Biosynthesis of Lipids VI

Suaib Luqman

Objectives

v To understand the biosynthesis of Tri acyl glycerol (TAG) or Triglyceride
v How do Phospholipids Synthesize
v What are the significance of their synthesis

Concept Map

. Description

Triacylglycerol also known as triacylglyceride (TAG) or Triglyceride (TG) is synthesis from a molecule of glycerol and three molecules of fatty acids. As an important and vital component of blood, TAG assist in the bidirectional transmission of glucose and adipose fat from the liver. Based on the source of oil, TAG may either be saturated or highly unsaturated. Saturated are those either having single bonds (C-C) between the carbon atoms or where carbon atoms are bonded to hydrogen (C-H) or unsaturated are those having double bonds (C=C) between carbon moieties plummeting the integer of hydrogen atoms bonded to carbon units.

TAG being the chief constituent of human skin oils is synthesized by coalescing a alcohol (glycerol) with a hydroxyl (HO-) group and organic acids (3 fatty acid) with a carboxyl (-COOH) group. Both organic acid and alcohol together form esters through the formation of ester bonds. Usually the 3 moieties of fatty acids are different and the chain length also vary with most of them have 16, 18, or 20 carbon moieties. Even number of carbon atoms is present in fatty acids of animals and plants shimmering the origin of the biosynthetic pathway from 2C (Acetyl CoA). In prokaryotes (e.g. bacteria) odd and branched chain fatty acids synthesis takes place. A complex mixture of idiosyncratic triglycerides is present in natural fats and due to this reason they liquefy over a wide temperature range. Extraordinarily, cocoa butter consists of a small number of triglycerides obtained from stearic, oleic and palmitic acids. The simplest TAGs are those where all the 3 fatty acids are indistinguishable. The TAGs stearin is obtained from stearic acid and palmitin is derived from palmitic acid. The TAGs can be acquired as polymorphs (three forms) such as α, β, and β’ vary in melting points.

In duodenum, lipids or fats cannot be absorbed in TAG form and hence hydrolysed by the pancreatic lipase that acts on ester bond resulting in the release of fatty acid and glycerol. One molecule of glycerol and one fatty acid (monoglycerides), sometimes diglycerides gets absorbed by the duodenum as TAGs have been broken down. In the intestine, after the secretion of bile and lipases, TAGs are ripped into free fatty acids and monoacylglycerol (MAG) by a procedure named as lipolysis. After lipolysis, TAGs consequently enthused to enterocytes and form chylomicrons together with protein and cholesterol. Through lymphatic system and blood, a number of tissues incarcerate the chylomicrons releasing TAGs to be utilized as energy source and stored in liver. As soon as energy requirement arises, glucagon signals the TAG breakdown by hormone sensitive lipase to discharge free fatty acids. The brain cannot exploit fatty acids as an energy source so it gets converted to a ketone and the glycerol gets transformed into glucose (gluconeogenesis) via Dihydroxyacetone phosphate (DHAP) and Glyceraldehyde 3-phosphate (G3P). Similarly, TAGs cannot pass through membranes liberally, hence breakdown by special enzyme (lipoprotein lipases) into glycerol and free fatty acids which further be taken up by cells via the fatty acid transporter (FAT).

TAGs are also vital constituent of chylomicrons and very-low-density lipoprotein (VLDL) and participate in metabolism as transporter and source of energy. A high level of TAGs in human blood is related to atherosclerosis, stroke and heart disease. The National Cholesterol Education Program has set up some guidelines for TAGs levels. The TAGs level can be tested after 8-12 h of fasting and it was observed that the level was higher after diet intake or eating.

TAGs Level	mg per dL	Mmol per L
Normal to Low Risk	<150	<1.70
Slightly Over Normal	150-199	1.70-2.25
Some Risk	200-499	2.26-5.65
High to Very High Risk	500 or higher	>5.65

As recommended by the American Heart Association, an optimal TAGs level of 1.1 mmol/L (100 mg/dL) or lesser perk up heart health. TAGs level also increases with carbohydrate rich diet (60%) and it correlates with a higher body mass index (BMI >28), insulin resistance and hyper triglyceridemia. In women also high glycemic index increases insulin production and boost TAGs level. Alcohol intake also bumps up TAGs level in blood. TAGs level can be reduced by the intake of flax seed oil and ώ-3 fatty acid from fish and other sources and through moderate exercise. Carnitine also lowers the TAGs level in blood and at some instance; fibrates have been used to fetch TAGs downward. Epanova, an ώ-3 carboxylic acid is developed by AstraZeneca for treating high TAGs level.

In oil paints, Linseed and related oils, rich in di and tri unsaturated fatty acid form, are used for coatings. In presence of oxygen, these oils also known as drying oils tend to generate heat and consolidate by polymerization offensive to the carbon backbone. During biodiesel manufacturing, TAGs undergo transesterification and the ensuing esters of fatty acid are used as fuel. The glycerin has been used in the pharmaceutical production and food manufacturing. Lysochromes (dye) are used for staining lipids, TAGs, fatty acids and lipoproteins. Other dye used include: Sudan Black B, Sudan IV, Oil Red O.

TAGs Biosynthesis

In adipocytes of the adipose tissues, fatty acids are primarily stored as TAGs for future use. Besides adipose tissues, glycerol is the key building block for TAGs synthesis in other tissues. In adipose tissues, the adipocytes lack glycerol kinase, so dihydroxyacetone phosphate (DHAP) serve as the precursor for TAG synthesis. In other tissues, DHAP can also dole out as a backbone precursor for the biosynthesis of TAG although to a much smaller level than glycerol. In TAGs, the glycerol backbone is activated by glycerol kinase via phosphorylation at C3 position. The DHAP deployment is conceded through either of the two pathways relying on whether the TAGs synthesis is carried out in endoplasmic reticulum and mitochondria or the peroxisomes and endoplasmic reticulum. The following sequential reaction takes place.

1. Glycerol 3 Phosphate dehydrogenase, an enzyme that requires NADH as the cofactor, converts DHAP to Glycerol 3 Phosphate.

2. Glycerol 3 Phosphate acyltransferase (GPAT) generates lysophosphatidic acid (monoacylglycerol phosphate structure) through esterification of a fatty acid to Glycerol 3 Phosphate. The transcription factor ChREBP controls the GPAT gene expression.

3. DHAP acyltransferase (a peroxisomal enzyme) acylates DHAP to Acyl DHAP followed by its reduction by Acyl DHAP reductase (NADPH as cofactor).

4. DHAP acyltransferase is intented to the peroxisomes via the peroxisome targeting sequence 2 (PTS2) motif recognition in the enzyme. Most of the peroxisomal enzymes include a PTS1 motif.

5. Acyl CoA synthetases activate fatty acids and 2 molecules of Acyl CoA are esterified to Glycerol 3 Phosphate to acquiesce 1, 2 Diacylglycerol Phosphate (normally recognized as phosphatidic acid).

6. Phosphatidic acid phosphatase (PAP1) removes phosphate to yield 1, 2 Diacylglycerol (a substrate for 3rd fatty acid addition).

7.Sometimes intestinal monoacylglycerols (MAGs) also dish up as substrates for 1, 2 diacylglycerols synthesis.

Transcriptional Regulation of TAG synthesis and role of Lipin Genes

Enzyme PAP1 play a crucial role in the homeostasis of phospholipid and TAG. The PAP1 gene was recognized as Smp2p in yeast (Saccharomyces cerevisiae) and the encoded protein was revealed to be the yeast ortholog of lipin 1 (mammalian protein). Ned 1p is recognized as ortholog of lipin 1 in the fission. In mammals, lipin 1 is the only recognized protein among 5 lipins and the gene LPIN1 was initially acknowledged in a mutant mouse with fatty liver dystrophy (mutation causing the disorder was found associated with LPIN1 gene). LPIN1, LPIN2, and LPIN3 are the three lipin genes found but only LPIN1 gene encodes 3 isoforms (lipin 1α, lipin 1β, and lipin 1γ) through alternative splicing. All the 5 lipin proteins own phosphatidic acid as the substrate and possess phosphatase activity which is Mn2+ or Mg2+ dependent. LPIN2 gene mutation has been linked to Majeed syndrome characterized by chronic recurrent osteomyelitis, congenital dyserythropoietic anemia, cutaneous inflammation and recurrent fever. Lipin proteins are also obligatory for fatty acid utilization and storage, co-ordination of secondary tissue glucose, development of mature adipocytes and endow with a transcriptional co-activator. The lipin proteins have protein-interaction domains (no DNA binding motifs) which allows them to function as transcriptional regulators by forming complex with nuclear receptor. They interact with PPARγ co-activator 1α (PGC 1α) and PPARα leading to better gene expression suggesting its significance in diabetes. Lipin 1 also interacts with the hepatocyte nuclear factor 4α (HNF 4α), glucocorticoid receptor and persuade the expression of PPARγ and CCAAT enhancer binding protein α (C-EBPα). Lipin 1α and lipin 1β functions in the differentiation of adipocyte where lipin 1α induces genes for differentiation while lipin 1β induces the lipid synthesizing genes expression like FAS (fatty acid synthase) and DGAT (diacylglycerol acyltransferase). The communication between lipin 1, PPARα and PGC 1α directs increased expression of Carnitine palmitoyl transferase 1, Acyl CoA oxidase and Medium chain acylCoA dehydrogenase (fatty acid oxidizing genes).

Biosynthesis of Phospholipids

Phospholipids are produced by the esterification of an alcohol (serine, ethanolamine and choline) to the phosphate of 1, 2 diacylglycerol 3 phosphate (phosphatidic acid). The alcohols contain nitrogen except glycerol and inositol and the fatty acid on C1 is saturated and on C2 is unsaturated of the glycerol backbone. The main classifications of phospholipids are: Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), Phosphatidylinositol (PI), Phosphatidylglycerol (PG), Cardiolipin (diphosphatidylglycerol, DPG). There are two known mechanism for the synthesis of phospholipids.

1. CDP activated polar head group for the attachment to the phosphate group of phosphatidic acid.

2. CDP activated 1, 2 diacylglycerol and an inactivated polar head group.

Phosphatidylcholine (PC)

Also known as lecithins, PC are neutral zwitterions at physiological pH. At C1 position, PC contain either stearic or palmitic acid and at C2 position linolenic or linoleic acid is present. Dipalmitoyllecithin is the chief (80%) phospholipid of pulmonary alveoli that hold palmitate at both C1 and C2 of glycerol. The synthesis occurs in the following steps.

1. Activation of choline by phosphorylation followed by coupling to CDP preceding its attachment to the phosphatidic acid.

2. Totting up of choline to CDP activated 1, 2 diacylglycerol.

3. Conversion of either PE or PS to PC. The switching of PS to PC first entails decarboxylation of PS to yield PE followed by a series of methylation reactions employing S-adenosylmethionine (SAM) as the methyl group donor.

Phosphatidylethanolamine (PE)

Like PC, at physiological pH, PE molecules are also neutral zwitterions. At C1 position, PE have either stearic or palmitic and at C2 position, a long chain unsaturated fatty acid (22:6, 20:4 and 18:2) is present. The PE synthesis crop up by two pathways.

1. Activation of ethanolamine by phosphorylation followed by coupling to CDP which is further transmitted from CDP ethanolamine to phosphatidic acid yielding PE.

2. Decarboxylation of PS to yield PE.

Phosphatidylserines (PS)

At physiological pH, PS carries a net charge of –1 and is poised of fatty acids alike to PE. The PS biosynthetic pathway engrosses a substitution of serine for ethanolamine in PE. This replacement arises when PE is in the membrane lipid bilayer. In addition, PS via decarboxylation reaction also dish up as a source of PE.

Phosphatidylinositol (PI)

At C1 position, PI exclusively contains stearic acid and at C2 position arachidonic acid is present. At physiological pH, PIs utterly compiled of inositol (non-phosphorylated) demonstrated a net charge of –1. PIs stay alive in membranes with an assortment of phosphate level esterified to the hydroxyl group of inositol. Phosphorylated inositol containing molecules are expressed as polyphosphoinositides (vital intracellular signal transducers originated from the plasma membrane). The PI synthesis rivets CDP activated 1, 2 diacylglycerol condensation with myo inositol followed by the phosphorylations of the hydroxyl group of inositol escorting to the polyphosphoinositides production. Phosphatidylinositol 4, 5 bisphosphate (PIP2) is an essentially imperative phospholipid of the membrane involved in the transmission signals of the cell growth and differentiation.

Phosphatidylglycerols (PG)

At physiological pH, PG displays a net charge of –1 and present in the mitochondrial membrane in high amount as a constituent of pulmonary surfactant. PG is also a well-known predecessor for cardiolipin synthesis and itself synthesized from CDP diacylglycerol and Glycerol 3 Phosphate. In addition to the above facts, an imperative role of PG is to dole out as the precursor for diphosphatidylglycerols (DPGs) synthesis.

Diphosphatidylglycerols (DPG)

At physiological pH, DPG show a net charge of –2 and are very acidic in nature. DPG is present as components of pulmonary surfactant principally in the inner mitochondrial membrane. Cardiolipins are one of the key class of DPGs synthesized by the condensation of CDP diacylglycerol with PG.

At C1 and C2 position of glycerol, fatty acid allocation within phospholipids is recurrently in flux due to the degradation and the incessant remodeling of phospholipid that transpires within the membranes. Phospholipases degrades phospholipid and there are different phospholipases that demonstrate substrate specificities for phospholipids at different positions. In most of the cases, the acyl group transferred to glycerol by Acyl transferases, is different from that of present in phospholipid when it inhabits within a membrane. In phospholipids, the acyl group remodeling is the consequence of phospholipase A1 (PLA1) and phospholipase A2 (PLA2) action. The resultant products are called Lysophospholipids and serve as substrates for Acyl transferases employing different Acyl CoA groups. In an swapping reaction catalyzed by lysolecithin : lecithin acyltransferase (LLAT), lysophospholipids also accepts Acyl groups from other phospholipids.

PLA2, an important class of enzyme (30 enzymes reported), acts on the C2 position of membrane phospholipids and releases arachidonic acid which serve as a substrate for eicosanoids biosynthesis. PLA2 exist in ten isozymic forms in the secretory pathway and abbreviated as sPLA2. sPLA2 are Ca2+-dependent, low molecular weight proteins involved in various processes such as inflammation, host defense and modification of eicosanoid generation. Another family phospholipases, the cPLA2 (cytosolic PLA2 family) embraces 3 isozymes with cPLA2-α being an indispensable factor of the initiation of arachidonic acid metabolism. Similar to that of sPLA2 enzymes, the cPLA2 are tightly regulated by Calcium ions and phosphorylation processes. Two PLA2 isozymes (iPLA2) are also recognized as Ca2+-independent for activity and are mainly involved with phospholipid remodeling. PAF, another class of PLA2 enzymes with four members, are being involved in the inactivation and hydrolysis of platelet activating factor. Like iPLA2 family, PAF hydrolyzing PLA2 isozymes are Ca2+-independent and the unique activity was called PAF acetylhydrolase (PAF AH). They also oxidized phospholipids and found to be allied with lipoprotein particles as lipoprotein associated PLA2 (Lp PLA2) family.

Plasmalogens

They are glycerol ether phospholipids with two basic types namely alkenyl ether (–O–CH=CH–) and alkyl ether (–O–CH2–). DHAP serves as the precursor for its synthesis and 3 most important classes of plasmalogens have been acknowledged: Serine, Ethanolamine and Choline plasmalogens. In cardiac tissue, choline plasmalogen is abundant and 1-O-1′-enyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor, PAF) has been recognized as an enormously powerful biological mediator, accomplished of stirring up cellular responses at low concentrations (< 10–11M). PAF functions as a moderator of anaphylactic shock, acute inflammatory reactions and hypersensitivity. In retort to antigen-IgE complexes formation, PAF is synthesized on the surfaces of basophils, eosinophils, macrophages, monocytes and neutrophils. In cells, the PAF synthesis and discharge leads to platelet aggregation and the liberation of serotonin. PAF also generates response in heart, liver, lung, smooth muscle and uterine tissues. In myelin tissue, ethanolamine plasmalogen is ubiquitous.

Summary

In this lecture we learnt about:

The Biosynthesis of TAGs
The role of Lipins
The Biosynthesis of Phospholipids

you can view video on Biosynthesis of Lipids VI

Weblinks

Books

Lehninger Principles of Biochemistry by David L. Nelson, ‎Albert L. Lehninger, ‎Michael M. Cox. 2008. https://books.google.co.in/books?isbn=071677108X
Phospholipid Biosynthesis by Edward A. Dennis, ‎Dennis E. Vance. 1992. Volume 209. https://books.google.co.in/books?id=ub0oAAAAYAAJ
Assay of Triglyceride Biosynthesis in Mice as Part of a …by Patricia Lee Simmons. 1977. https://books.google.co.in/books?id=2ehaGwAACAAJ
Essentials of Medicinal Chemistry by Andrejus Korolkovas. 2008. 2nd Edn. Page 516. https://books.google.co.in/books?isbn=8126516143
Triglycerides And Cholesterol Research by Linda T. Welson. 2006. https://books.google.co.in/books?isbn=1600211097