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Liver: Transport and Metabolic Functions I Cindy McKinney, Ph.D. Cell Biology and Physiology Block 5 Gastroenterology and Endocrinology Lecture Objectives • • • • • • • • • • • • Describe Functional anatomy of the Liver Describe Blood Supply to The Liver Define three arrangements of hepatocyte organization Describe portal ancinus (zonal) organization Describe zonal heterogenity of liver Describe the mechanisms that are involved in the biotransformation of compounds by the liver Define the processes involved in the synthesis of bile acids Describe bile composition Describe bile flow dynamics Describe metabolic functions of the liver Define ammonia handling by the liver Describe synthesis and storage of fat soluble vitamins Reading Required Reading: Guyton and Hall Textbook of Medical Physiology, John. E Hall, 12 th edition •Secretion of Bile by the Liver; Functions of the Biliary Tree, pp 783-786 •The Liver as an Organ, pp 837-842 Overview • Metabolically active—receives approximately 28% total body blood flow • Highly aerobic organ-extracts approximately 20% of O2 used by body • Directs synthesis and degradation: a) Carbohydrates b) Proteins c) Lipids Functional Anatomy of the Liver Functional unit of the liver is a “lobule” containing a branch of the hepatic vein at its center with cells ordered around it. Portal Triad at each corner of the lobule hexagon containing branches of: hepatic artery portal vein bile duct Note: Space of Disse—extracelluar gap PORTAL TRIAD Functional Anatomy of Liver • Hepatocytes (yellow) occupy 80% of the paraenchymal volume • Form a one cell thick epithelium that forms a functional barrier between two fluid compartments with distinct ionic compositions tiny canalicular lumen ---bile sinusoidal blood space---blood • Hepatocytes alter the composition of these fluid spaces by vectorial transport of solutes across their membranes • Apical and basolateral hepatocyte membranes ---many distinct characteristics Functional Anatomy of Liver Space of Disse: perisinusoidal space = an extracellular “gap” between endothelial cells that line the sinusoids and the basolateral membranes of the hepatocytes Hepatocytes have microvilli on basolateral side (in the Space of Disse) that project into space. This facilitates contact with solutes transported in sinusoidal blood. Single cell thickness of hepatocytes -- tight junctions and desmosomes between cells • apical membrane faces canalicular lumen • basolateral membrane faces the Space of Disse Bile Canaliculi Two adjacent hepatocytes juxtapose their groove-like apical membranes along their common face --- forming tiny canaliculus (1 μm diameter) Organization of the canaliculi: “chicken-wire” appearance Functional Anatomy of the Liver Other cell types in the liver—comprise 6% of cells • Endothelial cells (2.8%) ---lining of vascular sinusoids forming fenestrated structures allowing free movement of plasma solutes (not RBCs) into the Space of Disse • Kupffer cells--- fixed macrophages in sinusoidal vascular space ---remove particulates from circulation • Stellate Cells---located in Space of Disse; contain large fat droplets in their cytoplasm may be involved in pathogenesis of cirrhosis. Central role in Vitamin A storage. May be capable of transforming into proliferative, fibrogenic, and contractile myofibroblasts. Describe Blood Flow of Liver Hepatic artery=25% total circulation Portal vein=75% total circulation Blood from portal venules and hepatic arterioles drain into the network of hepatic sinusoids Blood from the sinusoids central veins hepatic vein Portal Triad (Bile duct, hepatic artery, portal vein) + Lymphatics + Nerves= “PORTAL TRACT” Describe Blood Flow of Liver • Hepatic artery forms peribiliary plexus where there is bi-directional exchange of solutes (electrolytes and bile acids etc) between the bile and the blood in the portal tract • Peribiliary plexus portal circulation absorbed substances from biliary ductportal vein • Portal vein drains back to systemic circulation (substances are reprocessed by hepatocytes) Describe Blood Flow of Liver Hepatocyte Organization Three Alternative Arrangements 1. Classic Hepatic Lobule (hexagon): central vein=core and lobule includes all hepatocytes drained by and single central vein. Boundaries =portal triads 2. Portal Lobule (Triangle): portal triad=core of hepatic lobule contains all the hepatocytes drained by a single bile duct. Boundaries = three central veins 3. Portal Acinus (Rhomboid): group of hepatocytes supplied by a single source of arterial blood. Small 3D mass of hepatocytes that are irregular in size and shape ---one axis is a line between two triads ( high pO2) and another axis formed by a line between the central vein (low pO2) Three Arrangements of Hepatocyte Organization Lowest O2, solutes modified by other hepatocytes Hepatocytes in intermediate layer Highest O2 and solutes Proposed by Rappaport---defined three zones of hepatocytes Zone I: hepatocytes perfused first (high pO2 and solutes) most resistant to effects of circulatory compromise or nutritional deficiency or other forms of cell injury, first to Regenerate Zone II: hepatocytes in intermediate layers Zone III: Most distal section of pericentral hepatocytes ---located near terminal central vein Exposed to progressively lesser concentrations of O2 and nutrients, receive solutes that May have been modified by hepatocytes in zone I and zone II Exact functional zones of hepatocytes in this organization are difficult to define Zonal Heterogeniety of Liver The functional zones defined by the portal acinus model allow for the presence of specialized microenvironments (provided by microcirculation of the zone) surrounding the hepatocytes. Consequently, some enzymes are selectively expressed in hepatocytes of one zone or another. NOTE: Reversing the blood flow, reverses the zones. Thus, the predominant enzyme activity is controlled by the microenvironment created by hepatic microcirculation at physical cell location. Zone I: Enzymes involved in oxidative energy metabolism with β-oxidation, AA metabolism, Ureagensis, gluconeogenesis, cholesterol synthesis and bile formation Zone II: Enzymes overlap Zones I and III Zone III: Enzymes involved in glycogen synthesis, glycolysis, liponeogensis, ketogenesis, xenobiotic metabolism (CYP 450) and glutamine formation. 1) detoxification mechanisms + involved in biotransformation of drugs 2) drug induced toxicity cell necrosis located in Zone III (example: Effects of Acetaminophen) Mechanisms of Biotransformation in Liver Liver detoxifies and metabolizes many endogenous AND exogenous compounds Reactions are divided in two phases: Phase I: oxidation and reduction reactions mediated via cytochrome P450 oxidases Phase II: conjugation of phase I metabolites with glucuronate, sulphate or glutathione RH Phase I Cyp 450s ROH Phase II RO-conjugate Phase I: a) Cytochrome P450 (heme protein) named because they absorb light at 450 nm when bound to CO—function to insert O2 into substrate a) Reside in ER (microsome fraction)----multi-gene family (150 isoforms known) b) Typically catalyze hydroxylation reactions c) Responsible for drug /chemical carcinogen metabolism, bile acid synthesis, activation and inactivation of vitamins d) Some products may be directly secreted if they are water soluble e) Usually require phase II biotransformation (conjugation) for excretion Phase II: Bilirubin Metabolism and Excretion Bilirubin=heme degradation RBCs (aged or damaged) are removed by macrophages of reticuloendothelial system heme biliverdin bilirubin bilirubin+ serum albumin Conjugated to glucuronic acid(s) Secreted by liver carried by bile to small intestine Bacterial in ileum and colon deconjugate urobilinogen (colorless) Some absorbed by enterocytes/plasma In colon converted to sterocobilin main pigment of feces Kidney filters plasma; oxidized to Urobilin (yellow) Mechanisms of Biotransformation in Liver Phase II: a) Conjugation reactions make compounds more water soluble (hydrophillic) leading to excretion in blood or bile b) Common products--- glucuronides, sulfates, and mercapturic acids c) Critical step in detoxification d) Three major mechanisms Conjugation to Glucuronate (UGT) Conjugation to Sulfate (-SO4) Conjugation to Glutathione (GSH) e) Other contributing mechanisms Methylation (examples catechols and thiols) Gray Baby Syndrome Acetylation (examples amines and hydrazines) Conjugation to glycine, taurine or glutamine ( bile acids) Clinical Note: Defects in conjugation enzymes (genetic or enzyme saturation) can be fatal Example: Gray Baby Syndrome Decrease in glucuronidation capacity in infants+ administration of chloramphenicol Results in lethargy, ashen gray appearance and circulatory collapse- COMA Phase II Biotransformation: Conjugation to Glucuronate Enzymes: Uridine Diphosphate Glucuronosyl Transferases (UGTs) • Two families based on substrate specificity • Located in ER of hepatocytes (microsomes) UGT Family 1: Four known members. Genes encoded on chromosome 2 Catalyze the conjugation of glucuronic acid with phenols or bilirubin UGT Family 2: At least five UGTs known. Genes encoded on chromosome 4. Catalyze the glucuronidation of steroids or bile acids. Clinical Note: UGT Family 1 essential for conjugation of bilirubin to form excreted in bile. Congenital deficit of UGT activity results in jaundice at birth and bilirubin encephalophy seen in patients with Crigler-Najjar Type I Syndrome (autosomal recessive –rare—incidence 1/106 live births Phase II Biotransformation: Conjugation to Glucuronate Clinical Note: Crigler-Najjar Syndrome Type II Can be treated with phenobarbitol; found in Amish, potential for neurologic damage (kernicturous-brain damage) by accumulation of bilirubin in brain Phase II Biotransformation: Conjugation to Sulfonate Enzyme= Sulfotransferases • Located in cytosol (NOT ER) • Catalyze sulfation of steroids, catechols and foreign compounds (alcohol, metabolites of carcinogenic hydrocarbons) • Location in cytosol suggests these sulfotransferases act cooperatively with UGTs. • Sulfate products are non-toxic and readily eliminated -exception: sulfate esters of carcinogens (these are recirculated) SO4 + 2 ATP 3’ Phosphoadenosine-5’-phosphate (PAPS) HNSO3H NH2 + PAPS sulfotransferases + PAP Phase II Biotransformation: Conjugation to Glutathione (GSH) • • • • • Enzyme=Glutathione-S-Transferases (GST) Located in cytosol Hepatocytes conjugate reduced GSH Conjugation at the cysteine residue in GSH Liver has the highest [GSH] (approximately 5 mM) ---90% in cytosol and 10% in mitochondria Substrates for GST modification: • Electrophillic metabolites from lipophillic compounds • Products of lipid peroxidation • Alkyl and aryl halides Excreted in bile and further modified by removal of glutamyl residue from glutatthione by ϒ-glutamyl transpeptidase located on surface of bile duct epithelium. Glutathione conjugates can be secreted in plasma where a ϒ-glutamyl transpeptidase (brush border of proximal tubule in kidney) removes the glutamyl reside from glutathione). Dipeptidase removes glycine residue to produce cysteine-S- conjugate. Final conjugate is excreted in urine or acteylated in the liver (mercapturic derivative) and excreted. Bile Formation Bile from hepatocytes Small canaliculi Small terminal ductules (canals of Herring) Perilobular bile ducts Interlobular bile ducts Septal ducts Cystic duct (gallbladder) Lobar ducts Biliary Tree --Canaliculi form an extensive meshwork surrounding and draining hepatocytes Left and right hepatic ducts Common hepatic bile duct COMMON BILE DUCT Enters into doudenal lumen at Sphincter of Oddi Pancreatic Duct (Ampulla of Vater) Bile Formation (Choleresis) Three steps to bile formation: 1) Active secretion of bile from hepatocytes into liver canaliculi 2) Bile transported with water rich fluid secreted from intrahepatic and extrahepatic ducts a) approximately 900 ml/day from steps 1-2 3) Between meals, approximately 50% of hepatic bile (estimate 450 ml/day) diverted to gallbladder GALLBLADDER ---stores and concentrates bile and remaining solutes (10-20 fold) a) isosmotically removes salts and water b) concentrates remaining solutes: bile salts, bilirubin, cholesterol, lecithin c) stores concentrated bile Approximately 500 ml bile /day reaches the duodenum via the Ampulla of Vater Mixture transferred to duodenum = “dilute” hepatic bile + “concentrated” gallbladder bile Bile Formation (Choleresis) Notes on bile formation: • Bile secretion =energy dependent process hydrostatic pressure in the hepatic canaliculi > sinusoidal perfusion pressure • • • • Bile formation requires: -an active, energy dependent secretion of inorganic and organic solutes -passive movement of water follows solutes Passive movement of water through interhepatic tight junctions carries other solutes along termed “Solvent Drag” Canalicular bile is an isosmotic fluid: passage of water carries other small ions Further down the biliary tree (ducts and gallbladder)---pore size between cells is significantly smaller----consequently the role of solvent drag in bile production Bile Composition • Hepatic bile and gallbladder bile are iso-osmotic with plasma (~300 mosmole/kg) Composition: water + inorganic electrolytes (Na+, Cl- , andHCO3-) + organic solutes (bilirubin, cholesterol, fatty acids, phospholipids) Functionally important solutes: 1) Micelle forming bile acids/salts: lipid digestion/ toxic substance excretion • 2) Phospholipids: solubilize cholesterol protect hepatocyte from • cytotoxicity of bile acids • 3) Immunoglobulin A: inhibits bacterial growth in bile Excretory products in bile: cholesterol, bile pigments, plants sterols, trace minerals, Lipophillic drugs/metabolites, oxidized glutathione, antigen-antibody complexes Bile Synthesis Cholesterol converted to “primary bile acids” (cholic and chenodeoxycholic acids) Rate limiting enzyme= 7α-dehydroxylase (P450 family in Smooth ER) - feedback inhibition by bile acids Principle route of cholesterol excretion and catabolism -essential factor in total body cholesterol balance Hepatocytes conjugate primary bile acids to glycine/taurine= bile saltssecreted into bile Secondary bile acids (deoxycholic and lithocholic acids) = breakdown products from bacterial action in the ileum and colon---recirculated to liver via enterohepatic circulation Will also be conjugated to glycine and taurine=bile salts Bile Flow Choliangiocyte secretions Constant flow Linear Δ w/bile acid secretions [Glutathione] generated osmotic flow Total bile flow sum of: • Canalicular flow from hepatocytes • Ductular flow from secretions of cholangiocytes lining bile ducts Example: ursodeoxycholic bile acid increases bile flow by direct stimulation of biliary excretion Canalicular flow: increases linearly following rate of bile acid secretion Two components: 1) bile acid independent: constant flow independent of bile acid secretion a) secretion of ORGANIC compounds drives the independent flow (example: High [glutathione] in bile generates a potent osmotic driving force for canalicular flow) 2) bile acid dependent: rising component that changes linearly with bile acid secretion a) micellar form of bile acids are predominant osmotic driving force for water movement in bile acid dependent flow b) bile acids increase electrolyte and water flow by stimulating the Na+-coupled co-transport mechanisms or modulating other solute transporters Metabolic Functions of Liver Synthesis and degradation of: 1. Carbohydrates -gluconeogensis -storage of glucose as glycogen -glycogenolysis 2. Proteins -synthesizes nonessential AA -synthesizes plasma proteins - urea formation from AA 3. Lipids Provides ENERGY to other systems by exporting -participates in FA oxidation GLUCOSE -synthesizes lipoproteins, KETO ACIDS (acetoacetate etc) cholesterol and phospholipids Critical for process of oxidation in peripheral tissues Liver: Carbohydrate Metabolism Between meals ----[Glucose] (Fasting state Insulin Glucagon ) Liver metabolism becomes a source of plasma glucose for other tissues a) de novo synthesis (gluconeogenesis) is one of liver’s important major functions 1) essential for maintaining normal plasma glucose concentrations b) Glycogenolysis also delivers glucose to plasma---breakdown of glycogen 1) stored liver glycogen may account for 7-10% total liver organ weight Note: Glycogenolysis in liver yields glucose as major product Glycogenolysis in muscle produces lactic acid as major product In Fed State: Liver serves as a reserve for glucose (Insulin Glucagon ) Used to synthesize glycogen Liver captures glucose from portal blood broken down to pyruvate Oxidized to H20 and CO2 (TCA Cycle) aerobic phase Glycolysis anaerobic phase Liver also converts glucose to glycogen—carbohydrate not stored as glycogen or oxidized for Energy is usually utilized to metabolize fat Liver Protein Synthesis Liver produces a wide variety of proteins that are exported to blood plasma Major PROTEIN products include: • major plasma proteins ---maintenance of colloidal osmotic pressure in plasma -maximum rate: 15-50 g/day synthesized • factors for hemostasis (blood clotting) and fibrinolysis (blood clot degradation) • Carrier proteins (bind and transport hormones and other substances in blood) • Pro-hormones and lipoproteins Examples: proteins synthesized by Liver: 1) Albumin -25% of all protein production in liver 2) a1-globulins- HDL and VHDL, cholesterol, glycoproteins, haptoglobins and mucoproteins 3) a2-globulins-ceruloplasmin, glycoproteins, macroglobulin, plasminogen, prothrombin 4) b-globulins-LDL, VLDL lipoproteins, transferrin 5) Blood clotting factors-Factors I, II, V, VII, VIII, IX, and X (vitamin K is needed for the synthesis of some factors) Liver Amino Acid Metabolism and Urea Formation Liver captures dietary amino acids ---absorbed by GI tract and carried to liver in the portal blood circulation Two mechanisms for hepatocyte capture located on both basolateral and apical hepatocyte surfaces: 1) Na+ dependent transporters (12 highly specific types) similar to small GI 2) Na+ independent transporters AAs in liver are used immediately for de novo synthesis of proteins OR degraded for energy AA degradation: deamination produces NH4 (ammonia) and α-keto acids enters TCA cycle enters the urea cycle ATP production Exits hepatocyte via urea channel (aquaporin 9) Travels via blood to kidneys and excreted Urea Cycle: Amino Acids AA Transporter Circulated in blood Removed by kidneys Water soluble product AA Transporter Ammonia Handling and the Urea Cycle • Ammonia small, neutral molecule derived from protein catabolism/ bacterial activity - derived from bacteria urease activity in colon (50%) -NH3 passively crosses colonic epithelium- travels via portal circulation to liver -approx 40-50% of NH3 comes from kidney • Like bilirubin and ammonia is toxic to CNS • Highly membrane permeable---no transporter required • Liver critical to prevention of ammonia accumulation in blood -Liver is only organ that can convert ammonia to urea via UREA CYCLE • Hepatocytes efficiently extract ammonia from portal and systemic circulation where it enters the urea cycle urea subsequently transported back to systemic circulation • Urea (small neutral molecule) filtered at the kidney glomerulus • Approx 50% of filtered urea is excreted in urine Clinical Notes: • Metabolic activity of liver acutely compromised—coma and death can result • Metabolic activity of liver chronically compromised: --gradual decline in mental acuity reflecting cumulative action of [ammonia] + [other toxins] that are not cleared via liver and kidney =Hepatic Encephalopathy Synthesis and Secretion of Glutathione (GSH) • • - Critical molecule in conjugations reactions detoxification GSH protective role against oxidative stress in a number of tissues RBCs have low [GSH] susceptible to oxidative stress induced hemolysis - protection comes from reducing capacity GSHGSSH • GSH release into blood via OATP-1 transporter ---exchanges GSH for organic solutes • GSH can also move into bile by moving across the canalicular membrane -mediated by MRP2 transporter and possibly another unknown one • 90% of GSH in circulation is synthesized in the liver GST Liver Failure: Ammonia Handling/Encephalopathy Urea cycle impaired: [NH3] in circulation and tissues Hepatic Encephalopathy: 1) Readily permeable NH3 crosses blood- brain barrier 2) Ammonia absorbed and metabolized by astrocytes---astrocytes swell 3) Increased activity () of inhibitory activity of γ-aminobutyric acid system (GABA) 4) Energy supply to other brain cells signs of confusion, dementia and coma However, ammonia levels are not always a direct correlate with the severity of encephalopathy Liver: Synthesis and Storage of Fat Soluble Vitamins D and K • Vitamin D synthesized by skin cells under the influence of UV light • Dietary Vitamin D comes from animal (D3) and plant (D2) sources • First step in activation is a cytochrome P450 mediated 25-hydoxylation of vitamin in the liver • 1-hydroxylation in the kidney completes activation to full biological activity full biological activity: 1,25 hydroxyvitamin D • Degradation is also mediated in liver with hydroxlation at carbon 24 by another cytochrome P450 • Vitamin D deficiency: Osteoporosis and rickets • Vitamin K—fat soluble obtained by action of intestinal bacteria • Essential for ϒ carboxylation of glutamate residues in coagulation factors II,VII, IX and X • Intestinal absorption of two forms K1 and K2 similar other fat soluble vitamins • Vitamin K deficiency (chronic) results in blood clotting abnormalities Synthesis and Storage of Fat Soluble Vitamins D and K Extrahepatic or intrahepatic cholestasis, fat malabsorption, biliary fistulas, dietary deficiency particularly in association with antibiotic therapy may result in Vitamin K deficiency.