Lipid and Amino Acid Metabolism

Lipid Digestion and Absorption

• Lipids serve many functions: energy storage/source, fat soluble vitamins act as coenzymes, prostaglandins and steroid hormones are necessary for homeostasis

Digestion

• Dietary fats consist mainly of triacylglycerols
  • Remainder consists of cholesterol, cholesteryl esters, phospholipids and free fatty acids.
• Digestion is minimal in the stomach and mouth.
  • Upon entry into the duodenum, emulsification occurs, which is the mixing of two normally immiscible liquids
  • Emulsion formation increases the surface area of the lipid: permits greater enzymatic interaction and procession
• Emulsification is aided by bile: bile salts, pigments and cholesterol
  • This is secreted by the liver and stored in the gallbladder
• The pancreas secretes pancreatic lipase, colipase & cholesterol esterase into the small intestine as well.
  • Enzymes hydrolyze the lipid components into 2-monoacylglycerol, free fatty acids, and cholesterol

Micelle Formation

• Emulsification is followed by absorption of fats by intestinal cells
• Micelles: consist of free fatty acids, cholesterol, 2-monoacylglycerol, and bile salts
  • These are clusters of amphipathic lipids that are soluble in aqueous intestinal environment
  • Water soluble spheres with lipid soluble interior
• Micelles are present from the duodenum all the way to the end of the ileum
  • Bile salts are recycled and restored at the end

Absorption

• Micelles diffuse into the brush border of the intestinal mucosal cells
• Digested lipids pass through the brush border and are absorbed into the mucosa and re-esterified to form triacylglycerols and cholesteryl esters
• Chylomicrons: packages of triacylglycerols, cholesteryl esters, apoproteins, fat-soluble vitamins, and other lipids
  • Leave the intestine via lacteals (lactic system vessels), and then reenter the bloodstream via the thoracic duct (long lymphatic vessel that empties into the left subclavian vein)
• Water soluble fatty acid chains can be absorbed by simple diffusion into the bloodstream.

Lipid Mobilization

• Hormone-sensitive lipase (HSL): hydrolyzes triacylglycerols which yields fatty acids and glycerol
  • This is activated by a fall in insulin levels or an increase in cortisol and epinephrine
• Released glycerol from fat may be transported to the liver for glycolysis or gluconeogenesis
  • HSL is effective within adipose cells
• Lipoprotein Lipase (LPL): is necessary for the metabolism of chylomicrons and very-low-density lipoproteins (VLDL)
  • This is an enzyme that can release free fatty acids from triacylglycerols in these lipoproteins

Lipid Transport

• Free fatty acids are transported through the blood in association with an albumin carrier protein
• Triacylglycerol and cholesterol are transported in the blood as lipoproteins:
  • Aggregates of apolipoproteins and lipids
  • These are named after according to their density
    • Density increases in proportion to the percentage of protein in the particle
• Chylomicrons are the least dense (high fat-to-protein ratio), followed by VLDL, followed by IDL, LDL and HDL

Chylomicrons

• Highly soluble in lymphatic fluid and blood
• Function in the transport of dietary triacylglycerols, cholesterol, and cholesteryl esters
• Assembly occurs in the intestinal lining. A newly formed chylomicron that contains lipids and apolipoproteins

VLDL (Very-Low-Density Lipoproteins)

• Metabolism is similar to chylomicrons
  • Difference is that VLDL is produced and assembled in the liver cells
• Main function is the transport of triacylglycerols
• Also contains fatty acid that are synthesized from excess glucose or retrieved from chylomicrons remnants

IDL (Intermediate-Density Lipoproteins)

• The resulting protein from when a triacylglycerols is removed from the VLDL
  • Sometimes referred to as a VLDL remnant
• Some IDL is reabsorbed by the liver by apolipoproteins and some is further processed in the bloodstream
  • Some pick up cholesteryl esters from HDL to become LDL
• Exists as a transition particle between triacylglycerol transport and cholesterol transport

LDL (Low-Density Lipoproteins)

• Majority of the cholesterol measured in blood is associated with LDL
• Normal role is to deliver cholesterol to tissues for biosynthesis
• Also important role in cell membranes and in the formation of bile salts
• Also required for steroid hormone synthesis: steroidogenesis

HDL (High-Density Lipoprotein)

• Synthesized in the liver and intestines and release as dense, proteins-rich molecules into the blood.
• Contains apolipoproteins used for cholesterol recovery
  • I.e. – the cleaning up of excess cholesterol from blood vessels for excretion.
• HDL can also deliver some cholesterol to steroidogenic tissues and can transfer necessary apolipoproteins to other lipoproteins

Apolipoproteins (apoproteins)

• Receptor molecules that are involved in signaling
• Do not need to know specific function of each apolipoprotein, just know that they are used for a diverse range of purposes.

Cholesterol Metabolism

• Plays a role in the synthesis of cell membranes, steroid hormones, bile acids, and vitamin D

Sources

• Derived mainly from LDL or HDL. May also be synthesized in the liver (de novo)
• De novo synthesis is driven by Acetyl-CoA and ATP
  • Citrate shuttle carries mitochondrial Acetyl-CoA into the cytoplasm, where synthesis occurs
  • NADPH supplies the reducing equivalents (from the PPP)
• Synthesis of mevalonic acid in the smooth endoplasmic reticulum is the rate-limiting step in cholesterol biosynthesis
  • Catalyzed by 3-hydroxy-3 methylglutaryl (HMG) CoA reductase
• Synthesis of cholesterol is regulated in many ways:
  • Increased levels of cholesterol inhibit further synthesis
  • Insulin promotes cholesterol synthesis
  • De Novo synthesis is also dependent on the regulation of HMG-CoA reductase.

Specific Enzymes

• Lecithin-cholesterol acyltransferase (LCAT): found in the bloodstream and is activated by HDL apoproteins
  • Adds a fatty acid to cholesterol. This produces soluble cholesteryl esters such as those in HDL
• Cholesteryl ester transfer protein (CETP): facilitates the transfer of cholesteryl esters from HDL to other lipoproteins like IDL

Fatty Acid and Triacylglycerols

• Fatty Acids: long chain carboxylix acids
  • Carboxyl carbon is carbon 1 and carbon 2 is known as the alpha-carbon

Nomenclature

• Total number of carbons is given along with the number of double bonds
  • E.g. – carbons: double bonds
  • Can be further specified by indicating the position and isomerism of the double bonds
• Saturated fatty acids: no double bonds while unsaturated fatty acids have one or more double bonds
• Humans can only synthesize a few fatty acids, the rest come from diet
  • a-Linolenic Acid & Linoleic acid: poly unsaturated fatty acids are important in maintain cell membrane fluidity,
• Omega (w) numbering system: used for unsaturated fatty acids
  • w designation describes the position of the last double bond in relation to the end of the chain
    • Also identifies the major precursor fatty acid
• Double bonds are usually in the cis configuration

Synthesis

• Fuel fatty acids are primarily supplied through diet, but excess carbohydrates and proteins can also be converted to fatty acids and stored as energy reserves
• Lipid and carbohydrate synthesis is termed non-template synthesis
  • Since they do not rely on the coding of a nucleic acid

Fatty Acid Biosynthesis

• This occurs in the livers, while its products are transferred to adipose tissue
• Adipose also has the ability to synthesize smaller quantities of fatty acids
• Synthesis is driven by the reactants: Acetyl-CoA carboxylase & fatty acid synthase
  • These are stimulated by insulin
• Palmitic acid (palmitate): primary end product of fatty acid synthesis

Acetyl-CoA Shuttling

• Acetyl-CoA accumulates in the mitochondrial matrix if a large fatty meal is eaten
  • It needs to be moved to the cytosol for fatty acid biosynthesis since it slows the citric acid cycle
• Acetyl-CoA is a product of the pyruvate dehydrogenase complex
  • It combines with oxaloacetate to form citrate, this begins the citric acid cycle
    • Limited by isocitrate dehydrogenase
• A buildup of acetyl-CoA causes a buildup of citrate
  • Citrate is able to diffuse across the mitochondrial membrane and be split up (by citrate lyase)
    • Splits into acetyl-CoA and oxaloacetate

Acetyl-CoA Carboxylase

• This is the rate limiting step of fatty acid synthesis
• Acetyl-CoA carboxylase requires ATP and biotin to function
  • Adds a CO₂ to Acetyl-CoA to form malonyl-CoA
• Enzyme is activated by insulin and citrate
• CO₂ is never incorporated into the fatty acid since it is removed during fatty acid synthase

Fatty Acid Synthase

• More appropriately called palmitate synthase since palmitate is the only fatty acid that humans can synthesize de novo
• Large multi-enzyme complex that is found in the cytosol
  • Induced by the liver after meals eaten that are high in carbohydrates (insulin induces the complex)
• Complex contains an acyl carrier protein (ACP)
  • This protein requires pantothenic acid (Vitamin B₅)
  • NADPH is required to reduce acetyl groups that are added to the fatty acid
• Palminate requires 8 acetyl-CoA groups
• Fatty acyl-CoA can be elongated and desaturated to a limited extent by the smooth endoplasmic reticulum
• Steps involved are: attachment to acyl carrier protein, bond formation between activated malonyl-CoA and the growing chain, reduction of a carbonyl group, dehydration and reduction of a double bond
  • Reaction occurs many times until a 16 carbon palmitate is created.
• Can usually be reversed by B-oxidation

Triacylglycerol Synthesis

• Formed by attaching three fatty acids (as fatty Acyl-CoA) to glycerol
• Storage form of fatty acids
• Occurs primarily in the liver and a little in the Adipose tissue
• Small amount also comes directly from the diet
• In the liver, triglycerides are packaged and sent to adipose tissue as VLDL
  • 
  • Action of Fatty Acid Synthase:
    • a. activation of growing chain
    • b. malonyl-CoA
    • c. bond formation between activated molecules
    • d. Reduction of carbonyl to a hydroxyl group
    • e.. dehydration
    • f. reduction to a saturated fatty acid

Oxidation

• Majority of fatty acid catabolism proceeds via b-oxidation
  • Occurs mainly in the mitochondria, but peroxisomal oxidation also can occur
  • Insulin inhibits indirectly while glucagon stimulates
• Branched chain fatty acids undergo a-oxidation
• w-oxidation occurs in the endoplasmic reticulum and produces dicarboxylic acids

Activation

• When metabolized, fatty acids first become activated by attachment to CoA
  • Known as Fatty-Acyl-CoA synthetase or fatty Acyl-CoA or Acyl-CoA

Fatty Acid Entry into Mitochondria

• Short chain (2-4 carbons) and medium chain (6-12 carbons) diffuse freely into mitochondria, where they can be oxidized
• Long chain (14-20 carbon) require transport into the mitochondria via a carnitine shuttle
• Carnitine acyltransferase I is the rate limiting enzyme of fatty acid oxidation
• Very long chain fatty acids (over 20 carbons) are not oxidized within the mitochondria

Beta-Oxidation in Mitochondria

• Reverses the process of fatty acid synthesis by oxidizing and releasing molecules of acetyl-CoA
• Pathway is a repetition of four steps, and each cycle releases one acetyl-CoA
  • Also reduces NAD⁺ and FAD
    • FADH₂ & NADH are oxidized in the electron transport chain to produce ATP
• In muscle and adipose tissue, acetyl-CoA enters the citric acid cycle
• In the liver, acetyl-CoA stimulates gluconeogenesis through activation of pyruvate carboxylase
• When fasting, the liver produces more acetyl-CoA from beta-oxidation than is used up in the citric acid cycle
  • A lot of the acetyl-CoA is used to form ketones bodies that are released into the bloodstream and transported to other tissues.
• Four steps of oxidation:
  • Oxidation of fatty acid to form a double bond
  • Hydration of the double bond to form a hydroxyl group
  • Oxidation of the hydroxyl group to form a carbonyl (beta-ketoacid)
  • Splitting of the beta-ketoacid into a shorter acyl-CoA and acetyl-CoA
• Odd-numbered fatty acids have a different final cycle as compared to the norm
  • Even numbered fatty acids yield two acetyl-CoA
  • Odd numbered fatty acids yield one acetyl-CoA and one propionyl-CoA
• Propionyl-CoA can be converted to glucose (i.e- only odd numbered fatty acids can be converted to glucose in humans)
  • Propionyl-CoA is converted to methylmalonyl-CoA by propionyl-CoA carboxylase
  • Requires biotin: Vitamin B₇
  • Methylmalonyl-CoA can then be converted to succinyl-CoA by methylmalonyl-CoA mutase. This steps requires cobalamin (Vitamin B₁₂)
  • Succinyl-CoA is used in the citric acid cycle as an intermediate, but can also be converted to malate to enter the gluconeogenic pathway in cytosol.
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• Unsaturated fatty acids require two additional enzymes to account for the double bonds
  • Enzyme must have at most one double bond in their active site
    • Bond must be located between carbons 2 & 3
  • Enoyl-CoA isomerase rearranges cis double bonds at the 3,4 position to trans double bonds at the 2,3 positions
    • This is the only extra step required so that monounsaturated fatty acids can undergo beta-oxidation
  • Polyunsaturated fatty acids require further reduction by using 2,4-dienoyl-CoA reductase
    • This converts two conjugated double bonds to just one double bond at the 3,4 position
    • Will then undergo the same rearrangement as above
  • 

Ketone Bodies

• When fasting, the liver converts excess acetyl-CoA (from beta oxidation) into ketone bodies acetoacetate and 3-hydroxybutyrate (b-hydroxybutyrate)
  • This can be used for energy in various tissues
  • Cardiac and skeletal muscle, and the renal cortex are able to metabolize above two products back into acetyl-CoA
  • If fasting lasts for longer than a week, concentration of ketones in blood becomes high enough to allow the brain to begin metabolizing them

Ketogenesis

• Occurs in the mitochondria of liver cells when excess acetyl-CoA accumulates in the fasting state
• HMG-CoA synthase forms HMG-CoA
• HMG-CoA lyase breaks down HMG-CoA into acetoacetate
• Acetoacetate can be reduced to 3-hydroxybutyrate and acetone (is not used for energy)

Ketolysis

• Acetoacetate is picked up from the blood and is activated in the mitochondria by succinyl-CoA acetoacetyl-CoA transferase (thiophorase)
  • Thiophorase is an enzyme that is only present in tissues outside of the liver
  • Liver lacks the enzyme so that it does not consume the ketones it produces
• 3-hydroxybutyrate is oxidized to acetoacetate

Ketolysis in the Brain

• After one week of fasting, brain derives two-thirds of its energy from ketone bodies
• When ketones are metabolized to acetyl-CoA, pyruvate dehydrogenase is inhibited
• This switch spares essential proteins which would ordinarily be used in gluconeogenesis to produce glucose.

Protein Catabolism

• Protein is rarely used as a source of energy since it is so important for other functions
• Only occurs in cases of extreme energy deprivation

Proteolysis

• The breakdown of proteins
  • Begins in the stomach with pepsin and continues with the pancreatic enzymes: trypsin, chymotrypsin and carboxypeptidases A/B
    • Secreted as zymogens (inactive form that is activated by something else)
• Protein digestion is completed in the small intestine by its brush-border enzymes: dipeptidase and aminopeptidase
• End products are amino acids, dipeptides, and tripeptides
  • Absorption of these products through the luminal membrane is accomplished through secondary active transport that is linked to sodium
• Once at the basal membrane, amino acids can be transported to the bloodstream through simple and facilitated diffusion
• Protein for energy can be used from the diet or from the body (during starvation)
  • Body protein is catabolized in the muscle and liver
  • Amino acids that are released from proteins usually lose their amino group through transamination or deamination
    • Remaining carbon skeleton is then used for energy
• Amino acids can be classified by their ability to turn into metabolic intermediates:
  • Glucogenic: can be converted to glucose through gluconeogenesis
    • Include all amino acids except leucine and lysine
  • Ketogenic: can be converted into acetyl-CoA and ketone bodies
    • Includes leucine, lysine, isoleucine, phenylalanine, threonine, tryptophan and tyrosine
• Amino groups that are removed through transamination must be disposed of safely since they can potentially be toxic
  • Urea Cycle: primary method of removing excess nitrogen from the body
    • Occurs in the liver