasome system of intracellular protein degradation.

Answer 96

• Before they are targeted for proteasomal degradation, most proteins are covalently modified with ubiquitin (Ub).
• Typically, three enzyme types are involved in this process — ubiquitin-activating (E1 - requires ATP), ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes.
• Proteins tagged with chains of four or more ubiquitins are shuttled to the proteasome by various proteins.
• In the proteasome, proteins are reduced to peptides, which are then released into the cytosol and further broken down by peptidases.

Question 97

What is Angelman Syndrome?

Answer 97

• Disease characterised by impaired intellectual development, seizures and a happy demeanour
• Disease of imprinting but can involve a mutation in UBE3A

Question 98

Give an example where ubiquitin-proteasome degradation is dysfunctional.

Answer 98

Parkison’s disease

• Degenerative disorder of the central nervous system that often impairs the sufferer’s motor skills, speech, and other functions
• Most cases are idiopathic – a small number are familial/genetic (e.g. autosomal recessive juvenile parkinsonism)
• The gene PARKIN (PARK2) encodes a ubiquitin ligase (E3) protein
• The loss of PARKIN activity probably disturbs the ubiquitin-proteasome system, which allows unwanted proteins to accumulate

Alzheimer’s disease - STILL DEBATED

• Ubiquitinylated proteins are found in the neurofibrillary tangles
• Defects in synaptic plasticity in AD may be attributed to the accumulation of ubiquitinylated proteins in pre- and post- synaptic processes