Lipid Metabolism-III

Serkan SAYINER, DVM PhD. Assist. Prof. Near East University, Faculty of Veterinary Medicine, Department of Biochemistry [email protected] Cholesterol Biosynthesis Cholesterol Biosynthesis ▪ Cholesterol is synthesized from the cytosolic acetyl- CoAs.

▪ Most animal cells have cholesterol, but the site of synthesis is liver.

▪ Cholesterol, which is synthesized in the liver and taken up with food, is transfered to body cells (peripheral tissues) by lipoproteins. Cholesterol Biosynthesis ▪ In the synthesis of cholesterol, the understanding of the acetyl CoA's incorporation into synthesis has been elucidated by radioisotope assays.

▪ Another part apparently is squalene, which has a linear 30 carbon and is an intermediate in cholesterol biosynthesis.

▪ While the squalene is forming, the 5-carbon isoprene units combine (that is terpene). Cholesterol Biosynthesis ▪ Cholesterol biosynthesis occurs in 4 steps.

1. Formation of Mevalonate (5 C)

2. Conversion of mevalonate to Isoprenoids (5 C)

3. Condensation of isoprenoids to squalene (30 C)

4. Conversion of Squalene to Cholesterol (27 C) Mevalonat Formation ▪ Cholesterol is synthesized from acetyl CoA which is transported from the mitochondria by the citrate transport system.

▪ These two major lipid biosynthetic pathways are metabolic pathways, as cytosolic acetyl-CoA is also used for the synthesis of fatty acids.

▪ The first step in the synthesis of cholesterol begins with the formation of hydroxymethylglutaryl-CoA (HMG- CoA) by combining 3 molecules of acetyl CoA. Mevalonat Formation ▪ The HMG-CoA reductase enzyme, which catalyzes the mevalonate reduction of HMG-CoA, is a rate-limiting enzyme in the cholesterol synthesis pathway.

▪ It is inactivated by phosphorylation and is interconvertible.

▪ In addition, the amount of enzyme in the cell is tried to be kept regularly. Mevalonat Formation ▪ Insulin enhances HMG-CoA reductase activity, while glucagon has an adverse effect.

▪ In addition, the long-chain acetyl CoA molecules inhibit HMG-CoA reductase by acting directly on the enzyme both on the allosteric effect and on the phosphorylating kinase enzyme. Mevalonat Formation ▪ The activity of HMG-CoA reductase is regulated by the cholesterol concentration.

▪ As the cholesterol concentration increases, the enzyme is allosterically inhibited and leads to the formation of cholesterol derivates.

▪ In addition, high levels of cholesterol leads to increased degradation and reduced enzyme synthesis. Conversion to Isoprenoid Units ▪ Mevalonate is converted to isopentenyl pyrophosphate by a second phosphorylation and decarboxylation as a result of a series of enzymatic reactions.

• With two phosphorylation and one decarboxylation event, it is converted into isopentenyl pyrophosphate, a molecule with 5 carbons. Squalene Formation ▪ An isomerase enzyme combines isopentenyl pyrophosphate with isomeric dimethylallyl pyrophosphate to form 10 carbon geranyl pyrophosphate. ▪ The ten carbon geranyl pyrophosphate associates with isopentenyl pyrophosphate to form the 15 carbon farnesyl pyrophosphate. ▪ Then the two molecules farnesyl combines to form 30 carbons to the squalene. Cholesterol Transformation ▪ This phase between squalene and cholesterol is very detailed and complex.

▪ The main ingredient, lanosterol, can accumulate approximately as much as the amount of cholesterol that is actively synthesized in the cell.

▪ At the stage between squalene and lanosterol, an oxygen atom is needed to form four steroid nuclei. Cholesterol Transformation ▪ Cyclization occurs in one step with electrons taken from neighboring double bonds.

▪ The conversion of lanosterol to cholesterol occurs in a very staged manner, which requires the methyl group, oxidation and decarboxylation.

▪ It is believed that many different enzymes are involved between lanosterol and cholesterol. However, the main way is not clear. Source: Engelgink, 2014

Acetyl-CoA

Relationship between cholesterol metabolism and other compounds in the cell ▪ Synthesized cholesterol and most of the intermediates formed during synthesis and isopentenyl pyrophosphate with five carbons form • Fat-soluble vitamins, • Terpenes, • Ubiquinone and • The source of many substances such as phytol, the side chain of chloroplast in plants. Relationship between cholesterol metabolism and other compounds in the cell ▪ Cholesterol is the main source of bile, which has significant in digestion.

▪ Vitamin D is effective in the absorption of calcium from the intestines.

▪ Testosterone and hormones like ApoA, B, C, 17β- estradiol are steroid hormones. Relationship between cholesterol metabolism and other compounds in the cell ▪ The source of the steroid hormones that regulate the electrolyte balance is also cholesterol.

▪ Another important feature of cholesterol is the ability to regulate membrane fluidity through cell membrane structure. Source: Engelgink, 2014 Plasma Cholesterol ▪ Two thirds of plasma cholesterol is esterified with long chain fatty acids, especially linoleic acid.

▪ Cholesterol esters are continuously hydrolyzed and re- synthesized.

▪ The hydrolysis takes place in the liver. The re-expression occurs mainly by transferring a fatty acid residue from the lecithin under the influence of lecithin cholesterol transferase (LCAT) in the plasma. Plasma Cholesterol ▪ Cholesterol is transported as free molecules or fatty acid esters in lipoproteins.

▪ Plasma cholesterol and triglycerides have been known for a number of years and therefore the severity of age and stress on vascular diseases has been known for many years.

▪ As a result of excess stress, adrenaline secretes excess free fatty acids from fat tissues, which increases the synthesis of low-density lipoproteins (LDL) in the liver. Functions of Cholesterol ▪ It allows some substances in the plasma to be transported as it is not saturated.

▪ It is the precursor of some steroid hormones.

▪ 7-dehydrocholesterol is transformed into Vitamin D3 by ultraviolet light at the skin. Lipoproteins Lipoproteins ▪ Some lipids combine with specific proteins to form lipoproteins.

▪ Plasma lipoproteins include lipids such as cholesterol and its esters, and triacylglycerols.

▪ Blood plasma lipoproteins are classified according to the particles of lipids and their concentrations which they contain. There are mainly 4 groups (+1 intermediate) and they contain 50-90% lipid. Lipoproteins ▪ Chylomicrons (CM): They carry the triacylglycerols to the tissues from intestine.

▪ Very Low Density Lipoproteins (VLDL): They contain triacylglycerols that are synthesized in the liver.

▪ Intermediate Density Lipoproteins (IDL): Intermediate lipoproteins. Lipoproteins ▪ Low Density Lipoproteins (LDL): They are formed by the breakdown of lipid fractions of very low-density lipoproteins. They allow cholesterol to be transported to tissues outside the liver. It regulates cholesterol metabolism in tissues outside the liver. It is the lipoprotein that contains the most cholesterol concentration.

▪ High Density Lipoproteins (HDL): Proteins and phospholipids are present in excess in this lipoprotein. It allows cholesterol to be transported from the various tissues to the liver. Lipoproteins

TG (%87) TG (%57)

Source: Engelgink, 2014 Lipoproteins ▪ Lipoproteins are spherical structures consisting of a neutral lipid core (triglyceride or cholesterol ester or both) and a shell consisting of apoproteins, phospholipids and free cholesterol (polar lipids) around it.

▪ Plasma lipoproteins are synthesized and secreted by the liver and intestine. Lipoproteins ▪ Proteins involved in the structure of lipoproteins are called apolipoproteins or apoproteins.

▪ These are classified as Apo-A, Apo-B, Apo-C and Apo-E. Each has subfractions.

▪ These apoproteins are synthesized and their lipoproteins differ. • The protein components of these structures organize the entry and exit of particulate lipids into specific locations. Lipoprotein Structure

Source: Engelgink, 2014 Classification of Lipoproteins ▪ Lipoproteins are classified according to their specificities in their differences in protein and lipid ratios.

▪ The most widely accepted classification of plasma lipoproteins is by ultracentrifugation.

▪ There are 5 lipoproteins. Classification of Lipoproteins ▪ Chylomicrons • The widest/lowest-density lipoproteins, triacylglycerols, cholesterol and other lipids in nutrients are transported to the fatty tissues of the intestines and to the liver. • It mainly contains 82% triglycerides. In addition, it contains 2% apoprotein, 7% phospholipid and 9% cholesterol. • Apo-A series, B-48, Apo-C and Apo E family apoproteins. • Chylomicrons are synthesized in the small intestine. • Their function is to carry the dietary lipids (triglycerides, cholesterol, fatty acids) to the tissues. Classification of Lipoproteins

• The chylomicrons synthesized in enterocytes first enter the lymph circulation then systemic circulation. • Triacylglycerols in the chylomicrons are hydrolyzed by lipases localized in the capillaries of adipose tissue and other peripheral tissues within minutes. • The chylomicron molecule, which undergoes triglyceride loss, is called the residue chylomicron. This molecule includes cholesterol, Apo-B and Apo-E molecules. • Residual chylomicrons are taken from the circulation by the liver. Classification of Lipoproteins ▪ Very low-density lipoproteins (VLDL) • VLDLs are synthesized in the liver. • VLDLs consist of 52% triglyceride, 18% phospholipid, 22% cholesterol, 8% apolipoprotein in structure. • Sphingomyelin and lecithin are the main phospholipids. • It contains 30-35% Apo-B100 as apolipoprotein. The rest are the C and E series. It contains A serie in trace amounts. • VLDL leaves endogenously synthesized triacylglycerols in adipose tissue. • Cholesterol residues are transported with low-density lipoproteins (LDL) rich in cholesterol esters. Classification of Lipoproteins ▪ Intermediate-density lipoprotein (IDL) • VLDLs become IDL (intermediate-density lipoprotein) (in other words VLDL residues) when TGs in VLDL start to hydrolyse with extrahepatic LPL effect.

• IDLs have equal cholesterol and triglycerides in cats and dogs, and there are two possible ways. 1. Cleared by the liver or 2. Converted to LDL. Classification of Lipoproteins ▪ Low-density lipoproteins (LDL) • The richest lipoprotein in means of cholesterol content. LDLs carry the cholesterol to extrahepatic tissues and allow it to be stored there. • It is a product of the catabolism of VLDL. • Its structure contains 47% cholesterol, 9% triglyceride, 21% apoprotein, 23% phospholipid. • The main apolipoprotein is Apo-B100. Total plasma contains 90-95% of Apo-B100. Classification of Lipoproteins

• VLDL leaves endogenously synthesized triacylglycerols in adipose tissue. Cholesterol residues are transported with low- density lipoproteins (LDL) rich in cholesterol esters. Most of the LDL cholesterol is linoleate esterified, which is a polyunsaturated fatty acid.

• The role of LDL is to carry cholesterol to peripheral tissues and regulate cholesterol synthesis in these locations. Classification of Lipoproteins ▪ High-density lipoproteins (HDL) • HDLs are mainly liver-derived lipoproteins. • The structure contains 45% apoprotein, 26% phospholipid, 8% triglyceride and 21% cholesterol. • 80% of the phospholipids in the structure are lecithin. This phospholipid plays an important role in the esterification of cholesterol with LCAT. • HDLs serve as a repository for Apo-A, Apo-E and Apo-C. • The role of HDL is to carry cholesterol from the peripheral tissues to the liver. Classification of Lipoproteins

• There are two subfractions of HDL.

1. HDL3: Take cholesterol from peripheral tissues.

2. HDL2: It is formed by esterifying cholesterol with HDL3 LCAT effect. HDL2 is also degraded by hepatic triglyceride lipase in the liver. Source: ApsuBiology Factors Affecting Lipoprotein Metabolism

▪ Starvation: Lipoprotein changes are induced in fasting conditions in pigs that are 3 days old or more, and under these conditions, the plasma VLDL concentration increases.

▪ Diet and Nutrition: In the absence of long chain essential fatty acids, lipoprotein transport from the liver, LPL activity, LCAT activity and fatty acid synthesis are affected. Factors Affecting Lipoprotein Metabolism

▪ Liver Disorders: Significant amounts of LDL cholesterol and phospholipid levels are observed in the fatty liver of animals.

▪ Pancreatitis: In acute pancreatitis, LPL inhibits VLDL and chylomicrons, which cause cancellation, and causes hyperlipidemia.

▪ Diabetes: Diabetes mellitus, secondary pancreatic disorders caused by insulin insufficiency, causes hyperlipidemia. Factors Affecting Lipoprotein Metabolism

▪ Exercise: Exercise in dogs causes changes in lipoprotein metabolism. Intensive physical exercise causes an increase in LPL activity.

▪ Hypothyroidism: It has been observed in a study conducted in an atrial setting that hypothyroidism changes lipoprotein concentrations. Experimental hypothyroidism induced by thyroidectomy significantly increased blood VLDL and VLDL subfractions at 4 weeks. Factors Affecting Lipoprotein Metabolism

▪ Life style: Lifestyle also affects the lipoprotein profile. The ratio of fat in the diet, the intensity of the exercise and the shelter conditions affect the amount of lipoprotein.

▪ Age: Age affects the lipoprotein profile in animals.

▪ Sex: Generally, HDL protein and cholesterol levels are lower in males than females. Factors Affecting Lipoprotein Metabolism

▪ Weight loss or gain: When plasma cholesterol and lipoprotein concentrations are compared with baseline values, these values increase in weight gain. Loss of lipoprotein fractions in the absence of HDL is observed. Ketone Bodies Ketone Bodies ▪ Further oxidation of Acetyl CoA, which occurs in the oxidation of fatty acids, follows two pathways in the liver.

▪ These are produced by the citric acid cycle. It is the way to the ketone bodies which are, • Acetoacetate, • β-hydroxybutyrate and • Acetone. Ketone Bodies ▪ Acetoacetate and β-hydroxybutyrate can not be more oxidized in the liver (adult).

▪ Transferred to the blood circulation and peripheral tissues, these tissues are oxidized and energized by the citric acid cycle.

▪ Keton bodies are an alternative fuel for cells. Due to their water-solubility properties, it is not necessary to carry within lipoproteins or with albumin. Ketone Bodies ▪ Acetyl CoAs present in the liver are produced when they reach the oxidative capacity in the liver, thus protecting the energy. ▪ Extrahepatic tissues such as skeletal muscle, heart and renal cortex use ketone bodies in proportion to the blood levels. ▪ Brain tissue can also use ketone bodies if the levels are high enough. Ketone Bodies 1. The first step in the formation of acetoacetate in liver mitochondria is the enzymatic condensation of two acetyl-CoA. This reaction is catalyzed by thiolase.

2. Then, Acetoacetyl CoA reacts again with an H2O and an Acetyl-CoA, and 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) forms. Ketone Bodies 3. In the subsequent reaction, acetoacetate and acetyl CoA occur. The reaction is catalyzed by 3-hydroxy-3- methylglutaryl CoA Lyase.

4. The resulting acetoacetate is reduced to β- hydroxybutyrate. • Acetoacetate also acts as a precursor molecule for acetone. It is spontaneously or enzymatically converted to acetone. Ketone Bodies ▪ Acetoacetate and β-hydroxybutyrate, formed by 2 enzymatic steps from 2 acetyl-CoA in the liver, pass into blood from liver cells and are transported to peripheral tissues.

▪ In peripheral tissues, β-hydroxybutyrate is oxidized to Acetoacetate by β-Hydroxybutyrate dehydrogenase.

▪ Acetoacetate is activated by transferring a CoA-SH from succinyl CoA and forming CoA-SH ester of acetoacetate. Ketone Bodies ▪ The resulting Acetoacetyl-CoA is degraded by the thiolase enzyme to 2 acetyl-CoA. ▪ The resulting acetyl-CoAs enter the citric acid cycle in the peripheral tissues and become fully oxidized. ▪ It is usually difficult to oxidize a small amount of acetone in the organism. ▪ The liver does not use ketone bodies, although it is the place where ketone bodies are synthesized in the organism. Ketone Bodies ▪ The concentration of ketone bodies in the blood of well- fed mammals normally does not exceed 0.2 mmol/L.

▪ In ruminants this ratio is somewhat higher due to the formation of β-hydroxybutyrate from butyric acid on the rumen wall.

▪ Increase in blood concentration of ketone bodies are called ketonemia, excration via urine is called ketonuria, the whole is called ketosis. Ketone Bodies ▪ Acetoacetate and β-hydroxybutyrate are both moderately strong acids.

▪ They are buffered when they are in blood or tissues.

▪ These outcrops, which are continuously excreted in large quantities, drain the alkali reservoir, causing the loss of buffering cations that lead to ketoacidosis. This can be particularly dangerous to diabetes mellitus. Ketone Bodies ▪The pathological features of ketosis occur due to diabetic mellitus, pregnancy toxemia and dairy cattle ketosis; non-pathological forms are observed in fasting, high fat feeding and long- term exercises. Origin and Utilization of Ketone Bodies

Source: Engelgink, 2014 Source: Engelgink, 2014 Source: Engelgink, 2014 Source: Engelgink, 2014 Source: Engelgink, 2014 Source: Engelgink, 2014 Source: Engelgink, 2014 Fatty Liver and Lipotropic Factor Fatty Liver and Lipotropic Factor ▪ In liver cells, the appearance of diffuse fat infiltration and degeneration is expressed as fatty liver syndrome (Fatty Liver Syndrome). Liver fat is called LIPOTROPISM.

▪ Methionine, Choline, Betaine and Inositol are substances that prevent fatty substances in the liver. These items are called LIPOTROPIC MATTERS. Fatty Liver and Lipotropic Factor ▪Fatty Liver Syndrome • The lipid ratio in the liver reaches 25-30%. • The diameter of the fat droplets is 2-10 microns. • It is characterized by the formation of fat cysts up to 100 microns in size. Fatty Liver and Lipotropic Factor ▪ Nutritional factors • Excess fat diet • Excess carbohydrate nutrition • Protein-poor nutrition • Hunger • Lipotropic substance deficiency • Insufficiency of essential fatty acids • Tiamin and biotin excess • Chronic alcoholism Fatty Liver and Lipotropic Factor ▪ Endocrine Disorders • Pituitary-related disorders • Cortical disorders • Thyroid disorders • Insulin disorders • Sex hormone disorders ▪ Other disorders • Central nervous system disorders • Obesity Fatty Liver and Lipotropic Factor ▪ Toxic Factors • Chemical factors o Carbon tetrachloride o Chloroform o Phosphorus

• Bacterial factors

• Anoxid factors o Anemia o The congestion Hormonal Control of Lipid Metabolism Hormonal Control of Lipid Metabolism ▪ Insulin reduces the concentration of unesterified fatty acids (NEFA) in the blood plasma by reducing the rate of separation of fat from adipose tissues.

▪ Stimulates Pentose-phosphate shunt for the use of glucose 6-phosphate via this pathway. Thus, the synthesis of NADPH and fatty acids increases. Hormonal Control of Lipid Metabolism ▪ Adrenaline stimulates the mobilization of fat from fat deposits, and thus increases the concentration of NEFA in the plasma.

▪ ACTH, TSH and glucagon hormones also stimulate fat mobilization from adipose tissue. • These hormones stimulate C-AMP as a secondary messenger, whereas Prostaglandin E1 shows the opposite of these effects. • Growth hormone also stimulates fat mobilization. • Glucocorticoid applications affect fat metabolism through carbohydrate metabolism. Question 1 ▪ Which of the following tissues can not use ketone bodies for energy purposes?

a. Kidney

b. Brain

c. Skeletal Muscle

d. Adult Liver

e. Intestine Answer: D Answer: Your Questions? Send to [email protected] References

▪ Ası. T. 1999. Tablolarla Biyokimya, Cilt 2 ▪ Engelking LR. 2014. Textbook of Veterinary Physiological Chemistry. 3rd edn. Academic Press. ▪ Eren Meryem. Prof.Dr. Ders Notları (Teşekkürlerimle) ▪ Fidancı Ulvi Reha. Prof. Dr. Ders Notları (Teşekkürlerimle) • http://80.251.40.59/veterinary.ankara.edu.tr/fidanci/Ders_Notlari/LM-Keton_Cisimleri.pdf ▪ Sözbilir Bayşu N, Bayşu N. 2008. Biyokimya. Güneş Tıp Kitapevleri, Ankara Next Chapter; Amino acid and Protein Metabolism-I For more on Biochemistry & Clinical Biochemistry and the world of laboratories follow www.biyokimya.vet @biyokimya.vet