• Allows organisms to be more efficient with resources and to adapt to the environment
• Important for cell differentiation (which results in many different cell types)

STEPS REGULATION OCCURS

1. Transcriptional Control

• When and how often a gene is transcribed
• Only point where the cell will not synthesize unneeded intermediates, thereby wasting resources

2. RNA Processing Control

• Control of the splicing and processing of RNA transcripts

3. RNA Transport and Localization Control

• Cells select which mRNAs are exported to the cytoplasm and where they are localized to

4. Translational Control

• Control which mRNAs get translated into protein

5. mRNA Degradation Control

• Cells can destabilize specific mRNAs in the cytoplasm to limit protein production

6. Protein Activity Control

• Cells can selectively activate, inactivate, localize or degrade proteins after they have been synthesized

DNA DAMAGE MECHANISMS

Endogenous Agents

• Spontaneous chemical reactions

1. Deamination: nucleotides lose amine groups
   • Cytosine –> uracil
   • Adenine –> hypoxanthine
2. Depurination: purine (adenine or guanine) released from DNA
   • Bond between deoxyribose and purine base spontaneously cleaves
   • Produces AP site (apurinic site)

Exogenous Agents

• Exposure to mutagens (chemicals or radiation)

1. Pyrimidine dimers: induced by UV light exposure
   • Cyclobutane ring forms between adjacent pyrimidines (often thymines)
   • Distorts the DNA double helix
2. Alkylation: addition of methyl/ethyl groups to nucleotides
   • -CH3 or –CH2CH3 add to nitrogenous bases at numerous positions
3. Bulky group addition: exposure to carcinogens
   • i.e. benzo(a)pyrene: aromatic, polycyclic structure can react with purines/pyrimidines at numerous positions
   • Cause distortions in DNA helix

Carcinogen

• Cancer-causing mutagen

CONSEQUENCES OF DNA DAMAGE
• Can increase frequency of mutations
• Mutations: nucleotide substitutions, deletions and insertions

CLINICAL CORRELATIONS

Skin melanomas

• Pyrimidine dimers produce helical distortions that result in skin cancers

Cigarette smoking

• Carcinogens in smoke form covalent bonds with DNA
• Disrupts H-bonding between nucleotides: causes frameshift
• Frameshift changes subsequent codons in DNA strand
• Constant exposure to carcinogens –> lung cancer

REPAIR MECHANISMS
• Mismatch-repair: fixes replication errors missed by DNA Polproofreading (cannot repair damage)
• Base excision repair: deamination, depurination and alkylation
• Nucleotide excision repair: pyrimidine dimers and bulky group addition

Amino acid pool

• Describes all free amino acids in the body
• Amino acids: body’s primary source of nitrogen

AMINO ACID STRUCTURE

• Central carbon bound to: -H, -COOH, -NH3, R-group
• R-group: acidic (-), basic (+), branched, etc.

AMINO ACID POOL: INPUTS AND OUTPUTS

Input

• Dietary proteins: absorbed as amino acids/peptides in small intestine
  – Pass through hepatic portal vein and immediately enter liver
  – Liver plays central role in fates of free amino acids
• De novo amino acid synthesis: use C-skeletons & alpha-amino group of other amino acids to synthesize new ones
• Protein turnover: breakdown of cellular proteins

Output

• Oxidation: excess amino acids degraded to alpha amino group & C-skeleton
  – Urea cycle: liver rids body of nitrogen waste
  – Energy production: C-skeletons enter glycolysis or citric acid cycle
  – Gluconeogenesis: occurs in liver
  – Storage: amino acids can’t be stored –> C-skeletons stored as glycogen/fats
• Synthesis of nitrogen-containing molecules
  – Amino acids
  – Neurotransmitters
  – Nucleotides (purines and pyrimidines)
  – Creatine, etc.
• Protein turnover: synthesis of new proteins

Protein turnover

Cyclic synthesis and breakdown of proteins in the body

• Produces and consumes free amino acids
• Protein synthesis: depletes pool of free amino acids
• Protein breakdown: replenishes pool

ESSENTIAL AMINO ACIDS

Must be obtained from diet

• “My Tall Vegan Friend Is Watering Kale Leaves” – M, T, V, F, I, W, K, L
• Glucogenic: Methionine, threonine, valine
• Glucogenic & ketogenic: Phenylalanine, isoleucine, tryptophan
• Ketogenic: lysine & leucine

Branched chain amino acids (BCAA)

• Valine, leucine, isoleucine
• Immediately shunt from liver to circulation
• Liver lacks an aminotransferase that muscle, adipose, kidney & brain have

Aminotransferases: family of enzymes involved in amino acid breakdown

NONESSENTIAL AMINO ACIDS

Synthesized endogenously (The rest of the amino acids)

• Glucogenic: alanine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine*, proline & serine
• Glucogenic and ketogenic: tyrosine
• Histidine is often considered essential because it is essential during the first 5 years of life but later becomes nonessential.

Note that there are many ways to categorize essential/nonessential amino acids

CLINICAL CORRELATION

Conditionally essential amino acids

• Cannot be synthesized under certain physiologic conditions
• Infants < 5 years old can’t synthesize: arginine, cysteine, histidine, tyrosine
• Immunosuppressed patients/postoperative infections: treat w/ arginine & glutamine supplements

LIPOPROTEIN METABOLISM

• Exogenous pathway: chylomicrons clear dietary lipids
• Endogenous pathway: VLDL and LDL transport/distribute endogenously synthesized lipids
• Reverse cholesterol transport: HDL clears excess plasma cholesterol

Enzymes

• Degrade triacylglycerol to glycerol and free fatty acids

1. Lipoprotein lipase (LPL), bound to the endothelial layer of peripheral capillaries.
2. Hepatic lipase, localizes within hepatic endothelial cells.

HIGH DENSITY LIPOPROTEIN (HDL)

• “Good cholesterol”
• Transports second most cholesterol (first LDL)
• Reservoir for lipoproteins

REVERSE CHOLESTEROL TRANSPORT
Step 1: Liver and small intestine synthesize nascent HDL

• Disc shape with ApoA-I and ApoA-II
• Heterogeneity exists: nascent HDL may have ApoA-I only, ApoA-II only or both
  Step 2: Nascent HDL picks up free cholesterol from peripheral tissues to become HDL3
  Step 3: Nascent HDL picks up LCAT from plasma as it becomes HDL3

Lecithin acyl transferase (LCAT) esterifies free cholesterol

HDL3

• Circular HDL particle with CE (esterifies free cholesterol with LCAT)
• Apolipoproteins on surface: ApoA-I, ApoA-II, ApoE & ApoC-II
• Circulating lipoproteins donate ApoE and ApoC-II to HDL3
  Step 4: HDL3 picks up more free cholesterol to become HDL2

HDL2

• Larger HDL particle with more CE (same lipoproteins as HDL3)
• Contains cholesterol ester transfer protein

Cholesterol ester transfer protein (CETP) transfers lipids between HDL and VLDL

• Activated by ApoA-II
  Step 5: CETP transfers CE (from HDL) to VLDL, and TAG (from VLDL) to HDL
• Endogenous pathway: VLDL eventually degrades to LDL (redistributes cholesterol)
  Step 6: HDL2 binds scavenger receptor (SR-B1) on liver
• HDL2 continues accumulating plasma cholesterol before binding
• Hepatic lipases degrade TAG & membrane phospholipids: HDL2 –> HDL3
• Liver converts excess cholesterol to bile salts (digestive elimination)

CLINICAL CORRELATION

Corneal clouding

• Symptom of LCAT or ApoA-I deficiency
• HDL cannot esterify cholesterol
• Leads to rapid HDL degradation: excess cholesterol deposits in cornea and peripheral vessels

LIPOPROTEIN METABOLISM

• Exogenous pathway: chylomicrons clear dietary lipids
• Endogenous pathway: VLDL and LDL transport/distribute endogenously synthesized lipids
• Reverse cholesterol transport: HDL clears excess plasma cholesterol

Enzymes

• Degrade triacylglycerol to glycerol and free fatty acids

1. Lipoprotein lipase (LPL), bound to the endothelial layer of peripheral capillaries.
2. Hepatic lipase, localizes within hepatic endothelial cells.

CHYLOMICRONS

• Fed state only
• Cleared ~ minutes
• 98% lipids (1.TAG 2. Free/esterified cholesterol)
• ApoB-48 = dietary lipoprotein marker

EXOGENOUS PATHWAY

• Only occurs in the fed state.
• Clears dietary lipids
  Step 1: Dietary lipids are packaged into a chylomicron
  Step 2: Chylomicron enters lymphatic system (empties into circulation via left subclavian vein)
  Step 3: HDL donates ApoE and ApoC-II to chylomicron
  Step 4: ApoC-II binds/activates LPL (peripheral tissues)
  Step 5: LPL degrades TAG into glycerol & FFA
• FA enter tissues & glycerol returns to liver
• Chylomicron = chylomicron remnant (low TAG, high CE and C)
  Step 6: ApoC-II returns to HDL
  Step 7: Apo E binds liver remnant receptor: chylomicron endocytosis
  Step 8: Remnant releases cholesterol in liver
• Remnant degraded by lysosomes

CLINICAL CORRELATION

Familial Hyperchylomicronemia

• Rare hereditary disease
• LPL or ApoC-II deficiency: cannot clear chylomicrons
• Fasting blood plasma: cloudy
• Lipemia retinalis: cloudy appearance of capillaries in the retina

LIPOPROTEIN FUNCTION

• Solubilize cholesterol and triacylglycerol (TAG) & transport them in plasma
• Deliver them to peripheral tissues

Triacylglycerol function

• Energy storage
• Membrane synthesis

Cholesterol function

• Component of cell membranes
• Precursor for steroid hormones and vitamin D

LIPOPROTEIN STRUCTURE

Apolipoprotein

• On surface of lipoprotein
• 3 functions:
  i. Structure: bind and solubilize hydrophobic lipids
  ii. Recognition: contain signals that target lipoproteins to cells (recognized by receptors)
  iii. Enzymatic: activate or act as coenzymes in lipid metabolism

Single layer of phospholipids

• Free cholesterol in layer

Hydrophobic core

• Esterified cholesterol and TAG

CLASSES OF LIPOPROTEINS

1. Chylomicrons (fed state only)

• Size: largest
• Origin: intestine (dietary)
• Composition: dietary lipids (mainly TAG)
• Density: least dense

2. Very low density lipoprotein (VLDL)

• Size: LDL < VLDL < chylomicron
• Origin: liver (endogenous lipids)
• Composition: Endogenously synthesized lipids (mainly TAG & phospholipids)
• Density: LDL > VLDL > chylomicron

3. Low density lipoprotein (LDL)

• Size: VLDL > LDL > HDL
• Origin: VLDL
• Composition: Major cholesterol carrier in body (greatest relative amount)
• Density: VLDL < LDL < HDL
• “Bad cholesterol” (atherosclerosis)
• Regulates de novo cholesterol biosynthesis

4. High density lipoprotein (HDL)

• Size: smallest
• Origin: intestine and liver
• Composition: picks up plasma cholesterol (released due to membrane turnover or cell death; carries less cholesterol than LDL)
• Density: most dense (greatest % of protein)
• Delivers cholesterol to liver for excretion (bile salts) and to tissues (steroid synthesis)
• “Good cholesterol”

CLINICAL CORRELATION

HDL:LDL cholesterol ratio

• Diagnostic used to determine a patient’s risk for developing heart disease
• Healthy ratio ~ 3.5

Hypercholesterolemia (high blood cholesterol)

• Common consequence of low HDL:LDL cholesterol ratio
• Can lead to atherosclerosis & heart attack

KETOGENESIS

• Ketone bodies synthesized in liver only (mitochondrial matrix)
• Occurs under 2 clinical conditions: prolonged starvation & uncontrolled diabetes
• Substrate: excess acetyl CoA (derived from fatty acid oxidation)
• Normal, healthy adult: excess acetyl CoA shunts to citric acid cycle or cholesterol biosynthesis

KETONE BODIES

1. Acetoacetate
2. Acetone

• Volatile
• Breathed out unused

3. Beta-hydroxybutyrate

KETOGENIC PATHWAY

• Fasting conditions (starvation or uncontrolled diabetes)

1. Oxaloacetate shunts into gluconeogenesis: slows down citric acid cycle
2. Acetyl CoA builds up and shunts into ketogenesis
3. 2 Acetyl CoA –> Acetoacetyl CoA (acetoacetyl CoA thiolase, reversible)
4. 1 Acetyl CoA + Acetoacetyl CoA –> HMG CoA (HMG CoA synthase)

• Thiolase & HMG CoA synthase also in cholesterol biosynthesis (ketogenic isozymes in matrix not cytosol)

5. HMG CoA –> Acetyl CoA + Acetoacetate (HMG CoA lyase)
6. Acetoacetate + NAD+ –> beta-hydroxybutyrate + NADH (beta-hydroxybutyrate dehydrogenase, reversible)

KETOSIS

• Spontaneous when [acetoacetate] is high
• Acetoacetate –> Acetone + CO2

RATE LIMITING STEP

• HMG CoA synthase: enzyme localized in liver
• Activated by: fasting, increased cAMP and increased lipolysis
• Inhibited by: feeding & insulin

TARGET CELL KETONE BODY USE

• Cells that can use ketone bodies
• Include: cardiac/skeletal muscle, renal cortex, intestinal mucosa, brain cells in starvation
• Ketone bodies can cross blood brain barrier: do NOT bind albumin (fatty acids do)
• Mobilized in matrix

Enzyme beta-ketoacyl CoA transferase

• NOT in liver (liver cannot mobilize ketone bodies)
• Acetoacetate + succinyl CoA –> acetoacetyl CoA + succinate (reversible)
• Remaining reactions are the reverse of ketogenesis

CLINICAL CORRELATION

Untreated diabetics

• Have fruity breath due to exhalation of acetone (ketosis)
• Decreased cellular glucose and CAC intermediates leads to inc. FA mobilization & acetyl CoA
• Excess acetyl CoA shunts into ketogenesis

BETA OXIDATION

• Occurs in liver and peripheral tissues (mitochondrial matrix)
• Occurs under low energy conditions (starvation, insulin:glucagon ratio is low)
• NOT reverse of fatty acid synthesis

TRIACYLGLYCEROL (TAG)

• Stored in adipose and liver (circulates as lipoprotein)
• TAG breaks down to glycerol and fatty acids
• Fatty acids exit adipose and travel to muscle for degradation (provide ATP when energy is low)

TYPES OF FATTY ACIDS

• Degradation pathways differ depending on chain length and saturation

Saturated long chain fatty acids

• Model pathway in diagram

Unsaturated fatty acids

• Already partially oxidized: yield less FADH2 and ATP
• Req. different enzymes to work with additional double bonds

Branched chain fatty acids

• Alpha-oxidation: produce acetyl CoA and propionyl CoA

Medium and short chain fatty acids

• Can be 6-12C long
• Do NOT need carnitine shuttle to enter matrix
• Req. medium-chain acyl CoA dehydrogenase (MCAD) for oxidation step

FATTY ACIDS DELIVERY (ADIPOSE TO MUSCLE)

1. Fatty acids travel from adipose to muscle cell
2. Fatty acid transporter transports fatty acids into cytosol

• Carnitine transporter moves carnitine into cytosol

3. Fatty acyl CoA synthetase: Fatty acid + CoA + ATP –> Fatty acyl CoA + AMP

Carnitine

• Specialized carrier that transports fatty acids within cell
• “Car” in carnitine ~ shuttle

CARNITINE SHUTTLE

1. Carnetine acyl-transferase I (CAT-1): Fatty acyl CoA + Carnitine –> Fatty acyl-carnitine + CoA
2. Fatty acyl-carnitine enters matrix
3. CAT-2 (embedded in inner membrane): Fatty acyl-carnitine –> Carnitine + Fatty acyl CoA

BETA-OXIDATION

• Breaks down fatty acids in 2C increments (acetyl CoA) + shorter chain fatty acid
• Req. four reactions per 2C increment
• All reactions occur at beta-carbon (beta-oxidation)

Each 2C increment

i. Oxidation

• Acyl CoA dehydrogenases (ACAD): family of chain-length specific enzymes
• Releases FADH2 (~ 2 ATP via oxidative phosphorylation)
  ii. Hydration
  iii. Another oxidation
• Releases NADH (~ 3 ATP via oxidative phosphorylation)
  iv. Release of 2C (acetyl CoA)
• Acetyl CoA can enter fatty acid synthesis or citric acid cycle
• Citric acid cycle: 1 ATP, 1 FADH2 & 3NADH (HIGH ENERGY YIELD!)

ODD CHAIN FATTY ACIDS

• Final products: 2C Acetyl CoA + 3C propionyl CoA

Propionyl CoA Breakdown

• Propionyl CoA carboxylase (ABC-carboxylase reaction):
  Propionyl CoA + ATP + biotin + CO2 –> Methylmalonyl CoA (4C)
• Methyl-malonyl CoA mutase (req. Vit B12 cobalamin): Methylmalonyl CoA –> Succinyl CoA

Product: Succinyl CoA (4C)

• Citric acid cycle intermediate
• Gluconeogenic intermediate: only fatty acid that can be converted to glucose

• ccurs in the liver and adipose tissue (cytosol)
• After a carbohydrate-rich meal (high insulin:glucagon ratio)
• Not just reverse of beta-oxidation: distinct enzymes and compartments

Reactions

1. Citrate Shuttle

• Acetyl CoA in mitochondrial matrix transported to cytosol as citrate
• Citrate synthase: citrate from oxaloacetate and acetyl CoA (also first CAC rxn)
• Citrate a marker of high intracellular energy (CAC intermediate)

2. Malonyl CoA Formation

• Acetyl CoA carboxylase: adds 1 carbon to acetyl CoA to form malonyl CoA
• ABC carboxylase reaction
• Citrate activates rxn
• Long-chain fatty acyl CoA (intermed. of FA breakdown): inhibits rxn

ABC carboxylase reactions: consume ATP, require biotin, consume CO2

• Malonyl CoA synthesis in fatty acid biosynthesis
• Gluconeogenesis: pyruvate carboxylase
• Odd chain fatty acid oxidation: propionyl carboxylase

3. Palmitate Synthesis

• Palmitate: 16-carbon fatty acid
• Catalyzed by fatty acid synthase
• ACP is carrier protein component of fatty acid synthase
• Series of 4 reactions:
  i. Condensation: acetyl-ACP + malonyl-ACP = 4-C intermediate + CO2
  ii. Reduction (NADPH)
  iii. Dehydration: water molecule released
  iv. Reduction (NADPH)
• First condensation = 4C molecule
• 6 increments x 2C (malonyl-ACP) = 12 C
• Total = 16 C palmitate

4. Palmitate Modification
   i. Elongation: in smooth ER or mitochondria

• 2-carbon increments using malonyl CoA (NOT malonyl-ACP)
• Each increment includes four rxn’s from palmitate synthesis
  ii. Desaturation: in smooth ER or peroxisomes
• Fatty acyl-CoA desaturase
• Short electron transport chain (requires O2 and NADPH)

CLINICAL CORRELATION
Essential fatty acids: linoleic acid and linolenic acid cannot be synthesized endogenously.

• Mammals cannot induce double bonds beyond C9

Blood cholesterol levels (average healthy adult)

< 200 mg/dL (cholesterol levels vary more than tightly controlled glucose levels)

• 2 sources of cholesterol: biosynthesis and dietary

REGULATORY POINTS

1. Cholesterol Biosynthesis

• Occurs in most tissues, but primarily in liver

2. LDL receptors

• LDL transports cholesterol throughout body

3. Bile acid biosynthesis

• Sole mechanism for cholesterol clearance

1. CHOLESTEROL BIOSYNTHESIS

HMG CoA Reductase Regulation

i. Transcription

• SREBP-2 binds sterol regulatory element (SRE) and enhances transcription
• SREBP-2/SCAP release from Golgi inhibited by excess intracellular cholesterol
  ii. Translation
• Mevalonate (biosynthetic intermediate) produces non-sterol metabolites that block RNA translation in the cytosol
  iii. Proteolysis
• Excess cholesterol stimulates reductase proteolysis by ubiquitination
  iv. Covalent modification
• Insulin promotes dephosphorylation and activation of reductase
• Glucagon promotes phosphorylation and inactivation of reductase (AMP-dependent pathway)

Acyl CoA cholesterol acyltransferase (ACAT)

Esterifies cholesterol in hepatic cells: “There is A CAT in the liver”

2. LDL RECEPTORS

LDL: main cholesterol carrier in the body

• Excess cholesterol inhibits LDL-receptor synthesis

3. BILE ACID BIOSYNTHESIS

HDL: picks up plasma cholesterol released by cell death or membrane turnover

• Contains lecithin cholesterol acyl transferase (LCAT): esterifies cholesterol
• Delivers esterified cholesterol to liver cell
• Liver converts esterified cholesterol to bile salts: secreted into intestine

CLINICAL CORRELATIONS

Diabetics taking exogenous insulin

Have high levels of HMG CoA reductase in hepatocytes

Statins

Class of drugs that inhibit HMG CoA reductase: combat high cholesterol

Cholestyramine

Bile acid binding resin: binds bile salts in intestine and prevents reabsorption

• Initiates feedback mechanism: increases bile production, cholesterol and LDL receptor synthesis
• Combats high cholesterol