Biochemistry Department. Lipid Digestion and Lipoprotein Metabolism (1) 2 lectures Prof. Reem Sallam Dr. Marwa Mahmoud

Transcription

1 Biochemistry Department Lipid Digestion and Lipoprotein Metabolism (1) 2 lectures Prof. Reem Sallam Dr. Marwa Mahmoud

2 Intended Learning Outcomes By the end of this lecture, the student should be able to: Understand the process of digestion of dietary lipids including, the organs involved, the enzymes required, and the end products. Implement the basic science knowledge of the process of lipid digestion to understand the clinical manifestations of diseases that involve defective lipid digestion and/or absorption (maldigestion and malabsorption syndrome)

3 Dietary Lipids Dietary lipids intake is ~81 g/day Triacylglycerol is ~ 90% The remainder includes (10%): Cholesterol Cholesterol ester Phospholipids Glycolipids Free fatty acids

4 Acid lipases Acid-stable lipases Lingual and gastric lipases ph optimums 4 6 Their target is: TAG, particularly containing FA of short- or medium-chain length (fewer than 12 carbons, such as are found in milk Important role in: lipid digestion in neonates, for whom milk fat is the primary source of calorie in individuals with pancreatic insufficiency, e.g. cystic fibrosis (a disease with a near or complete absence of pancreatic lipase)

5 Lipid Digestion: Sites, Enzymes & End products

6 1- Digestion of Lipids Begins in the Stomach The effects of lingual and gastric lipases on TAG: Little significance in adults (Why?) Important for digestion of milk fat in neonates and infants (Explain)

7 2- Digestion of Lipids in Small Intestine Digestion of lipids is preceded by emulsification Digestion in small intestine is hormonally controlled: 1. Cholecystokinin (CCK) 2. Secretin

8 Emulsification of Dietary Lipids in duodenum Emulsification occurs in the duodenum. Increases the surface area of lipid droplets, therefore the digestive enzymes can effectively act (at the interface of the droplet and the surrounding aqueous solution) Mechanisms: 1. Mechanical mixing by peristalsis 2. Detergent effect of bile salts: Bile salts interact with lipid particles and aqueous duodenal contents, stabilizing the particles as they become smaller, and preventing them from coalescing

9 Pancreatic Enzymes for Digestion of Lipids Pancreatic Lipase and co-lipase Cholesterol esterase Phospholipase A2 Lysophospholipase

10 Pancreatic lipase : Found in high conc. in pancreatic secretion (2-3% of total proteins) Is highly efficient catalytically only severe pancreatic deficiency, such as that seen in cystic fibrosis, results in significant malabsorption of fat Gastric and pancreatic lipases are Inhibited by Orlistat, an antiobesity drug decreasing fat absorption, resulting in loss of weight. igestion of TAG by Pancreatic Lipase (an esterase) & Colipase

11 Colipase Pancreatic protein Binds the lipase at a ratio of 1:1 anchors it at the lipid-aqueous interface. It restores activity to lipase in the presence of inhibitory substances like bile acids that bind the micelles. Is secreted as the zymogen, procolipase, & activated in the intestine by trypsin.

12 Digestion of Cholesterol Ester by Cholesterol Esterase Most dietary cholesterol is present in the free form 10 15% is present in the esterified form. CE are hydrolyzed by pancreatic cholesteryl ester hydrolase (cholesterol esterase) cholesterol + FFA The enzyme activity is greatly increased in the presence of bile salts.

13 Digestion of Phospholipids (PL) by Phospholipase A2 & Lysophospholipase H 2 O Fatty acid PL Lysophospholipid Phosphophlipase A2 H 2 O Fatty acid Lysophospholipid Glycerylphosphoryl base Lysophosphophlipase excreted in the feces, further degraded, or absorbed. phospholipases A is secreted as proenzyme, and is activated by Trypsin. Like cholesteryl ester hydrolase, it requires bile salts for optimum activity.

14 Main End Products of Lipid Digestion IN JEJUNUM 1. 2-Monoacylglycerol 2. Cholesterol 3. Free fatty acids

15 Absorption of Lipids by Intestinal Mucosal Cells Mixed micelles: Disc-shaped clusters of amphipathic lipids. Arranged with their hydrophobic groups on the inside and their hydrophilic groups on the outside. Mixed micelle includes end products of lipid digestion, bile salts and fat-soluble vitamins Short- and medium-chain fatty acids are water soluble and do not require mixed micelle for absorption by intestinal cells

16 Absorption of Lipids by Intestinal Mucosal Cells Mixed Micelle: 2-Monoacylglycerol Cholesterol FFA Bile salts Fat-soluble vitamins

17 Re-synthesis of Lipids by Intestinal Mucosal Cells Activation of long chain FA into acyl CoA by FA-CoA synthetase (thiokinase) In the endoplasmic reticulum: Synthesis of TAG from monoacylglycerol by the enzyme complex, TAG synthase Synthesis of Cholesterol ester from cholesterol Synthesis of phospholipids from glycerylphosphoryl base Short- and medium-chain fatty acids are not converted into their CoA derivatives. Instead, they are released into portal circulation, carried by serum albumin

18 Re-synthesis of Lipids and Assembly of Chylomicrons by Intestinal Mucosal Cells

19 Assembly of Chylomicrons by Intestinal Mucosal Cells Assembly of chylomicrons: Newly synthesized TAG and cholesterol ester are packaged as lipid droplets surrounded by thin layer of: Apolipoprotein B-48 (apo B-48) Phospholipids Free cholesterol

20 Secretion of Chylomicrons by Intestinal Mucosal Cells Secretion of chylomicrons: By exocytosis into lymphatic vessels around villi of small intestine (lacteals) then enter into systemic circulation Milky-appearance of serum after lipid-rich meal

21 Abnormalities in Lipid Digestion/Absorption Liver and gall bladder diseases Pancreatic insufficiency e.g., chronic pancreatitis, cystic fibrosis, surgical removal of the pancreas Intestinal diseases: e.g., Intestinal resection (shortened bowel) incomplete digestion & absorption of fat & protein abnormal appearance of lipids (steatorrhea) & undigested protein in the feces (Malabsorption syndrome)

22 Maldigestion/Malabsorption of Lipids

23 Cystic Fibrosis (CF) This is the most common lethal genetic disease in Caucasians of Northern European ancestry Autosomal recessive disorder Caused by mutations to the gene for the CF transmembrane conductance regulator (CFTR) protein that functions as a chloride channel on epithelium of Pancreas, lungs, testes, and sweat glands Defective CFTR decreased secretion of chloride & increased reabsorption of sodium and water thick, viscous secretions

24 Cystic Fibrosis (CF) In the pancreas, the decreased hydration thickened secretions such that pancreatic enzymes are not able to reach the intestine pancreatic insufficiency. Consequences: pancreatic insufficiency, chronic lung infections, progressive pulmonary disease, and male infertility. Treatment: replacement therapy with enzymes Supplementation with fat-soluble vitamins Treat the associated chronic lung infections

25 Take home message Dietary lipids are relatively hydrophobic Lipid digestion begins in stomach Emulsification of lipids occurs in duodenum, helped by peristalsis and bile salts Intestinal digestion of lipids by pancreatic enzymes Lipid absorption by formation of mixed micelles

26 Take home message Re-synthesis of TAGs, cholesterol ester and PLs inside the intestinal mucosal cells Assembly and secretion of chylomicrons into lymphatic lacteals and then into systemic circulation Short- and medium-chain fatty acids: Do not require micelle for absorption Do not participate in re-synthesis of TAGs & PLs Released directly from intestinal cells into portal circulation

27 Take home message Liver diseases, pancreatic insufficiency, or intestinal diseases incomplete digestion and absorption of fat & protein steatorrhea & appearance of undigested proteins in the feces (Malabsorpton syndrome)

28 References Lippincott s Illustrated Reviews, Biochemistry, Denise R. Ferrier, Lippincott Williams & Wilkins, a Wolters Kluwer business, 6th edition, 2014, Chapter 15: Page

29 Lipoprotein (1)

30 Intended Learning Outcomes By the end of this lecture, the students are expected to be able to: Recall the general characteristics of lipids solubility Analyze the common lipoprotein particles structure List different types of lipoprotein particles and compare their structural criteria and functions Discuss the metabolism of chylomicrons and very-lowdensity lipoprotein (VLDL) particles Recognize diseases related to disturbed metabolism of chylomicrons and VLDL

31 Introduction Because lipid compounds are relatively water insoluble, they are transported in plasma (aqueous) as Lipoproteins. Lipoproteins functions: 1.To keep their component lipids soluble as they transport them in the plasma 2.To provide an efficient mechanism for transporting their lipid contents to (and from) the tissues. In humans, this transport system is less perfect than in other animals gradual deposition of lipid especially cholesterol in tissues e.g. atherosclerosis

32 Lipoproteins and Related Clinical Problems Atherosclerosis and hypertension Coronary heart diseases Lipoproteinemias (hypo- and hyper-) Fatty liver

33 Lipoproteins Structure 1. Neutral Lipid core (containing TAG & CE) According to the type of lipoprotein. Different lipid components in various combinations 2. A shell of amphipathic components (oriented with their polar portions exposed on the surface): Apolipoproteins (Apo-A, B, C, D, E) Phospholipids Nonesterified (free) cholesterol

34 Lipoproteins Structure The origin of TAG & cholesterol carried by the lipoproteins are diet (exogenous source) or de novo synthesis (endogenous source).

35 Types of Lipoproteins What s different in various types of lipoproteins? They differ in: Lipid & protein composition Site of origin Size Density Electrophoretic mobility Their actual composition is somewhat variable, because they interchange lipids and apolipoproteins with each other.

36 Size and density of lipoprotein particles Chylomicrons: lowest in density and largest in size Contain the highest % of lipid and the lowest % of protein VLDLs and LDLs: successively denser than chylomicrons having higher ratios of protein to lipid HDL: the densest The highest % of proteins

37 Chylomicrons Types and Composition of Lipoproteins Very low density Lipoprotein (VLDL) Low density Lipoprotein (LDL) High density Lipoprotein (HDL)

38 LP separation on the basis of their density by ultracentrifugation.

39 LP separation on the basis of their electrophoretic mobility.

40 Spherical macromolecules complexes of lipids & specific proteins (apolipoproteins) Outer coat: - Apoproteins - Phospholipids - Cholesterol (Unesterified) Inner core: - TG - Cholesterol ester (CE) Lipoprotein Structure

41 Apolipoproteins Functions: 1. Providing recognition sites for cell-surface receptors 2. Serving as activators or coenzymes for enzymes involved in lipoprotein metabolism. 3. Some are essential structural components of LP particles and cannot be removed. 4. Some are transferred freely between lipoproteins.

42 Apolipoproteins Classes +/- (subclasses) according to the structure & function: 1.A (A-I) 2.B 3.C (apo C-I, apo C-II, apo C-III) 4.D 5.E Functions of all of the apolipoproteins are not yet known

43 Plasma Lipoproteins For triacylglycerol transport (TG-rich): - Chylomicrons: TG of dietary origin - VLDL: TG of endogenous (hepatic) synthesis For cholesterol transport (cholesterol-rich): LDL: Mainly free cholesterol HDL: Mainly esterified cholesterol

44 Chylomicrons Assembled in intestinal mucosal cells Lowest density Largest size Highest % of lipids and lowest % proteins Highest triacylglycerol (~ 90% of the lipids in a chylomicron) Carry dietary lipids (TAG, cholesterol, fat-soluble vitamins, cholesteryl esters) to peripheral tissues Responsible for physiological milky appearance of plasma (up to 2 hours after meal)

45 Chylomicrons Metabolism Nascent = Functionally incomplete FA are stored (by the adipose), used for energy (by the muscle), or transported by serum albumin until uptake e.g., in lipid synthesis, or gluconeogenesis

46 RNA Editing in the synthesis of apo-b 100 & apo-b 48 CAA TAA apo-b gene 5 3 TRANSCRIPTION No editing Nonsense Posttranscriptional CAA UAA codon RNA EDITING apo-b mrna CAA UAA UAA UAA 5 A n 5 A n TRANSLATION Apo-B 100 Protein In Liver 48% Apo-B 48 Protein In Small Intestine

47 Synthesis of chylomicrons Apolipoprotein B-48: unique to chylomicrons Its synthesis begins on the rough ER it is glycosylated as it moves through the RER and Golgi. Assembly of chylomicrons: The enzymes involved in TAG, cholesterol, & phospholipid synthesis are located in the SER. Assembly of the apolipoproteins and lipid into chylomicrons requires microsomal TAG transfer protein (MTP) MTP loads apo B-48 with lipid before transition from the ER to the Golgi In Golgi: particles are packaged in secretory vesicles fuse with the plasma membrane release LP enter the lymphatic thoracic duct blood

48 Modification of chylomicrons 1. Nascent chylomicron in the blood receives apolipoprotein E (which is recognized by hepatic receptors) and apo C (including apo C-II, which is necessary for the activation of lipoprotein lipase) from circulating HDL 2. Degradation of TAG by LPL: LPL is extracellular, anchored by heparan sulfate to the capillary walls of most tissues, but predominantly those of adipose tissue and cardiac and skeletal muscle. The highest concentration is in heart (the heart uses FA to provide much of the energy needed for its function) Is activated by apo C-II on circulating lipoprotein particles It hydrolyzes the TAG FA + glycerol Adult liver does not have this enzyme. (Liver has a lipase on the surface of endothelial cells of the liver, that is particularly important in HDL metabolism)

49 Regulation of lipoprotein lipase activity Tissue-specific LPL isozymes synthesis occurs in adipose tissue, heart & skeletal muscle. Regulation of LPL expression is by: Nutritional state Hormones In the fed state insulin LPL synthesis in adipose tissue and LPL synthesis in muscle tissue. In the fasting state insulin LPL synthesis in muscle tissue.

50 Formation & fate of chylomicron remnants When > 90% of the TAG in chylomicrons is degraded Chyloicron size Chyloicron density the apo C is returned to HDL. = chylomicron remnant chylomicron remnant is rapidly removed from the circulation by the liver (by receptors that recognize apo E) Chylomicron remnants endocytosis fusion with a lysosome degradation releasing amino acids, free cholesterol, and FA. The receptor is recycled.

51 CHYLOMICRON- RELATED DISEASES: Type 1 hyperlipoproteinemia, or familial lipoprotein lipase deficiency Deficiency of lipoprotein lipase or its activator (apo C-II) a dramatic accumulation of chylomicron-tag in the plasma (hypertriacylglycerolemia) even in the fasted state (> 1000 mg/dl) Patients are at increased risk for acute pancreatitis.

52 Very Low Density Lipoproteins VLDLs Assembled and secreted from the liver directly to the blood High TAG ~60% (endogenous, hepatic origin) Carry lipids from liver to peripheral tissues (where the TAG is degraded by LPL, as in the chylomicrons) Nascent VLDL: contains Apo B-100 Mature VLDL: contains Apo B-100, Apo C-II & Apo E (from HDL)

53 VLDL Metabolism

54 Metabolism and Fate of VLDL When most of the TAG in VLDL is degraded 1. size 2. density 3. the apo C & apo E are returned to HDL 4. some TAG are transferred from VLDL to HDL in an exchange with CE from HDL to VLDL (by cholesteryl ester transfer protein: CETP) These modifications the VLDL is converted to IDL or VLDL remnants then to LDL

55 Apo E IDLs can also be taken up by cells through receptor-mediated endocytosis that uses apo E as the ligand. Normally Apo E is present in 3 isoforms: E-2: the least common, it binds poorly to receptors E-3: the most common E-4 Individuals homozygotic for apo E-2 are deficient in the clearance of chylomicron remnants and IDL from the circulation familial type III hyperlipoproteinemia (familial dysbetalipoproteinemia, or broad beta disease): hypercholesterolemia & premature atherosclerosis Apo E-4 isoform increased susceptibility to and decreased age of onset of late-onset Alzheimer disease, doubling the lifetime risk (Not yet understood why).

56 Modifications of Circulating VLDLs 1- Degradation of TG by lipoprotein lipase, VLDLs become Smaller in size More dense 2- Apo C & Apo E return back to HDL 3- Some TG are transferred from VLDL to HDL in exchange with cholesterol ester (By cholesterol ester transfer protein) VLDL IDL (returns Apo E to HDL) LDL

57 Lipid-Transfer Protein

58 VLDLs-Related Diseases 1. Hypolipoproteinemia: Abetalipoproteinemia Defect in the microsomal TAG transfer protein (MTP) Apo B cannot be loaded with lipid few VLDLs or chylomicrons are formed accumulation of TAG in liver and intestine.

59 VLDLs-Related Diseases, continued 2- Fatty Liver (hepatic steatosis) Imbalance between hepatic synthesis of TAG and secretion of VLDLs accumulation of TAG in liver In conditions such as: obesity and Type 2 diabetes mellitus

60 VLDLs-Related Diseases, continued 3- Hyperlipoproteinemia (type I and type III) Type I Hyperlipoproteinemia Familial Lipoprotein lipase deficiency (rare, autosomal recessive disease). Due to deficiency of LPL or its Activator (Apo C-II) dramatic accumulation ( 1000 mg/dl) of chylomicrons in plasma (chylomicronemia) Usually associated with acute abdomen due to acute pancreatitis plasma TAG even in the fasted state i.e fasting hypertriacylglycerolemia

61 VLDLs-Related Diseases, continued Type III Hyperlipoproteinemia Also called dysbetalipoproteinemia, or broad beta disease): Individuals homozygous for apo E-2 are deficient in the clearance of chylomicron remnants and IDL from the circulation hypercholesterolemia & premature atherosclerosis

62 Take home message Chylomicrons are assembled in the intestine and carry exogenous (dietary) lipids. Originally, chylomicrons contain only one apolipoprotein (apo B-48). Afterward, they acquire apo C-II and apo E from HDL. Nascent VLDL are produced in the liver, they contain apo B-100, and carries endogenous (hepatic) TAG to the peripheral tissues. Mature VLDL contains also apo C-II and apo E from HDL. Lipoprotein lipase degrades TAG within the chylomicrons and the VLDL.

63 Take home message VLDL receives cholesteryl esters from HDL by cholesteryl ester transfer protein. VLDL is converted to IDL, then to LDL. Type I hyperlipoproteinemia or familial lipoprotein lipase deficiency is due to deficiency of lipoprotein lipase or its coenzyme (apo C-II). Individuals homozygous for apo E-2 isoforms defective uptake of chylomicrons remnants and IDL by the liver and causes Type III hyperlipoproteinemia.

64 References Lippincott s Illustrated Reviews, Biochemistry, Denise R. Ferrier, Lippincott Williams & Wilkins, a Wolters Kluwer business, 6th edition, 2014, chapter 18: Pages: